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pubmed23n0001_364 | Evaluation of combined pharmacological and psychotherapeutic treatment in patients with functional abdominal disorders. | 78 patients suffering from various functional abdominal complaints have been trated in a 2 x 2 double-blind design: (a) psychotherapy with Ro 5-3350 (TH/Ro); (b) psychotherapy with placebo (TH/P); (c) Ro 5-3350 without psychotherapy (NIH/Ro); (d) placebo without psychotherapy (NTH/P). Results show that a considerable amount of improvement cannot be ascribed to the two critical factors or the interaction of both, but are due to unspecific influences in the course of treatment. Some of the results concerning the combination of TH and the psychotropic drug pose interesting questions for further research and bare implications for double-blind trials of psychotropic drugs. The results suggest that possibly properties of any psychotropic drug have to be related to a doctor-patient relationship within which the personal problems of the patient are dealt with. In order to evaluate such properties, special methodological precautions have to be taken. These will be briefly discussed. | Evaluation of combined pharmacological and psychotherapeutic treatment in patients with functional abdominal disorders. 78 patients suffering from various functional abdominal complaints have been trated in a 2 x 2 double-blind design: (a) psychotherapy with Ro 5-3350 (TH/Ro); (b) psychotherapy with placebo (TH/P); (c) Ro 5-3350 without psychotherapy (NIH/Ro); (d) placebo without psychotherapy (NTH/P). Results show that a considerable amount of improvement cannot be ascribed to the two critical factors or the interaction of both, but are due to unspecific influences in the course of treatment. Some of the results concerning the combination of TH and the psychotropic drug pose interesting questions for further research and bare implications for double-blind trials of psychotropic drugs. The results suggest that possibly properties of any psychotropic drug have to be related to a doctor-patient relationship within which the personal problems of the patient are dealt with. In order to evaluate such properties, special methodological precautions have to be taken. These will be briefly discussed. | 725 |
pubmed23n0001_370 | Colorimetric determination of 5-aminosalicylic acid and its N-acetylated metabolite on urine and feces. | A simple and convenient colorimetric method is described for the quantitative determination of 5-aminosalicylic acid (5-ASA) and N-acetyl-5-ASA in urine and feces after oral administration of salicylazosulfa-pyridine (SASP), the drug of choice in the treatment of ulcerative colitis. N-acetyl-5-ASA is extracted directly from the acidified biological specimen, deacetylated, and the liberated 5-ASA subjected to a modified Bratton-Marshall reaction. The 5-ASA present in the specimen must be acetylated with acetic anhydride prior to extraction. The violet colored product of the Bratton-Marshall reaction has a lambdamax of 560 nm and conforms to Beer's law over the concentration range of 0-70 umg/ml. Average recoveries (+/- S.D., N = 6) OF 5-ASA added to rat and human urine and rat fecal homogenates were 91.6 +/- 4.9%, 102 +/- 6.0%, and 71.0 +/- 4.8%, respectively. Interference by SASP and its sulfapyridine metabolities is negligible. As demonstrated, the colorimetric method is of sufficient sensitivity for application in most metabolic and pharmacokinetic studies conducted with SASP in laboratory animals and man. | Colorimetric determination of 5-aminosalicylic acid and its N-acetylated metabolite on urine and feces. A simple and convenient colorimetric method is described for the quantitative determination of 5-aminosalicylic acid (5-ASA) and N-acetyl-5-ASA in urine and feces after oral administration of salicylazosulfa-pyridine (SASP), the drug of choice in the treatment of ulcerative colitis. N-acetyl-5-ASA is extracted directly from the acidified biological specimen, deacetylated, and the liberated 5-ASA subjected to a modified Bratton-Marshall reaction. The 5-ASA present in the specimen must be acetylated with acetic anhydride prior to extraction. The violet colored product of the Bratton-Marshall reaction has a lambdamax of 560 nm and conforms to Beer's law over the concentration range of 0-70 umg/ml. Average recoveries (+/- S.D., N = 6) OF 5-ASA added to rat and human urine and rat fecal homogenates were 91.6 +/- 4.9%, 102 +/- 6.0%, and 71.0 +/- 4.8%, respectively. Interference by SASP and its sulfapyridine metabolities is negligible. As demonstrated, the colorimetric method is of sufficient sensitivity for application in most metabolic and pharmacokinetic studies conducted with SASP in laboratory animals and man. | 747 |
pubmed23n0001_378 | Determination of inorganic pyrophosphatase in rat odontoblast layer by a radiochemical method. | The enzyme inorganic pyrophosphatase (PPiase, EC 3.6.1.1) from the odontoblastic layer of rat incisors has been studied by means of a radiochemical micromethod. The enzyme was incubated with 32P-pyrophosphate in tris-HCl buffer at 37 degrees C. The reaction was linear with time for at least 45 min, and the pH optimum was found to be 8.8, independent of the amount of pyrophosphate present. Heating the enzyme at 56 degrees C inhibited the enzyme activity rapidly, Mg2+ ions activated the enzyme by 15% at an ion concentration of 4 mM, while higher concentrations were inhibitory. Ca2+ ions and PO43-ions inhibited the enzyme at all concentrations. F- ions did not affect the PPiase at concentrations below 8 mM, whereas higher concentrations had an inhibiting effect. Urea was found to inhibit the enzyme at concentrations above 1.5 M, while EDTA was a strong inhibitor at very low concentrations. The characteristics of PPiase agree well with the properties of the enzyme nonspecific alkaline phosphatase (EC 3.1.3.1.) studied earlier. | Determination of inorganic pyrophosphatase in rat odontoblast layer by a radiochemical method. The enzyme inorganic pyrophosphatase (PPiase, EC 3.6.1.1) from the odontoblastic layer of rat incisors has been studied by means of a radiochemical micromethod. The enzyme was incubated with 32P-pyrophosphate in tris-HCl buffer at 37 degrees C. The reaction was linear with time for at least 45 min, and the pH optimum was found to be 8.8, independent of the amount of pyrophosphate present. Heating the enzyme at 56 degrees C inhibited the enzyme activity rapidly, Mg2+ ions activated the enzyme by 15% at an ion concentration of 4 mM, while higher concentrations were inhibitory. Ca2+ ions and PO43-ions inhibited the enzyme at all concentrations. F- ions did not affect the PPiase at concentrations below 8 mM, whereas higher concentrations had an inhibiting effect. Urea was found to inhibit the enzyme at concentrations above 1.5 M, while EDTA was a strong inhibitor at very low concentrations. The characteristics of PPiase agree well with the properties of the enzyme nonspecific alkaline phosphatase (EC 3.1.3.1.) studied earlier. | 784 |
pubmed23n0001_385 | Gastric emptying of liquids after different vagotomies and pyloroplasty. | Gastric emptying of five liquid meals which differ in their physicochemical properties have been measured in control dogs and dogs that have received a Heinecke-Mikulicz pyloroplasty alone, proximal gastric vagotomy without drainage, selective gastric vagotomy and pyloroplasty and truncal vagotomy and pyloroplasty. The first two phases of emptying have been computed by the method of least squares to obtain a logarithmic-linear pattern and are expressed as relative rates: The initial post-ingestion process is characterized by beta or the average relative rate of emptying in the first ten minutes, the basic or exponential rate as beta and the change in rate from the initial to basic pattern as deltabeta. Each measure of gastric emptying was statistically analyzed to determine specific differences in rates between the operations studied. We confirmed the earlier claims that pyloroplasty alone does not change the emptying rate of liquid meals. Each measure or phase of emptying varies consistently across the operations from meal to meal tested. Initial emptying after all three vagotomies is significantly faster than control with progressive rate increases as proximal gastric vagotomy is compared with selective gastric vagotomy with pyloroplasty and with truncal vagotomy with pyloroplasty, probably indicative of gastric fundal loss of accommodation to volume distention after denervation. The basic exponential pattern of emptying is not lost after any of the operations studied. The basic rate after proximal gastric vagotomy and selective gastric vagotomy with pyloroplasty is nearly identical, slightly delayed from the control rate and significantly slower than the more rapid rate after truncal vagotomy with pyloroplasty. Possible explanations for these are discussed and imply a particular importance of the hepatic and celiac vagal fibers, sectioned only with truncal vagotomy, in the regulation of gastric emptying of liquids. | Gastric emptying of liquids after different vagotomies and pyloroplasty. Gastric emptying of five liquid meals which differ in their physicochemical properties have been measured in control dogs and dogs that have received a Heinecke-Mikulicz pyloroplasty alone, proximal gastric vagotomy without drainage, selective gastric vagotomy and pyloroplasty and truncal vagotomy and pyloroplasty. The first two phases of emptying have been computed by the method of least squares to obtain a logarithmic-linear pattern and are expressed as relative rates: The initial post-ingestion process is characterized by beta or the average relative rate of emptying in the first ten minutes, the basic or exponential rate as beta and the change in rate from the initial to basic pattern as deltabeta. Each measure of gastric emptying was statistically analyzed to determine specific differences in rates between the operations studied. We confirmed the earlier claims that pyloroplasty alone does not change the emptying rate of liquid meals. Each measure or phase of emptying varies consistently across the operations from meal to meal tested. Initial emptying after all three vagotomies is significantly faster than control with progressive rate increases as proximal gastric vagotomy is compared with selective gastric vagotomy with pyloroplasty and with truncal vagotomy with pyloroplasty, probably indicative of gastric fundal loss of accommodation to volume distention after denervation. The basic exponential pattern of emptying is not lost after any of the operations studied. The basic rate after proximal gastric vagotomy and selective gastric vagotomy with pyloroplasty is nearly identical, slightly delayed from the control rate and significantly slower than the more rapid rate after truncal vagotomy with pyloroplasty. Possible explanations for these are discussed and imply a particular importance of the hepatic and celiac vagal fibers, sectioned only with truncal vagotomy, in the regulation of gastric emptying of liquids. | 800 |
pubmed23n0001_387 | Gastric fibrinolysis. | Gastric juice from 15 normals, 20 patients with gastric ulcer and 14 patients with erosive haemorrhagic gastroduodenitis was investigated in respect of its activity on unheated and heated fibrin plates and its content of FDP and plasminogen or plasmin with immunochemical methods. Gastric juice from normals showed no activity on unheated and heated fibrin plates, and no FDP or plasminogen could be demonstrated. In the patients with gastric ulcer the gastric juice showed little or no fibrinolytic activity on fibrin plates except in 2, who had regurgitation of duodenal juice and neutral pH of the juice. These patients had equally high activity on heated as on unheated plates and no plasmin could be demonstrated. It was shown that this activity was not due to fibrinolysis, but to non-specific proteolytic activity (probably trypsin). The patients with erosive haemorrhage gastroduodenitis exhibited quite a different picture. The gastric juice from these patients showed extremely high activity on fibrin plates, the activity was higher on unheated than on heated plates. The activity was inhibited in vitro by addition of EACA and in vivo after administration of AMCA. The occurence of plasmic could be demonstrated directly immunologically in the gastric juice. By comparsion of plasmin and trypsin in various assays it could further be improved that the gastric juice in these cases contained plasminogen activator and plasmin. The patients with erosive haemorrhagic gastroduodenitis showed no increase in fibrinolysis in the blood, but low values for plasminogen and alpha2-M, and the serum contained FDP. These findings in the blood and gastric juice were interpreted as signs of local fibrinolysis in the stomach and duodenum. There is reason to assume that this gastric fibrinolysis contributes substantially to the bleeding tendency. The effect of administration of AMCA on fibrinolytic activity and the haemorrhage lends support to the assumption of such a mechanism. | Gastric fibrinolysis. Gastric juice from 15 normals, 20 patients with gastric ulcer and 14 patients with erosive haemorrhagic gastroduodenitis was investigated in respect of its activity on unheated and heated fibrin plates and its content of FDP and plasminogen or plasmin with immunochemical methods. Gastric juice from normals showed no activity on unheated and heated fibrin plates, and no FDP or plasminogen could be demonstrated. In the patients with gastric ulcer the gastric juice showed little or no fibrinolytic activity on fibrin plates except in 2, who had regurgitation of duodenal juice and neutral pH of the juice. These patients had equally high activity on heated as on unheated plates and no plasmin could be demonstrated. It was shown that this activity was not due to fibrinolysis, but to non-specific proteolytic activity (probably trypsin). The patients with erosive haemorrhage gastroduodenitis exhibited quite a different picture. The gastric juice from these patients showed extremely high activity on fibrin plates, the activity was higher on unheated than on heated plates. The activity was inhibited in vitro by addition of EACA and in vivo after administration of AMCA. The occurence of plasmic could be demonstrated directly immunologically in the gastric juice. By comparsion of plasmin and trypsin in various assays it could further be improved that the gastric juice in these cases contained plasminogen activator and plasmin. The patients with erosive haemorrhagic gastroduodenitis showed no increase in fibrinolysis in the blood, but low values for plasminogen and alpha2-M, and the serum contained FDP. These findings in the blood and gastric juice were interpreted as signs of local fibrinolysis in the stomach and duodenum. There is reason to assume that this gastric fibrinolysis contributes substantially to the bleeding tendency. The effect of administration of AMCA on fibrinolytic activity and the haemorrhage lends support to the assumption of such a mechanism. | 807 |
pubmed23n0001_391 | Effects of oxygen saturation and pCO2 on brain uptake of glucose analogues in rabbits. | The effect of oxygen saturation and PCO2 on brain uptake of glucose analogues was studied in rabbits. Using a modified Oldendorf technique, 14C-labeled glucose analogues with a 3H2O reference standard were introduced into the cerebral circulation via the common carotid artery, and the radioactivity of the ipsilateral cerebral cortex was counted and expressed in terms of a brain uptake index (BUI). Severe hypoxia (oxygen saturation less than or equal to 18%) resulted in approximately a 40% decrease in the BUI of 2-deoxy-D-glucose and a 45% decrease in the BUI of 3-0-methyl-D-glucose. Severe hypercapnia (PCO2 = 100 mm Hg) caused a 45% decrease in the BUI of both of these glucose analogues. Hypercapnia superimposed on severe hypoxia had no additional effect. Hypocapnia (PCO2 = 15 mm Hg) increased the BUI of 3-0-methyl-D-glucose by 35% of the control value, and this increase was extremely sensitive to competitive inhibition. When BUI values were plotted against pH rather than PCO2 for the same experiments, there was a good correlation with the calculated linear regression. These results are compared with previous findings on pathologically induced changes in brain uptake of glucose analogues, and the possible role of blood flow is considered in detail. | Effects of oxygen saturation and pCO2 on brain uptake of glucose analogues in rabbits. The effect of oxygen saturation and PCO2 on brain uptake of glucose analogues was studied in rabbits. Using a modified Oldendorf technique, 14C-labeled glucose analogues with a 3H2O reference standard were introduced into the cerebral circulation via the common carotid artery, and the radioactivity of the ipsilateral cerebral cortex was counted and expressed in terms of a brain uptake index (BUI). Severe hypoxia (oxygen saturation less than or equal to 18%) resulted in approximately a 40% decrease in the BUI of 2-deoxy-D-glucose and a 45% decrease in the BUI of 3-0-methyl-D-glucose. Severe hypercapnia (PCO2 = 100 mm Hg) caused a 45% decrease in the BUI of both of these glucose analogues. Hypercapnia superimposed on severe hypoxia had no additional effect. Hypocapnia (PCO2 = 15 mm Hg) increased the BUI of 3-0-methyl-D-glucose by 35% of the control value, and this increase was extremely sensitive to competitive inhibition. When BUI values were plotted against pH rather than PCO2 for the same experiments, there was a good correlation with the calculated linear regression. These results are compared with previous findings on pathologically induced changes in brain uptake of glucose analogues, and the possible role of blood flow is considered in detail. | 821 |
pubmed23n0001_397 | [Properties of NAD-glycohydrolase of the nuclei of the liver cells of rats]. | Certain properties of the rat liver cell nuclei NAD-glycohydrolase (EC 3.2.2.5) were investigated. It is established that its highest activity is at 37 degrees with activation energy equal to 9480 cal/M and with factor Q10 equal to 1.5. The enzyme pH optimum in 0.2 M tris acetate is equal to 6.5 and in 0.2 potassium phosphate - 7.5. It was shown that the enzyme manifests its strict specificity only with beta-NAD, and it hardly decomposes NADP without affecting NADH, NADPH and NMN. The apparent Km value of the enzyme with respect to NAD is established. Isonicotinic acid hydrazide, nicotinamide and to the less extent nicotinic acid inhibit the enzymatic activity of nuclei. EDTA, EGTA, p-CMB, mercaptoethanol do not cause any changes in the rat liver cells nuclei NADase activity. | [Properties of NAD-glycohydrolase of the nuclei of the liver cells of rats]. Certain properties of the rat liver cell nuclei NAD-glycohydrolase (EC 3.2.2.5) were investigated. It is established that its highest activity is at 37 degrees with activation energy equal to 9480 cal/M and with factor Q10 equal to 1.5. The enzyme pH optimum in 0.2 M tris acetate is equal to 6.5 and in 0.2 potassium phosphate - 7.5. It was shown that the enzyme manifests its strict specificity only with beta-NAD, and it hardly decomposes NADP without affecting NADH, NADPH and NMN. The apparent Km value of the enzyme with respect to NAD is established. Isonicotinic acid hydrazide, nicotinamide and to the less extent nicotinic acid inhibit the enzymatic activity of nuclei. EDTA, EGTA, p-CMB, mercaptoethanol do not cause any changes in the rat liver cells nuclei NADase activity. | 830 |
pubmed23n0001_412 | Simultaneous determination of 5'-nucleotidase and alkaline phosphatase activities in serum. | A simple method is described for the simultaneous determination of alkaline phosphatase (EC 3.1.3.1) and 5'-nucleotidase (EC 3.1.3.5) in serum. The method is based on the determination of inorganic phosphorus released by the action of the two enzymes on adenosine-5'-monophosphate at pH 9.5 (200 mmol/l tris-buffer) in the presence and absence of L-cysteine. This amino acid at a concentration of 2--10 mmol/l was found to be a specific inhibitor for alkaline phosphatase but with no effect on 5'-nucleotidase activity. | Simultaneous determination of 5'-nucleotidase and alkaline phosphatase activities in serum. A simple method is described for the simultaneous determination of alkaline phosphatase (EC 3.1.3.1) and 5'-nucleotidase (EC 3.1.3.5) in serum. The method is based on the determination of inorganic phosphorus released by the action of the two enzymes on adenosine-5'-monophosphate at pH 9.5 (200 mmol/l tris-buffer) in the presence and absence of L-cysteine. This amino acid at a concentration of 2--10 mmol/l was found to be a specific inhibitor for alkaline phosphatase but with no effect on 5'-nucleotidase activity. | 859 |
pubmed23n0001_420 | [Sympathico-adrenal system activity in a primary immune response]. | Experiments were carried out on linear mice immunized with sheep erythrocytes; it was found that the primary immune respose developed against the background of significant changes in the state of the sympathico-adrenal system, whose activity was determined by the dynamics of catecholamines in the blood and in the tissues of a number of organs, including the thymus, the spleen and the lymph nodes. By comparing the value of specific and neurohumoral indices it was revealed that the neurohumoral shifts preceded the maximal development of the immune response. On the example of studying the catecholamine dynamics the opinion on a close association between the state of the regulatory mechanisms and the effector formations responsible for the formation of specific immunological reactions was confirmed. It is suggested that a full-value immunological response developed on condition of activation of the sympathico-adrenal system. | [Sympathico-adrenal system activity in a primary immune response]. Experiments were carried out on linear mice immunized with sheep erythrocytes; it was found that the primary immune respose developed against the background of significant changes in the state of the sympathico-adrenal system, whose activity was determined by the dynamics of catecholamines in the blood and in the tissues of a number of organs, including the thymus, the spleen and the lymph nodes. By comparing the value of specific and neurohumoral indices it was revealed that the neurohumoral shifts preceded the maximal development of the immune response. On the example of studying the catecholamine dynamics the opinion on a close association between the state of the regulatory mechanisms and the effector formations responsible for the formation of specific immunological reactions was confirmed. It is suggested that a full-value immunological response developed on condition of activation of the sympathico-adrenal system. | 877 |
pubmed23n0001_421 | [Covalent binding of peroxidase to CH- and AH-Sepharose 4 B]. | The properties of peroxidase insolubilized by covalent binding to CH- and AH-Sepharose 4 B in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are described. CH-Sepharose 4 B bound peroxidase yields an enzyme preparation with a residual specific activity of 60.6%. When bound to AH-Sepharose 4 B, the residual specific activity is to 78%. The reasons of these differences in the catalytic activity of the two insolubilized enzyme preparations are discussed. By covalent binding on CH- and AH-Sepharose 4 B, peroxidase exibits no changes in its pH optimum; it virtually keeps the same activity after being used ten times. Insolubilized peroxidase preparations, dried and reimbibed after being stored for 6 weeks at room temperature still display 50% of the initial specific activity of the insolubilized enzyme. Stored in acetate buffer, the enzyme preparations maintain their activity during all this interval. | [Covalent binding of peroxidase to CH- and AH-Sepharose 4 B]. The properties of peroxidase insolubilized by covalent binding to CH- and AH-Sepharose 4 B in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are described. CH-Sepharose 4 B bound peroxidase yields an enzyme preparation with a residual specific activity of 60.6%. When bound to AH-Sepharose 4 B, the residual specific activity is to 78%. The reasons of these differences in the catalytic activity of the two insolubilized enzyme preparations are discussed. By covalent binding on CH- and AH-Sepharose 4 B, peroxidase exibits no changes in its pH optimum; it virtually keeps the same activity after being used ten times. Insolubilized peroxidase preparations, dried and reimbibed after being stored for 6 weeks at room temperature still display 50% of the initial specific activity of the insolubilized enzyme. Stored in acetate buffer, the enzyme preparations maintain their activity during all this interval. | 878 |
pubmed23n0001_428 | Acute otitis media. A clinical bacteriological and serological study of children with frequent episodes of acute otitis media. | A series of episodes of acute otitis media was studied with reference to bacterial findings and specific serological responses in 48 children with histories of frequent episodes before. D. pneumoniae and H. influenzae were the most frequently isolated pathogens. Re-isolations after therapy were often made in episodes with slow healing or therapeutic failure. Most children harboured pathogens in nasopharynx even when they had no signs of respiratory tract infections. Homologous relapses were seen only in few cases and never with pneumococcus type 3 and only once with H. influenzae type b. Specific serological responses were demonstrable generally in children over 2 years of age. D. pneumococcus type 3 and H. influenzae type b generally provoked antibody response. No levels indicating immunoglobulin deficiencies could be found in the children. | Acute otitis media. A clinical bacteriological and serological study of children with frequent episodes of acute otitis media. A series of episodes of acute otitis media was studied with reference to bacterial findings and specific serological responses in 48 children with histories of frequent episodes before. D. pneumoniae and H. influenzae were the most frequently isolated pathogens. Re-isolations after therapy were often made in episodes with slow healing or therapeutic failure. Most children harboured pathogens in nasopharynx even when they had no signs of respiratory tract infections. Homologous relapses were seen only in few cases and never with pneumococcus type 3 and only once with H. influenzae type b. Specific serological responses were demonstrable generally in children over 2 years of age. D. pneumococcus type 3 and H. influenzae type b generally provoked antibody response. No levels indicating immunoglobulin deficiencies could be found in the children. | 885 |
pubmed23n0001_439 | Inpatient and outpatient patterns of psychotropic drug prescribing by nonpsychiatrist physicians. | The authors found that among 228 general hospital patients, minor tranquilizers were prescribed most often and with the least justification and that major tranquilizers were prescribed sparingly and by and large judiciously. Antidepressants were given less often than would be justified by the incidence of depressive illness among these patients. Nonrecognition of depression in patients with somatic complaints and autonomic signs of depression contributed to this lack of treatment. | Inpatient and outpatient patterns of psychotropic drug prescribing by nonpsychiatrist physicians. The authors found that among 228 general hospital patients, minor tranquilizers were prescribed most often and with the least justification and that major tranquilizers were prescribed sparingly and by and large judiciously. Antidepressants were given less often than would be justified by the incidence of depressive illness among these patients. Nonrecognition of depression in patients with somatic complaints and autonomic signs of depression contributed to this lack of treatment. | 915 |
pubmed23n0001_442 | Characterization of a caprine herpesvirus. | A virus, which was isolated from kids (Capra hircus) affected with a relatively severe generalized infection, was found to contain DNA and to have a buoyant density of 1.2820 g/cm3. The virus was sensitive to the action of lipid solvents and trypsin and was rapidly inactivated at pH 3.0 and at temperatures of 50 and 56 C. The virion, an icosahedron consisting of a nucleoid surrounded by a double membrane, measured approximately 135 nm in diameter. On the basis of its chemical and physical properties, the virus is considered a herpesvirus. | Characterization of a caprine herpesvirus. A virus, which was isolated from kids (Capra hircus) affected with a relatively severe generalized infection, was found to contain DNA and to have a buoyant density of 1.2820 g/cm3. The virus was sensitive to the action of lipid solvents and trypsin and was rapidly inactivated at pH 3.0 and at temperatures of 50 and 56 C. The virion, an icosahedron consisting of a nucleoid surrounded by a double membrane, measured approximately 135 nm in diameter. On the basis of its chemical and physical properties, the virus is considered a herpesvirus. | 933 |
pubmed23n0001_447 | The virus hypothesis in systemic lupus erythematosus. | Type-C viruses are currently the prime etiologic candidates in systemic lupus erythematosus. On the basis of knowledge gained from studies of experimental and human models of chronic viral disease, there are possible pathogenetic roles of a virus in systemic lupus erythematosus. Experimental attempts at implicating specific viruses have been predominantly negative, but evidence for enhanced type-C-virus expression has recently been reported. | The virus hypothesis in systemic lupus erythematosus. Type-C viruses are currently the prime etiologic candidates in systemic lupus erythematosus. On the basis of knowledge gained from studies of experimental and human models of chronic viral disease, there are possible pathogenetic roles of a virus in systemic lupus erythematosus. Experimental attempts at implicating specific viruses have been predominantly negative, but evidence for enhanced type-C-virus expression has recently been reported. | 938 |
pubmed23n0001_450 | A hepatoma-associated alkaline phosphatase, the Kasahara isozyme, compared with one of the isozymes of FL amnion cells. | It was found that a human hepatoma-associated ALP (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.1) shared electrophoretic mobility, inactivation by urea, inhibition by inorganic phosphate, ethylenediaminetetraacetate, and amino acids (L-phenylalanine, L-leucine and L-homoarginine), heat stability, sensitivity to neuraminidase, pH optimum, Km value, and antigen site with fast moving ALP isozymes of FL cell strain derived from human amniotic membrane. However, 40-week-old fresh amniotic membrane lacked this isozyme. Instead, it had a placental type ALP consisting of minor components. The other ALP isozyme of FL cells had properties common to hepatoma ALP with regard to L-phenylalanine sensitivity, inhibition by ethylenediaminetetraacetate, inactivation by urea, and antigen site, but differed from it in electrophoretic mobility, sensitivity to L-leucine and L-homoarginine, and the presence of another antigen site. It was more heat stable and more sensitive to inhibition by inorganic phosphate than Hepatoma AP. The possible regulatory mechanism between the hepatoma-type ALP and the placental type ALP in the amnion cells is considered. | A hepatoma-associated alkaline phosphatase, the Kasahara isozyme, compared with one of the isozymes of FL amnion cells. It was found that a human hepatoma-associated ALP (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.1) shared electrophoretic mobility, inactivation by urea, inhibition by inorganic phosphate, ethylenediaminetetraacetate, and amino acids (L-phenylalanine, L-leucine and L-homoarginine), heat stability, sensitivity to neuraminidase, pH optimum, Km value, and antigen site with fast moving ALP isozymes of FL cell strain derived from human amniotic membrane. However, 40-week-old fresh amniotic membrane lacked this isozyme. Instead, it had a placental type ALP consisting of minor components. The other ALP isozyme of FL cells had properties common to hepatoma ALP with regard to L-phenylalanine sensitivity, inhibition by ethylenediaminetetraacetate, inactivation by urea, and antigen site, but differed from it in electrophoretic mobility, sensitivity to L-leucine and L-homoarginine, and the presence of another antigen site. It was more heat stable and more sensitive to inhibition by inorganic phosphate than Hepatoma AP. The possible regulatory mechanism between the hepatoma-type ALP and the placental type ALP in the amnion cells is considered. | 948 |
pubmed23n0001_451 | Gamma-glutamyl transpeptidase. A sensitive indicator of renal ischaemic injury in experimental animals and renal homograft rejection in man. | The sites of ischaemic injury within the kidney are reviewed and the diagnostic value of measurements of plasma and urinary enzymes in renal ischaemic injury and in renal homotransplant rejection in experimental animals and man is examined. Gamma-glutamyl transpeptidase (gamma-GT) is an enzyme primarily located in the brush border of the proximal convoluted tubule of the kidney. Its unique localization in the cells most easily damaged by ischaemia and its ease of assay provide the rationale for its use in the measurement and diagnosis of renal ischaemic injury. gamma-GT activity was measured in dogs undergoing varying periods of renal ischaemia and under conditions of local renal hypothermia and was shown to be a sensitive indicator of ischaemic injury. Twenty consecutive patients undergoing renal homotransplantation were studied by daily estimation of their 24-h urinary gamma-GT activity; excellent correlation was obtained between raised levels of this enzyme and the clinical diagnosis of transplant rejection. | Gamma-glutamyl transpeptidase. A sensitive indicator of renal ischaemic injury in experimental animals and renal homograft rejection in man. The sites of ischaemic injury within the kidney are reviewed and the diagnostic value of measurements of plasma and urinary enzymes in renal ischaemic injury and in renal homotransplant rejection in experimental animals and man is examined. Gamma-glutamyl transpeptidase (gamma-GT) is an enzyme primarily located in the brush border of the proximal convoluted tubule of the kidney. Its unique localization in the cells most easily damaged by ischaemia and its ease of assay provide the rationale for its use in the measurement and diagnosis of renal ischaemic injury. gamma-GT activity was measured in dogs undergoing varying periods of renal ischaemia and under conditions of local renal hypothermia and was shown to be a sensitive indicator of ischaemic injury. Twenty consecutive patients undergoing renal homotransplantation were studied by daily estimation of their 24-h urinary gamma-GT activity; excellent correlation was obtained between raised levels of this enzyme and the clinical diagnosis of transplant rejection. | 949 |
pubmed23n0001_458 | Nephrotic syndrome in indian children. | A clinicopathological study of 206 Indian children with nephrotic syndrome showed a primary renal cause in 195 (96%), of which 77% were boys. In 126 children (96 boys, 30 girls) onset of the disorder occurred before the age of 5 years. Renal biopsy showed minimal lesions in 150 patients (77%); in 85 of these biopsy was done 3 months to 16 years after onset of the nephrotic syndrome. Significant renal histological abnormalities in 45 cases were labelled as mesangiocapillary 8, mesangioproliferative 4, proliferative with extensive crescents 2, membranous 3, focal segmental glomerulosclerosis 9, focal global glomerulosclerosis 2, advanced nonspecific 8, and mild proliferative 9. Nephritic manifestations were mainly associated with significant renal lesions, which were more frequently encountered when the onset of disease was after the age of 5 years. Clearance of proteinuria with corticosteroid therapy was practically confined to patients with minimal or mild renal histological changes. Our findings suggest that the pattern of idiopathic nephrotic syndrome in Indian children is similar to that reported from Western countries. | Nephrotic syndrome in indian children. A clinicopathological study of 206 Indian children with nephrotic syndrome showed a primary renal cause in 195 (96%), of which 77% were boys. In 126 children (96 boys, 30 girls) onset of the disorder occurred before the age of 5 years. Renal biopsy showed minimal lesions in 150 patients (77%); in 85 of these biopsy was done 3 months to 16 years after onset of the nephrotic syndrome. Significant renal histological abnormalities in 45 cases were labelled as mesangiocapillary 8, mesangioproliferative 4, proliferative with extensive crescents 2, membranous 3, focal segmental glomerulosclerosis 9, focal global glomerulosclerosis 2, advanced nonspecific 8, and mild proliferative 9. Nephritic manifestations were mainly associated with significant renal lesions, which were more frequently encountered when the onset of disease was after the age of 5 years. Clearance of proteinuria with corticosteroid therapy was practically confined to patients with minimal or mild renal histological changes. Our findings suggest that the pattern of idiopathic nephrotic syndrome in Indian children is similar to that reported from Western countries. | 973 |
pubmed23n0001_461 | A reinvestigation of the sites of transcription and translation of Euglena chloroplastic phenylalanyl-tRNA synthetase. | An attempt was made to determine the sites of chloroplast phenylalanyl-tRNA synthetase transcription and translation. Inhibitors of bacterial RNA and protein synthesis were added to logarithmic and stationary phase cultures of Euglena gracilis wild-type B. Logarithmic phase cultures were sensitive to both types of inhibitors. In stationary phase cultures plastid synthetase was reduced by RNA but not by protein synthesis inhibitors. The effect of the antibiotics on the mitochondrial enzyme was also noted. Several possible explanations of these resuults are discussed. | A reinvestigation of the sites of transcription and translation of Euglena chloroplastic phenylalanyl-tRNA synthetase. An attempt was made to determine the sites of chloroplast phenylalanyl-tRNA synthetase transcription and translation. Inhibitors of bacterial RNA and protein synthesis were added to logarithmic and stationary phase cultures of Euglena gracilis wild-type B. Logarithmic phase cultures were sensitive to both types of inhibitors. In stationary phase cultures plastid synthetase was reduced by RNA but not by protein synthesis inhibitors. The effect of the antibiotics on the mitochondrial enzyme was also noted. Several possible explanations of these resuults are discussed. | 976 |
pubmed23n0001_468 | [Morphological manifestations and morphogenesis of the graft vs. host reaction in the brain of F1 hybrids following the administration of parental lymphoid cells and its effect on tumor inoculation]. | It was established that intracerebral introduction of 10 min parental cells of the spleen brought about in hybrids (CBAXC57Bl/6) F1 infiltrative-destructive changes (development of lymphocytic infiltrations, dystrophy in the nerve cells, myelin fibres and neurogliacytes) the intensity of which was found to be considerably higher in cases of injection of parental spleen cells from specifically sensitized donors. Spleen cells of mice CBA produced much greater changes as compared with those produced by spleen cells of mice C57Bl/6. Inoculation into the mice brain of 10 mln of spleen cells together with tumour in a dose of 1500 cells produced a clear cut inhibitory effect on the tumour growth, this effect being more pronounced following introduction of sensitized cells of the spleen of mice CBA. | [Morphological manifestations and morphogenesis of the graft vs. host reaction in the brain of F1 hybrids following the administration of parental lymphoid cells and its effect on tumor inoculation]. It was established that intracerebral introduction of 10 min parental cells of the spleen brought about in hybrids (CBAXC57Bl/6) F1 infiltrative-destructive changes (development of lymphocytic infiltrations, dystrophy in the nerve cells, myelin fibres and neurogliacytes) the intensity of which was found to be considerably higher in cases of injection of parental spleen cells from specifically sensitized donors. Spleen cells of mice CBA produced much greater changes as compared with those produced by spleen cells of mice C57Bl/6. Inoculation into the mice brain of 10 mln of spleen cells together with tumour in a dose of 1500 cells produced a clear cut inhibitory effect on the tumour growth, this effect being more pronounced following introduction of sensitized cells of the spleen of mice CBA. | 984 |
pubmed23n0001_471 | Molecular nature of beta-galactosidase from different tissues in two strains of the house mouse. | One inbred mouse strain, C57BL/Kl, has high galactosidase activities in all tissues while another strain, DBA/2/Kl, has low activities determined by the Bgs locus. Beta-Galactosidase from these two strains was partly purified by a five-step procedure: acidification, ammonium sulfate precipitation, gel filtration at two pHs, and isoelectric focusing. No qualitative differences were found between the enzyme preparations from the two strains. They had identical heat inactivation curves, pH optima, molecular weight, and isoelectric points, and the Km values were very similar. It thus seems that this genetic difference in enzyme activity probably cannot be explained by a variation of the galactosidase-specific activity but rather reflects a difference in number of enzyme molecules. Eight different isoenzymes were separated from liver, kidney, and spleen. Each isoenzyme has a different electrophoretic mobility and there is a stepwise increase in molecular weight from 143,000 to 380,000 beginning with the protein having the lowest isoelectric point. A likely interpretation is that the isoenzymes bind a smaller polypeptide in varying numbers in addition to the enzymatic polypeptide per se. | Molecular nature of beta-galactosidase from different tissues in two strains of the house mouse. One inbred mouse strain, C57BL/Kl, has high galactosidase activities in all tissues while another strain, DBA/2/Kl, has low activities determined by the Bgs locus. Beta-Galactosidase from these two strains was partly purified by a five-step procedure: acidification, ammonium sulfate precipitation, gel filtration at two pHs, and isoelectric focusing. No qualitative differences were found between the enzyme preparations from the two strains. They had identical heat inactivation curves, pH optima, molecular weight, and isoelectric points, and the Km values were very similar. It thus seems that this genetic difference in enzyme activity probably cannot be explained by a variation of the galactosidase-specific activity but rather reflects a difference in number of enzyme molecules. Eight different isoenzymes were separated from liver, kidney, and spleen. Each isoenzyme has a different electrophoretic mobility and there is a stepwise increase in molecular weight from 143,000 to 380,000 beginning with the protein having the lowest isoelectric point. A likely interpretation is that the isoenzymes bind a smaller polypeptide in varying numbers in addition to the enzymatic polypeptide per se. | 988 |
pubmed23n0001_474 | Influence of some physical factors on survival of Marek's disease vaccine virus. | Cell-associated Marek's disease (MD) vaccine was suspended at dilutions normally used for vaccination in seven commercially available diluents and in tryptose phosphate broth. The stability of diluted vaccines was determined by assay in cell cultures subjected to 0 to 37 C for 0 to 90 minutes. Optimum holding temperatures for MD vaccine virus survival varied with the specific diluents employed. Some diluents afforded greatest survival when dilution was at 0 C and held at 0 C, while others performed best when dilution was at 25 C followed by cooling and holding at 0 C. Diluents which allowed greatest survival when tested at 37 C also performed well under other temperature regimes. Spectinomycin dihydrochloride pentahydrate and various buffering compounds were added to commercial diluents used for diluting MD vaccine. Additives producing osmolality of 745 mOsm/kg and higher markedly reduced vaccine virus survival. The adverse effects of high osmotic pressure were accentuated by extended holding time, elevated incubation temperature, and physical manipulations including mechanical mixing or expressing through a syringe and needle. Satisfactory MD vaccine virus survival was afforded by a commercial diluent especially formulated to accommodate the pH osmolality changes produced by adding spectinomycin dihydrochloride pentahydrate. | Influence of some physical factors on survival of Marek's disease vaccine virus. Cell-associated Marek's disease (MD) vaccine was suspended at dilutions normally used for vaccination in seven commercially available diluents and in tryptose phosphate broth. The stability of diluted vaccines was determined by assay in cell cultures subjected to 0 to 37 C for 0 to 90 minutes. Optimum holding temperatures for MD vaccine virus survival varied with the specific diluents employed. Some diluents afforded greatest survival when dilution was at 0 C and held at 0 C, while others performed best when dilution was at 25 C followed by cooling and holding at 0 C. Diluents which allowed greatest survival when tested at 37 C also performed well under other temperature regimes. Spectinomycin dihydrochloride pentahydrate and various buffering compounds were added to commercial diluents used for diluting MD vaccine. Additives producing osmolality of 745 mOsm/kg and higher markedly reduced vaccine virus survival. The adverse effects of high osmotic pressure were accentuated by extended holding time, elevated incubation temperature, and physical manipulations including mechanical mixing or expressing through a syringe and needle. Satisfactory MD vaccine virus survival was afforded by a commercial diluent especially formulated to accommodate the pH osmolality changes produced by adding spectinomycin dihydrochloride pentahydrate. | 987 |
pubmed23n0001_475 | Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs. | The isolation is described of pure cultures of three non-methane-utilizing methylotrophic bacteria which, together with the previously described Bacillus PM6, have a very limited range of growth substrates; these organisms are designated "restricted facultative' methylotrophs. Two of these isolates, W6A and W3A1, grow only on glucose out of 50 non-C1 compounds tested, whereas the third isolate S2A1 and Bacillus PM6 grow on betaine, glucose, gluconate, alanine, glutamate, citrate and nutrient agar, but not on any of a further 56 non-C1 compounds. Crude sonic extracts of trimethylamine-grown and glucose-grown W6A and W3A1 isolates, and of trimethylamine-grown C2A1 (an obligate methylotroph) contain (i) no detectable 2-oxogltarate dehydrogenase activity, (ii) very low or zero specific activities of succinate dehydrogenase and succinyl-CoA synthetase and (iii) NAD+-dependent isocitrate dehydrogenase activity. Extracts of trimethylamine-grown PM6 and S2A1 methylotrophs have (i) very low 2-oxoglutarate dehydrogenase specific activities, (ii) comparatively high specific activities of succinate dehydrogenase, malate dehydrogenase and succinyl-CoA synthetase and (iii) NADP+-dependent isocitrate dehydrogenase activity but no NAD+-dependent isocitrate dehydrogenase activity. The activities of most of these enzymes are increased during growth on glucose, alanine, glutamate or citrate, but only very low 2-oxoglutarate dehydrogenase activities are present under all growth conditions. The restricted facultative methylotrophs grow on certain non-C1 compounds in the absence of 2-oxoglutarate dehydrogenase and, in some cases, of other enzymes of the tricarboxylic acid cycle; these lesions cannot therefore be the sole cause of obligate methylotrophy. | Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs. The isolation is described of pure cultures of three non-methane-utilizing methylotrophic bacteria which, together with the previously described Bacillus PM6, have a very limited range of growth substrates; these organisms are designated "restricted facultative' methylotrophs. Two of these isolates, W6A and W3A1, grow only on glucose out of 50 non-C1 compounds tested, whereas the third isolate S2A1 and Bacillus PM6 grow on betaine, glucose, gluconate, alanine, glutamate, citrate and nutrient agar, but not on any of a further 56 non-C1 compounds. Crude sonic extracts of trimethylamine-grown and glucose-grown W6A and W3A1 isolates, and of trimethylamine-grown C2A1 (an obligate methylotroph) contain (i) no detectable 2-oxogltarate dehydrogenase activity, (ii) very low or zero specific activities of succinate dehydrogenase and succinyl-CoA synthetase and (iii) NAD+-dependent isocitrate dehydrogenase activity. Extracts of trimethylamine-grown PM6 and S2A1 methylotrophs have (i) very low 2-oxoglutarate dehydrogenase specific activities, (ii) comparatively high specific activities of succinate dehydrogenase, malate dehydrogenase and succinyl-CoA synthetase and (iii) NADP+-dependent isocitrate dehydrogenase activity but no NAD+-dependent isocitrate dehydrogenase activity. The activities of most of these enzymes are increased during growth on glucose, alanine, glutamate or citrate, but only very low 2-oxoglutarate dehydrogenase activities are present under all growth conditions. The restricted facultative methylotrophs grow on certain non-C1 compounds in the absence of 2-oxoglutarate dehydrogenase and, in some cases, of other enzymes of the tricarboxylic acid cycle; these lesions cannot therefore be the sole cause of obligate methylotrophy. | 991 |
pubmed23n0001_483 | Determination of polyadenylate-rich ribonucleic acid in the nucleus and in the cytoplasm of plasmacytoma cells. | A number of parameters affecting the adsorption of rRNA and poly(A)-containing RNA to Millipore filters were investigated separately. Binding of both types of RNA to the filter was dependent on the concentration of RNA, pH and Mg2+ concentration of the reaction mixture. Both types of RNA bound to the filter optimally at slightly acid pH values. The binding of poly(A)-containing RNA to the filter exhibited a broad pH-dependence compared with that of rRNA. The ratio of poly(A)-rich RNA/rRNA retained by the filter was maximal between pH7 and 8. The presence of 1 mM-EDTA or a high concentration of NaCl (over 0.5M) decreased the affinity of RNA for the filter. The amount of poly(A)-containing RNA in the nucleus and in the cytoplasm of a plasmacytoma cell line (MPC-11) labelled with [32P]Pi was determined by the Millipore-filter technique under conditions that minimized contamination by rRNA. These data were compared with the estimations made by oligo(dT)-cellulose chromatography. The results obtained by these two methods were in good agreement for RNA labelled for short periods (up to 2h). In long labelling and pulse-chase experiments, however, contamination of the filter by rRNA of increasing specific radioactivity in the cytoplasm gave an erroneous value for poly(A)-containing RNA by the Millipore-filter technique. Determinations made on the nuclear fraction by these two methods did not show significant variation in short- and long-term labelling experiments. | Determination of polyadenylate-rich ribonucleic acid in the nucleus and in the cytoplasm of plasmacytoma cells. A number of parameters affecting the adsorption of rRNA and poly(A)-containing RNA to Millipore filters were investigated separately. Binding of both types of RNA to the filter was dependent on the concentration of RNA, pH and Mg2+ concentration of the reaction mixture. Both types of RNA bound to the filter optimally at slightly acid pH values. The binding of poly(A)-containing RNA to the filter exhibited a broad pH-dependence compared with that of rRNA. The ratio of poly(A)-rich RNA/rRNA retained by the filter was maximal between pH7 and 8. The presence of 1 mM-EDTA or a high concentration of NaCl (over 0.5M) decreased the affinity of RNA for the filter. The amount of poly(A)-containing RNA in the nucleus and in the cytoplasm of a plasmacytoma cell line (MPC-11) labelled with [32P]Pi was determined by the Millipore-filter technique under conditions that minimized contamination by rRNA. These data were compared with the estimations made by oligo(dT)-cellulose chromatography. The results obtained by these two methods were in good agreement for RNA labelled for short periods (up to 2h). In long labelling and pulse-chase experiments, however, contamination of the filter by rRNA of increasing specific radioactivity in the cytoplasm gave an erroneous value for poly(A)-containing RNA by the Millipore-filter technique. Determinations made on the nuclear fraction by these two methods did not show significant variation in short- and long-term labelling experiments. | 999 |
pubmed23n0001_484 | The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. The tryptic peptides. | The NADP-specific glutamate dehydrogenase of Neurospora crassa was digested with trypsin, and peptides accounting for 441 out of the 452 residues of the polypeptide chain were isolated and substantially sequenced. Additional experimental detail has been deposited as Supplementary Publication SUP 50052 (11 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem J. (1975) 145, 5. | The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. The tryptic peptides. The NADP-specific glutamate dehydrogenase of Neurospora crassa was digested with trypsin, and peptides accounting for 441 out of the 452 residues of the polypeptide chain were isolated and substantially sequenced. Additional experimental detail has been deposited as Supplementary Publication SUP 50052 (11 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem J. (1975) 145, 5. | 1,000 |
pubmed23n0001_485 | The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptides from digestion with a staphylococcal proteinase. | The extracellular proteinase of Staphylococcus aureus strain V8 was used to digest the NADP-specific glutamate dehydrogenase of Neurospora crassa. Of 35 non-overlapping peptides expected from the glutamate content of the polypeptide chain, 29 were isolated and substantially sequenced. The sequences obtained were valuable in providing overlaps for the alignment of about two-thirds of the sequences found in tryptic peptides [Wootton, J. C., Taylor, J, G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 739-748]. The blocked N-terminal peptide of the protein was isolated. This peptide was sequenced by mass spectrometry, and found to have N-terminal N-acetylserine by Howard R. Morris and Anne Dell, whose results are presented as an Appendix to the main paper. The staphylococcal proteinase showed very high specificity for glutamyl bonds in the NH4HCO3 buffer used. Partial splits of two aspartyl bonds, both Asp-Ile, were probably attributable to the proteinase. No cleavage of glutaminyl or S-carboxymethylcysteinyl bonds was found. Additional experimental detail has been deposited as Supplementary Publication SUP 50053 (5 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K, from whom copies may be obtained under the terms given in Biochem. J. (1975) 1458 5. | The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptides from digestion with a staphylococcal proteinase. The extracellular proteinase of Staphylococcus aureus strain V8 was used to digest the NADP-specific glutamate dehydrogenase of Neurospora crassa. Of 35 non-overlapping peptides expected from the glutamate content of the polypeptide chain, 29 were isolated and substantially sequenced. The sequences obtained were valuable in providing overlaps for the alignment of about two-thirds of the sequences found in tryptic peptides [Wootton, J. C., Taylor, J, G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 739-748]. The blocked N-terminal peptide of the protein was isolated. This peptide was sequenced by mass spectrometry, and found to have N-terminal N-acetylserine by Howard R. Morris and Anne Dell, whose results are presented as an Appendix to the main paper. The staphylococcal proteinase showed very high specificity for glutamyl bonds in the NH4HCO3 buffer used. Partial splits of two aspartyl bonds, both Asp-Ile, were probably attributable to the proteinase. No cleavage of glutaminyl or S-carboxymethylcysteinyl bonds was found. Additional experimental detail has been deposited as Supplementary Publication SUP 50053 (5 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K, from whom copies may be obtained under the terms given in Biochem. J. (1975) 1458 5. | 1,001 |
pubmed23n0001_486 | The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptic and chymotryptic peptides and the complete sequence. | Peptic and chymotryptic peptides were isolated form the NADP-specific glutamate dehydrogenase of Neurospora crassa and substantially sequenced. Out of 452 residues in the polypeptide chain, 265 were recovered in the peptic and 427 in the chymotryptic peptides. Together with the tryptic peptides [Wootton, J. C., Taylor, J. G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 749-755], these establish the complete sequence of the chain, including the acid and amide assignments, except for seven places where overlaps are inadequate. These remaining alignments are deduced from information on the CNBr fragments obtained in another laboratory [Blumenthal, K. M., Moon, K. & Smith, E. L. (1975), J. Biol. Chem. 250, 3644-3654]. Further information has been deposited as Supplementary Publication SUP 50054 (17 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1975) 145, 5. | The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptic and chymotryptic peptides and the complete sequence. Peptic and chymotryptic peptides were isolated form the NADP-specific glutamate dehydrogenase of Neurospora crassa and substantially sequenced. Out of 452 residues in the polypeptide chain, 265 were recovered in the peptic and 427 in the chymotryptic peptides. Together with the tryptic peptides [Wootton, J. C., Taylor, J. G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 749-755], these establish the complete sequence of the chain, including the acid and amide assignments, except for seven places where overlaps are inadequate. These remaining alignments are deduced from information on the CNBr fragments obtained in another laboratory [Blumenthal, K. M., Moon, K. & Smith, E. L. (1975), J. Biol. Chem. 250, 3644-3654]. Further information has been deposited as Supplementary Publication SUP 50054 (17 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1975) 145, 5. | 1,002 |
pubmed23n0001_504 | The purification and characterization of the low molecular weight human folate binding protein using affinity chromatography. | The low molecular weight folate binding protein (FABP) has been purified 1000-fold to a specific activity of 7.2 gamma g of pteroylglutamic acid (PGA) bound per mg of protein. This purified FABP represents two protein bands that bind PGA on polyacrylamide disc gel electrophoreis, elutes from DEAE-cellulose in 0.001 M phosphate buffer, stains positive with PAS, Elutes from concanavalin A Sepharose affinity columns with methyl alpha-mannoside, and shows three major peaks (pl =6.8, 7.5, 8.2) by isotric focusing. The binding of PGA to purified FABP dependent on pH and is inhibited by urea... | The purification and characterization of the low molecular weight human folate binding protein using affinity chromatography. The low molecular weight folate binding protein (FABP) has been purified 1000-fold to a specific activity of 7.2 gamma g of pteroylglutamic acid (PGA) bound per mg of protein. This purified FABP represents two protein bands that bind PGA on polyacrylamide disc gel electrophoreis, elutes from DEAE-cellulose in 0.001 M phosphate buffer, stains positive with PAS, Elutes from concanavalin A Sepharose affinity columns with methyl alpha-mannoside, and shows three major peaks (pl =6.8, 7.5, 8.2) by isotric focusing. The binding of PGA to purified FABP dependent on pH and is inhibited by urea... | 1,084 |
pubmed23n0001_506 | Conformational properties of bovine plasma albumin with a cleaved internal peptide bond. | As shown previously, proteinases frequently associated with plasma albumin samples catalyze a very limited and specific cleavage of the albumin molecule when it exists in the F conformational state near pH 3.7. The primary proteolytic product, BPA, has a molecular weight similar to or identical with that of the parent protein but yields two large fragments of molecular weight approximately 46000 and 23000 on reduction. Evidence is presented here that cleavage occurs within the disulfide loop between Cys390 and Cys434 with no detectable loss of small peptides, the amino acid composition of BPA being identical with that of the parent protein within experimental error. Cleavage exposes a new amino-terminal phenylalanine residue and may occur at the Glx392-Phe393 bond although the possibility exists that it occurs at another X-Phe bond in the unsequenced region of residues 400-402. The damaged protein has a somewhat altered secondary structure as judged from optical rotatory dispersion and circular dichroism measurements, probably an approximate 15% loss in helicity. The hydrodynamic volume is increased by approximately 20%. However, various physical studies indicate the tertiary structure to be strikingly similar to that of the native protein. Of most significance is the fact that the protein still undergoes the N-F and N-B transitions, although in both cases they occur at somewhat more moderate pH than in the parent protein. Moreover a sensitivity of the N-B transition to Ca2+ is still seen and binding behavior toward the dye 8-anilino-1-naphthalenesulfonic acid is essentially unaltered. The results are best understood in terms of the concept of a multidomain structure which has been suggested frequently for plasma ablumin. Bond cleavage damages one domain but leaves the overall structure essentially unaltered except for some weakening of the interaction between domains. | Conformational properties of bovine plasma albumin with a cleaved internal peptide bond. As shown previously, proteinases frequently associated with plasma albumin samples catalyze a very limited and specific cleavage of the albumin molecule when it exists in the F conformational state near pH 3.7. The primary proteolytic product, BPA, has a molecular weight similar to or identical with that of the parent protein but yields two large fragments of molecular weight approximately 46000 and 23000 on reduction. Evidence is presented here that cleavage occurs within the disulfide loop between Cys390 and Cys434 with no detectable loss of small peptides, the amino acid composition of BPA being identical with that of the parent protein within experimental error. Cleavage exposes a new amino-terminal phenylalanine residue and may occur at the Glx392-Phe393 bond although the possibility exists that it occurs at another X-Phe bond in the unsequenced region of residues 400-402. The damaged protein has a somewhat altered secondary structure as judged from optical rotatory dispersion and circular dichroism measurements, probably an approximate 15% loss in helicity. The hydrodynamic volume is increased by approximately 20%. However, various physical studies indicate the tertiary structure to be strikingly similar to that of the native protein. Of most significance is the fact that the protein still undergoes the N-F and N-B transitions, although in both cases they occur at somewhat more moderate pH than in the parent protein. Moreover a sensitivity of the N-B transition to Ca2+ is still seen and binding behavior toward the dye 8-anilino-1-naphthalenesulfonic acid is essentially unaltered. The results are best understood in terms of the concept of a multidomain structure which has been suggested frequently for plasma ablumin. Bond cleavage damages one domain but leaves the overall structure essentially unaltered except for some weakening of the interaction between domains. | 1,086 |
pubmed23n0001_510 | Adenosine triphosphate: nicotinamide mononucleotide adenylyltransferase of pig liver. Purification and properties. | Adenosine triphosphate : nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) has been purifiec approximately 3500-fold from an extract of pig liver nuclei to a specific activity of 40 mumol of NAD+ per min per mg protein. The enzyme was found to have a molecular weight of 203 000, a frictional ratio of 1.6 and an isoelectric point of approximately 5. Michaelis constants for ATP and NMN were 0.11 mM and 0.12 mM, respectively. | Adenosine triphosphate: nicotinamide mononucleotide adenylyltransferase of pig liver. Purification and properties. Adenosine triphosphate : nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) has been purifiec approximately 3500-fold from an extract of pig liver nuclei to a specific activity of 40 mumol of NAD+ per min per mg protein. The enzyme was found to have a molecular weight of 203 000, a frictional ratio of 1.6 and an isoelectric point of approximately 5. Michaelis constants for ATP and NMN were 0.11 mM and 0.12 mM, respectively. | 1,090 |
pubmed23n0001_511 | Phospholipase A2 isoenzyme from porcine pancreas. Purification and some properties. | Porcine pancreas synthesizes a prephospholipase A2 which occurs in a 5 : 95 ratio compared with the more abundant zymogen of the same enzyme (phosphatide-acylhydrolase; EC 3.1.1.4). These two prephospholipases could be well separated by CM-cellulose chromatography. Both the active and the zymogen form of the isoenzyme were isolated and purified. The activation peptides of both prephospholipases appeared to be identical, while the active enzymes showed a few interesting differences. The most striking differences were the loss of one histidine and one methionine in the isoenzyme, corresponding to residues 24 and 27, respectively, in alpha-phospholipase A2. The positional and stereo specificity of both enzymes are the same, but the specific activity of the beta-phospholipase A2 is lower. The molecular weight of the isoenzyme was estimated to be about 14 000, while the isoelectric points were 5.1 and 5.9 for the isoprecursor and active isoenzyme, respectively. | Phospholipase A2 isoenzyme from porcine pancreas. Purification and some properties. Porcine pancreas synthesizes a prephospholipase A2 which occurs in a 5 : 95 ratio compared with the more abundant zymogen of the same enzyme (phosphatide-acylhydrolase; EC 3.1.1.4). These two prephospholipases could be well separated by CM-cellulose chromatography. Both the active and the zymogen form of the isoenzyme were isolated and purified. The activation peptides of both prephospholipases appeared to be identical, while the active enzymes showed a few interesting differences. The most striking differences were the loss of one histidine and one methionine in the isoenzyme, corresponding to residues 24 and 27, respectively, in alpha-phospholipase A2. The positional and stereo specificity of both enzymes are the same, but the specific activity of the beta-phospholipase A2 is lower. The molecular weight of the isoenzyme was estimated to be about 14 000, while the isoelectric points were 5.1 and 5.9 for the isoprecursor and active isoenzyme, respectively. | 1,091 |
pubmed23n0001_517 | Aminopeptidases in webbing clothes moth larvae. Properties and specificities of the enzymes of intermediate electrophoretic mobility. | The major group of aminopeptidases (EC 3.4.11.-) of intermediate electrophoretic mobility, from Tineola bisselliella larvae, hav been fractionated into six bands by preparative polyacrylamide gel electrophoresis and the properties of these fractions investigated. They resemble each other in their pH optima of 8.2, their molecular weight of 240 000, their responses to various active site inhibitors and metal cations, and their specificities towards seventeen L-amino-acyl-beta-naphthylamide substrates. The derivatives of methionine, leucine, alanine, lysine, arginine and glutamic acid were those most rapidly hydrolysed. They appear to be true aminopeptidases hydrolysing amino acid amides, dipeptides and oligopeptides from the N-terminal end. | Aminopeptidases in webbing clothes moth larvae. Properties and specificities of the enzymes of intermediate electrophoretic mobility. The major group of aminopeptidases (EC 3.4.11.-) of intermediate electrophoretic mobility, from Tineola bisselliella larvae, hav been fractionated into six bands by preparative polyacrylamide gel electrophoresis and the properties of these fractions investigated. They resemble each other in their pH optima of 8.2, their molecular weight of 240 000, their responses to various active site inhibitors and metal cations, and their specificities towards seventeen L-amino-acyl-beta-naphthylamide substrates. The derivatives of methionine, leucine, alanine, lysine, arginine and glutamic acid were those most rapidly hydrolysed. They appear to be true aminopeptidases hydrolysing amino acid amides, dipeptides and oligopeptides from the N-terminal end. | 1,097 |
pubmed23n0001_521 | [The fragmentation and reconstruction of the oligomycin-sensitive ATPase system of liver mitochondria]. | The protein and proteolipid complexes and oligomycin insensitive soluble ATPase were prepared from rat liver mitochondria. The incubation of soluble ATPase with protein and proteolipid complexes resulted in restoration of ATPase sensitivity to oligomycin at room temperature. The process of reconstruction depended on pH, incubation time, temperature and other conditions. | [The fragmentation and reconstruction of the oligomycin-sensitive ATPase system of liver mitochondria]. The protein and proteolipid complexes and oligomycin insensitive soluble ATPase were prepared from rat liver mitochondria. The incubation of soluble ATPase with protein and proteolipid complexes resulted in restoration of ATPase sensitivity to oligomycin at room temperature. The process of reconstruction depended on pH, incubation time, temperature and other conditions. | 1,103 |
pubmed23n0001_524 | [Wheat protein disulfide reductase]. | Proteindisulphide reductase is isolated and partially purified from wheat seedlings and some properties of the enzyme are studied: pH optimum is 7.4; temperature optimum - 37 degrees C; Km = 2.6-10(-4)M for the substrate (wheat albumin); Km = 7.5-10(-5) M for coenzyme (NADP-H). The enzyme is specific for NADP-H and is not active in the presence of NAD-H. Maximal activity of proteindisulphide reductase is developed in anaerobic conditions. A technique of the estimation of proteindisulphide reductase activity using wheat albumin as a substrate is worked out. The enzyme activity decreases regularly in the corn ripening and increases under germination. It is accompanied by the respective increase or decrease in the amount of disulphide bonds in gluten protein and by changes of physico-chemical characteristics of gluten. Incubation of gluten with the enzyme preparation affects reological properties of gluten (it becomes weaker) and decreases the gluten viscosity of gluten solution. | [Wheat protein disulfide reductase]. Proteindisulphide reductase is isolated and partially purified from wheat seedlings and some properties of the enzyme are studied: pH optimum is 7.4; temperature optimum - 37 degrees C; Km = 2.6-10(-4)M for the substrate (wheat albumin); Km = 7.5-10(-5) M for coenzyme (NADP-H). The enzyme is specific for NADP-H and is not active in the presence of NAD-H. Maximal activity of proteindisulphide reductase is developed in anaerobic conditions. A technique of the estimation of proteindisulphide reductase activity using wheat albumin as a substrate is worked out. The enzyme activity decreases regularly in the corn ripening and increases under germination. It is accompanied by the respective increase or decrease in the amount of disulphide bonds in gluten protein and by changes of physico-chemical characteristics of gluten. Incubation of gluten with the enzyme preparation affects reological properties of gluten (it becomes weaker) and decreases the gluten viscosity of gluten solution. | 1,106 |
pubmed23n0001_530 | [Multiple forms of rabbit muscle phosphofructokinase revealed by means of specific elution]. | The modified procedure for rabbit skeletal muscle phosphofructokinase (PFK) purification is worked out utioizing the method of specific elution from DEAE-cellulose in 0.1 M tris-EDTA-phosphate pH 8.0 with 10 mM citrate. By the latter procedure PFK can be resolved into fractions A, B and C which are eluted specifically, with 0.3 M buffer and with 1.5 M NaCl respectively. Rechromatography of each fraction reveals their interconvertibility. The preparations are characterized by disc electrophoresis and velocity sedimentation. The results of formalinization experiments demonstrate that high concentrations of formaldehyde dissociate PFK u to the 5.3 S component. The presence of the 19.3 S component in the formalinized preparations evidences against the possibility that the middle component, presented at the schlieren patterns of PFK at pH 8.0, is and artifact of superposition. Complex profiles of protein distribution observed in different transport experiments are discussed from the point of view of slow equilibrium of oligomers and conformers characteristic to PFK over the pH range from 6 to 9. | [Multiple forms of rabbit muscle phosphofructokinase revealed by means of specific elution]. The modified procedure for rabbit skeletal muscle phosphofructokinase (PFK) purification is worked out utioizing the method of specific elution from DEAE-cellulose in 0.1 M tris-EDTA-phosphate pH 8.0 with 10 mM citrate. By the latter procedure PFK can be resolved into fractions A, B and C which are eluted specifically, with 0.3 M buffer and with 1.5 M NaCl respectively. Rechromatography of each fraction reveals their interconvertibility. The preparations are characterized by disc electrophoresis and velocity sedimentation. The results of formalinization experiments demonstrate that high concentrations of formaldehyde dissociate PFK u to the 5.3 S component. The presence of the 19.3 S component in the formalinized preparations evidences against the possibility that the middle component, presented at the schlieren patterns of PFK at pH 8.0, is and artifact of superposition. Complex profiles of protein distribution observed in different transport experiments are discussed from the point of view of slow equilibrium of oligomers and conformers characteristic to PFK over the pH range from 6 to 9. | 1,113 |
pubmed23n0001_533 | [The effect of oxidazable substrates and ATP on the sensitivity of certain energy-dependent functions submitochondrial particles to phospholipases A, C and D]. | The effect of NADH, succinate and ATP on the sensitivity of a number of energy-dependent functions of submitochondrial particles ot phospholipases A, C and D has been studied. It has been shown that in the conditions of oxidation of NADH and succinate by oxygen and also of ATP hydrolysis, the decrease in the phosphorylating activity of the particles under the action of phospholipases C and D accelerates. No such acceleration has been observed with phospholipase A. For other two functions, i. e. reverse electron transfer (ATP-dependent NAD+ reduction by succinate) and ATP-dependent transhydrogenase reaction the results proved to be different. Oxidizable substrates and ATP promoted the maintenance of these functions in the presence of phospholipase A, but did not retard their suppression by phospholipases C and D. The effects of NADH, succinate and ATP on the sensitivity of different energy-dependent functions of submitochondrial particles to phospholipases A, C and D could be removed by the uncoupling agent carbonyl cyanide-m-chlorophenyl hydrazone. The conclusion is made that the effects revealed are associated with an increase in the sensitivity of coupling sites II PAND/OR III to phospholipases C and D and with a decrease in the sensitivity of sites I and IV to phospholipase A on energization of submitochondrial particles. | [The effect of oxidazable substrates and ATP on the sensitivity of certain energy-dependent functions submitochondrial particles to phospholipases A, C and D]. The effect of NADH, succinate and ATP on the sensitivity of a number of energy-dependent functions of submitochondrial particles ot phospholipases A, C and D has been studied. It has been shown that in the conditions of oxidation of NADH and succinate by oxygen and also of ATP hydrolysis, the decrease in the phosphorylating activity of the particles under the action of phospholipases C and D accelerates. No such acceleration has been observed with phospholipase A. For other two functions, i. e. reverse electron transfer (ATP-dependent NAD+ reduction by succinate) and ATP-dependent transhydrogenase reaction the results proved to be different. Oxidizable substrates and ATP promoted the maintenance of these functions in the presence of phospholipase A, but did not retard their suppression by phospholipases C and D. The effects of NADH, succinate and ATP on the sensitivity of different energy-dependent functions of submitochondrial particles to phospholipases A, C and D could be removed by the uncoupling agent carbonyl cyanide-m-chlorophenyl hydrazone. The conclusion is made that the effects revealed are associated with an increase in the sensitivity of coupling sites II PAND/OR III to phospholipases C and D and with a decrease in the sensitivity of sites I and IV to phospholipase A on energization of submitochondrial particles. | 1,116 |
pubmed23n0001_538 | Profiles for pH, temperature, and dissolved O2 levels in enzyme production: monitoring in small-scale fermentors. | The profiles thus established may be utilized for investigations of an organism's relationship to its microenvironment including metabolic shifts and pathways. Areas of maximum respiratory activity, enzyme production, enzyme degradation, and attainment of the stationary phase are quite evident; however, duration and magnitude of the various phenomena may change with nutrient, temperature, and aeration efficiency. Practical application of this simplified method would include: a) determination of environmental conditions existing during maximum growth or enzyme synthesis and application of these conditions to feedback control; b) estimation of requirements for pH, oxygen, and heat removal capacities needed for scale-up; c) specific points during the fermentation at which samples should be analyzed to yield maximum information on depletion of nutrients and its effects on microbial activity. | Profiles for pH, temperature, and dissolved O2 levels in enzyme production: monitoring in small-scale fermentors. The profiles thus established may be utilized for investigations of an organism's relationship to its microenvironment including metabolic shifts and pathways. Areas of maximum respiratory activity, enzyme production, enzyme degradation, and attainment of the stationary phase are quite evident; however, duration and magnitude of the various phenomena may change with nutrient, temperature, and aeration efficiency. Practical application of this simplified method would include: a) determination of environmental conditions existing during maximum growth or enzyme synthesis and application of these conditions to feedback control; b) estimation of requirements for pH, oxygen, and heat removal capacities needed for scale-up; c) specific points during the fermentation at which samples should be analyzed to yield maximum information on depletion of nutrients and its effects on microbial activity. | 1,123 |
pubmed23n0001_539 | The specificity of heterophil antibodies in patients and healthy donors with no or minimal signs of infectious mononucleosis. | Over several years sera were collected from 14 heterophil-positive students or patients who did not fulfill minimal hematologic criteria for infectious mononucleosis (I.M.) The specificity of these heterophil reactions for I.M. was investigated by determining antibodies to Epstein-Barr virus-determined antigens, i.e., to viral capsid antigens (VCA), early antigens (EA), and EBV-associated nuclear antigens (EBNA). On the basis of detectable anti-EA and/or the early absence and late emergence of anti-EBNA, four of these 14 individuals showed evidence of a current or very recent primary Epstein-Barr virus infection. The other ten patients showed antibody patterns indicative of Epstein-Barr virus infections in the past, and no firm conclusions could be drawn with regard to the specificity of their heterophil reactions. It was assumed, however, that some represented atypical clinical forms of EBV infection and that timing of specimen collection was a factor in explaining the paucity of Downey cells. In three patients, the absorbed heterophil-positive reactions persisted with little change in titer for at least 22 mo and thus might represent false-positive tests. | The specificity of heterophil antibodies in patients and healthy donors with no or minimal signs of infectious mononucleosis. Over several years sera were collected from 14 heterophil-positive students or patients who did not fulfill minimal hematologic criteria for infectious mononucleosis (I.M.) The specificity of these heterophil reactions for I.M. was investigated by determining antibodies to Epstein-Barr virus-determined antigens, i.e., to viral capsid antigens (VCA), early antigens (EA), and EBV-associated nuclear antigens (EBNA). On the basis of detectable anti-EA and/or the early absence and late emergence of anti-EBNA, four of these 14 individuals showed evidence of a current or very recent primary Epstein-Barr virus infection. The other ten patients showed antibody patterns indicative of Epstein-Barr virus infections in the past, and no firm conclusions could be drawn with regard to the specificity of their heterophil reactions. It was assumed, however, that some represented atypical clinical forms of EBV infection and that timing of specimen collection was a factor in explaining the paucity of Downey cells. In three patients, the absorbed heterophil-positive reactions persisted with little change in titer for at least 22 mo and thus might represent false-positive tests. | 1,126 |
pubmed23n0001_542 | Antiarrhythmic, haemodynamic and metabolic effects of 3alpha-amino-5alpha-androstan-2beta-ol-17-one hydrochloride in greyhounds following acute coronary artery ligation. | 1 The antiarrhythmic, haemodynamic and metabolic effects of a new amino steroid, ORG6001, have been investigated in experimental acute myocardial infarction in anaesthetized greyhounds. 2 ORG6001 administered either intravenously (2-10 mg/kg) or orally (50 mg/kg) significantly reduced the incidence of ventricular ectopic beats in the first 30 min after ligation of the left anterior descending coronary artery. 3 In dogs pretreated with ORG6001, metabolic changes indicative of myocardial ischaemia (lactate production and potassium efflux) were less marked than those occurring in control animals. 4 Antiarrhythmic doses of ORG6001 caused only minimal transient haemodynamic effects. 5 These results suggest that ORG6001 may possess distinct advantages over presently-used antiarrhythmic drugs in the prevention and treatment of the early arrhythmias which occur after myocardial infarction. | Antiarrhythmic, haemodynamic and metabolic effects of 3alpha-amino-5alpha-androstan-2beta-ol-17-one hydrochloride in greyhounds following acute coronary artery ligation. 1 The antiarrhythmic, haemodynamic and metabolic effects of a new amino steroid, ORG6001, have been investigated in experimental acute myocardial infarction in anaesthetized greyhounds. 2 ORG6001 administered either intravenously (2-10 mg/kg) or orally (50 mg/kg) significantly reduced the incidence of ventricular ectopic beats in the first 30 min after ligation of the left anterior descending coronary artery. 3 In dogs pretreated with ORG6001, metabolic changes indicative of myocardial ischaemia (lactate production and potassium efflux) were less marked than those occurring in control animals. 4 Antiarrhythmic doses of ORG6001 caused only minimal transient haemodynamic effects. 5 These results suggest that ORG6001 may possess distinct advantages over presently-used antiarrhythmic drugs in the prevention and treatment of the early arrhythmias which occur after myocardial infarction. | 1,133 |
pubmed23n0001_549 | Properties of a free and a solubilized form of bound alpha,alpha-trehalase purified from honey bee thorax. | The free and bound forms of alpha,alpha-trehalase (EC 3.2.1.28) of the honey bee thorax were separated and the bound enzyme was solubilized by raising the pH to 8.0 for 10 h. Both enzymes were purified. They were homogeneous as determined by several electrophoretic criteria. It was found that the two enzymes had very similar Km's (each about 0.89 mM), Vm's (53.2 and 54.3 U/mg for free and solubilized, respectively), inhibition characteristics, specificities (both only hydrolyzed alpha,alpha-trehalose), pH maxima (each had maxima at about 3.5 and 6.5), molecular weights (65,000), isoelectric points (5.1), reactivities to sulfhydryl reagents, electrophoretic mobilities, activation energies (about 12.8 kcal/mol), and similar stabilities to heat, pH, and urea. Some significant differences between the two enzymes were, however, found: the solubilized alpha,alpha-trehalase floated at 70% saturation of ammonium sulfate while the free alpha,alpha-trehalase did not; the solubilized alpha,alpha-trehalase did not dissociate into subunits as readily as did the free one; and the solubilized alpha,alpha-trehalase was found to bind more readily to a hydrophobic grouping than the free enzyme. In addition to these comparisons, three new findings relating to thorax alpha,alpha-trehalases are reported. (1) Thorax alpha,alpha-trehalases are strongly inhibited by beta-glucosides (Ki values of about 8 x 10(-4) M); (2) under certain conditions thorax alpha,alpha-trehalases from honey bees dissociated into subunits of one-half the normal molecular weight; (3) honey bee thorax alpha,alpha-trehalases have unusual biphasic pH activity profiles. | Properties of a free and a solubilized form of bound alpha,alpha-trehalase purified from honey bee thorax. The free and bound forms of alpha,alpha-trehalase (EC 3.2.1.28) of the honey bee thorax were separated and the bound enzyme was solubilized by raising the pH to 8.0 for 10 h. Both enzymes were purified. They were homogeneous as determined by several electrophoretic criteria. It was found that the two enzymes had very similar Km's (each about 0.89 mM), Vm's (53.2 and 54.3 U/mg for free and solubilized, respectively), inhibition characteristics, specificities (both only hydrolyzed alpha,alpha-trehalose), pH maxima (each had maxima at about 3.5 and 6.5), molecular weights (65,000), isoelectric points (5.1), reactivities to sulfhydryl reagents, electrophoretic mobilities, activation energies (about 12.8 kcal/mol), and similar stabilities to heat, pH, and urea. Some significant differences between the two enzymes were, however, found: the solubilized alpha,alpha-trehalase floated at 70% saturation of ammonium sulfate while the free alpha,alpha-trehalase did not; the solubilized alpha,alpha-trehalase did not dissociate into subunits as readily as did the free one; and the solubilized alpha,alpha-trehalase was found to bind more readily to a hydrophobic grouping than the free enzyme. In addition to these comparisons, three new findings relating to thorax alpha,alpha-trehalases are reported. (1) Thorax alpha,alpha-trehalases are strongly inhibited by beta-glucosides (Ki values of about 8 x 10(-4) M); (2) under certain conditions thorax alpha,alpha-trehalases from honey bees dissociated into subunits of one-half the normal molecular weight; (3) honey bee thorax alpha,alpha-trehalases have unusual biphasic pH activity profiles. | 1,144 |
pubmed23n0001_550 | Immunotherapy for cancer: an overview. | Immunotherapy of cancer is of interest to oncologists because it is specifically directed to cancer cells, sparing normal cells. While it is ineffective in most patients, especially those with widespread metastatic disease, it occasionally produces good results. Each of the available methods has inherent problems and, recently, attempts have been made to overcome some of these. There is a strong case for small-scale experimental trials in highly selected groups of patients who are intensively investigated for their immunologic status in relation to their tumour. Despite the lack of success in general, immunotherapy still appears to have a future as an adjunct to existing therapy in order to control as much as to cure residual tumour. | Immunotherapy for cancer: an overview. Immunotherapy of cancer is of interest to oncologists because it is specifically directed to cancer cells, sparing normal cells. While it is ineffective in most patients, especially those with widespread metastatic disease, it occasionally produces good results. Each of the available methods has inherent problems and, recently, attempts have been made to overcome some of these. There is a strong case for small-scale experimental trials in highly selected groups of patients who are intensively investigated for their immunologic status in relation to their tumour. Despite the lack of success in general, immunotherapy still appears to have a future as an adjunct to existing therapy in order to control as much as to cure residual tumour. | 1,145 |
pubmed23n0001_555 | Quantitative fractionation of alkaline phosphatase isoenzymes according to their thermostability. | Continuous monitoring of heat denaturation of a mixture of alkaline phosphatase isoenzymes at 60 degrees C and pH 7.5 permits the simultaneous direct identification and quantitation of three isoenzymes: the placental isoenzyme, the L-phenylalanine-sensitive intestinal isoenzyme, and the liver isoenzyme (hepatocytic). The isoenzyme that is principally of bone origin cannot be identified as such without the help of other diagnostic aids and the patient's medical history. All human tissues contain alkaline phosphatase, many organs more than one of the isoenzymes. Liver alkaline phosphatase, which constitutes 40-50% of normal serum alkaline phosphatase activity, was measured in the serum of persons with various liver diseases. Its activity exceeded normal in all types of liver disease; in 80% of cases this increase was accompanied by increased gamma-glutamyl-transferase activity, but the quantitative correlationship (r = 0.54) was not as good as expected if both enzymes come from the same source and are indices of liver dieases. Liver alkaline phosphatase activity increases in the blood early in liver disease, before most liver tests show abnormalities. The other major isoenzyme of normal serum probably represents a mixture of isoenzymes from bone and reticulo-endothelial and vascular tissues, which all contain the same "very heat-labile" alkaline phosphatase. Cord blood and children's sera contain mostly this very heat-labile isoenzyme. | Quantitative fractionation of alkaline phosphatase isoenzymes according to their thermostability. Continuous monitoring of heat denaturation of a mixture of alkaline phosphatase isoenzymes at 60 degrees C and pH 7.5 permits the simultaneous direct identification and quantitation of three isoenzymes: the placental isoenzyme, the L-phenylalanine-sensitive intestinal isoenzyme, and the liver isoenzyme (hepatocytic). The isoenzyme that is principally of bone origin cannot be identified as such without the help of other diagnostic aids and the patient's medical history. All human tissues contain alkaline phosphatase, many organs more than one of the isoenzymes. Liver alkaline phosphatase, which constitutes 40-50% of normal serum alkaline phosphatase activity, was measured in the serum of persons with various liver diseases. Its activity exceeded normal in all types of liver disease; in 80% of cases this increase was accompanied by increased gamma-glutamyl-transferase activity, but the quantitative correlationship (r = 0.54) was not as good as expected if both enzymes come from the same source and are indices of liver dieases. Liver alkaline phosphatase activity increases in the blood early in liver disease, before most liver tests show abnormalities. The other major isoenzyme of normal serum probably represents a mixture of isoenzymes from bone and reticulo-endothelial and vascular tissues, which all contain the same "very heat-labile" alkaline phosphatase. Cord blood and children's sera contain mostly this very heat-labile isoenzyme. | 1,158 |
pubmed23n0001_557 | Improved separation of creatine kinase cardiac isoenzyme in serum by batch fractionation. | I describe a simple, single-tube batch fractionation procedure for separating MM and MB isoenzymes of creatine kinase on a macroporous strong anion exchanger (AG MP-1, Bio-Rad Laboratories). The isoenzymes can be separated in less than 3 min, with a resulting dilution of the serum with no more than an equal volume of buffer. Without sample concentration or spectrofluorometric measurement, the procedure detects 4 U of MB isoenzyme per liter. Sensitivity is limited by the sensitivity and precision of the method of measurement. The CV for the fractionation can be held to less than 4.0% at 65 U of MB per liter. Current fractionation methods are compared to the proposed procedure. With use of a discrete analyzer (Du Pont aca) the mean MB activity in a population free of heart disease was 3.2 +/- 3.0 U/liter (range, 0 to 8 U/liter). The kinetics and stability of isolated isoenzymes are reported, indicating that advisability of storing or pre-incubating samples with mercaptoethanol. | Improved separation of creatine kinase cardiac isoenzyme in serum by batch fractionation. I describe a simple, single-tube batch fractionation procedure for separating MM and MB isoenzymes of creatine kinase on a macroporous strong anion exchanger (AG MP-1, Bio-Rad Laboratories). The isoenzymes can be separated in less than 3 min, with a resulting dilution of the serum with no more than an equal volume of buffer. Without sample concentration or spectrofluorometric measurement, the procedure detects 4 U of MB isoenzyme per liter. Sensitivity is limited by the sensitivity and precision of the method of measurement. The CV for the fractionation can be held to less than 4.0% at 65 U of MB per liter. Current fractionation methods are compared to the proposed procedure. With use of a discrete analyzer (Du Pont aca) the mean MB activity in a population free of heart disease was 3.2 +/- 3.0 U/liter (range, 0 to 8 U/liter). The kinetics and stability of isolated isoenzymes are reported, indicating that advisability of storing or pre-incubating samples with mercaptoethanol. | 1,160 |
pubmed23n0001_567 | Seasonal variations in the composition of urine in relation to calcium stone-formation. | 1. A retrospective cross-sectional study was carried out on data derived from single 24 h urine collections from 246 male idiopathic calcium stone-formers. 2. The daily urine volume and pH and the exretions of calcium, oxalate, phosphate, creatinine and magnesium were related to the time of year when the urine was collected, and the saturation of urine with calcium oxalate and octocalcium phosphate calculated for each month. 3. There were significant seasonal variations in the urinary excretion of calcium and oxalate, each showing a maximum during the summer months and a minimum in the winter. There was no significant seasonal variation in urinary pH, volume, creatinine, phosphate or magnesium. 4. There was a significant increase in the saturation of urine with calcium oxalate and a trend towards higher saturation levels of octo-calcium phosphate in the summer. These changes were dependent only on the seasonal variation in urinary calcium and oxalate and not on urine volume. 5. A retrospective study of the seasonal incidence of stone episodes among these 246 stone-formers showed that the rate of stone passage per month was 50% higher in the summer than in the winter. There was no significant seasonal variation in the incidence of stones removed surgically. | Seasonal variations in the composition of urine in relation to calcium stone-formation. 1. A retrospective cross-sectional study was carried out on data derived from single 24 h urine collections from 246 male idiopathic calcium stone-formers. 2. The daily urine volume and pH and the exretions of calcium, oxalate, phosphate, creatinine and magnesium were related to the time of year when the urine was collected, and the saturation of urine with calcium oxalate and octocalcium phosphate calculated for each month. 3. There were significant seasonal variations in the urinary excretion of calcium and oxalate, each showing a maximum during the summer months and a minimum in the winter. There was no significant seasonal variation in urinary pH, volume, creatinine, phosphate or magnesium. 4. There was a significant increase in the saturation of urine with calcium oxalate and a trend towards higher saturation levels of octo-calcium phosphate in the summer. These changes were dependent only on the seasonal variation in urinary calcium and oxalate and not on urine volume. 5. A retrospective study of the seasonal incidence of stone episodes among these 246 stone-formers showed that the rate of stone passage per month was 50% higher in the summer than in the winter. There was no significant seasonal variation in the incidence of stones removed surgically. | 1,172 |
pubmed23n0001_579 | Further studies of metyrapone effects upon anilide hydroxylation. | The enhancing effect of metyrapone upon the p-hydroxylation of acetanilide has been confirmed with the use of a new gas-chromatographic method for the determination of acetaminophen. This effect has been shown not to be due to inhibition of hydrolysis of acetaminophen or interference with its determination, or to preferential formation of other phenolic metabolites. This effect of metyrapone is remarkably substrate-specific: phenol formation from the homologues of acetanilide, formanilide and propionanilide, and that from the sulfonamide analog of acetanilide, methanesulfonanilide, is inhibited by metyrapone over the concentration range in which acetanilide hydroxylation is enhanced. The same substrate specificity was observed when the modifier was acetophenone. alpha,alpha'-Dipyridyl, however, enhances phenol formation from all three carbonacylanilides, but does not affect that from methanesulfonanilide. | Further studies of metyrapone effects upon anilide hydroxylation. The enhancing effect of metyrapone upon the p-hydroxylation of acetanilide has been confirmed with the use of a new gas-chromatographic method for the determination of acetaminophen. This effect has been shown not to be due to inhibition of hydrolysis of acetaminophen or interference with its determination, or to preferential formation of other phenolic metabolites. This effect of metyrapone is remarkably substrate-specific: phenol formation from the homologues of acetanilide, formanilide and propionanilide, and that from the sulfonamide analog of acetanilide, methanesulfonanilide, is inhibited by metyrapone over the concentration range in which acetanilide hydroxylation is enhanced. The same substrate specificity was observed when the modifier was acetophenone. alpha,alpha'-Dipyridyl, however, enhances phenol formation from all three carbonacylanilides, but does not affect that from methanesulfonanilide. | 1,226 |
pubmed23n0001_583 | Biliary excretion of colchicine in newborn rats. | The 24-hr LD50 of colchicine in newborn rats is 0.24 mg/kg, which is about 1/10 that observed in the adult. The 24-hr LD50 of colchicine was relatively constant in rats over 25 days of age. In an attempt to determine the mechanism of the increased sensitivity of the newborn rat to the toxic action of colchicine, the distribution of 3H after the administration of 3H-colchicine (0.1 mg/kg) was measured in 10- and 35-day-old rats. The concentration of 3H was higher in all tissues of the newborn than the adult after ip administration, suggesting an immaturity in the pathway for colchicine elimination. After iv administration, radioactivity disappeared much more slowly from the plasma of the newborn rat than from the adult. This was due to a lower capacity of the liver of the newborn to concentrate colchicine and to excrete it into the bile. Development of the hepatic excretory mechanism responsible for excretion of colchicine occurred at the same age as did the increase in LD50. These results suggest that colchicine is more toxic in the newborn because the drug remains in the body for a longer time due to immaturity of the liver excretory process. | Biliary excretion of colchicine in newborn rats. The 24-hr LD50 of colchicine in newborn rats is 0.24 mg/kg, which is about 1/10 that observed in the adult. The 24-hr LD50 of colchicine was relatively constant in rats over 25 days of age. In an attempt to determine the mechanism of the increased sensitivity of the newborn rat to the toxic action of colchicine, the distribution of 3H after the administration of 3H-colchicine (0.1 mg/kg) was measured in 10- and 35-day-old rats. The concentration of 3H was higher in all tissues of the newborn than the adult after ip administration, suggesting an immaturity in the pathway for colchicine elimination. After iv administration, radioactivity disappeared much more slowly from the plasma of the newborn rat than from the adult. This was due to a lower capacity of the liver of the newborn to concentrate colchicine and to excrete it into the bile. Development of the hepatic excretory mechanism responsible for excretion of colchicine occurred at the same age as did the increase in LD50. These results suggest that colchicine is more toxic in the newborn because the drug remains in the body for a longer time due to immaturity of the liver excretory process. | 1,230 |
pubmed23n0001_585 | Uptake and disposition of aldrin and dieldrin by isolated perfused rabbit lung. | The uptake, metabolism, and release of aldrin and dieldrin by the lungs were studied by use of isolated perfused rabbit lungs that were artificially ventilated and perfused through the pulmonary artery. Both recirculating and single-pass experiments were conducted using an artificial medium as perfusate. Aldrin accumulated in the lung from the perfusate through two distinct phases of uptake: a rapid phase involving simple diffusion and nonspecific binding and a slower phase representing its metabolic turnover as dieldrin. Dieldrin was not metabolized but accumulated in the lungs by a saturable and a nonsaturable process. Single-pass experiments with aldrin indicated that the initial velocity of uptake could be fitted to one component and a constant representing the rate of metabolism. Uptake of dieldrin was biphasic: one phase independent of the perfusate concentration and the other saturable with respect to the perfusate concentration. By the application of Michaelis-Menten kinetics, the maximum amount of dieldrin accumulation attributable to the saturable component was calculated to be 0.64 mumol/lung. Our results indicate that the accumulation of these chlorinated xenobiotics takes place through the processes of simple diffusion followed by nonspecific tissue binding. There was no evidence for irreversible binding of aldrin or dieldrin, its epoxide, in the lung. While the lung plays a role in metabolizing aldrin to dieldrin followed by a transient storage, neither substrate has the potential for long-term storage in the lung. | Uptake and disposition of aldrin and dieldrin by isolated perfused rabbit lung. The uptake, metabolism, and release of aldrin and dieldrin by the lungs were studied by use of isolated perfused rabbit lungs that were artificially ventilated and perfused through the pulmonary artery. Both recirculating and single-pass experiments were conducted using an artificial medium as perfusate. Aldrin accumulated in the lung from the perfusate through two distinct phases of uptake: a rapid phase involving simple diffusion and nonspecific binding and a slower phase representing its metabolic turnover as dieldrin. Dieldrin was not metabolized but accumulated in the lungs by a saturable and a nonsaturable process. Single-pass experiments with aldrin indicated that the initial velocity of uptake could be fitted to one component and a constant representing the rate of metabolism. Uptake of dieldrin was biphasic: one phase independent of the perfusate concentration and the other saturable with respect to the perfusate concentration. By the application of Michaelis-Menten kinetics, the maximum amount of dieldrin accumulation attributable to the saturable component was calculated to be 0.64 mumol/lung. Our results indicate that the accumulation of these chlorinated xenobiotics takes place through the processes of simple diffusion followed by nonspecific tissue binding. There was no evidence for irreversible binding of aldrin or dieldrin, its epoxide, in the lung. While the lung plays a role in metabolizing aldrin to dieldrin followed by a transient storage, neither substrate has the potential for long-term storage in the lung. | 1,232 |
pubmed23n0001_596 | Purification and properties of isoenzymes of cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures. | Two isoenzymes of an NADP+ -dependent cinnamyl alcohol dehydrogenase and an NAD+ - dependent aliphatic alcohol dehydrogenase were extracted from cell suspension cultures of soybean (Glycine max L., var. Mandarin) which form lignin during growth. These enzymes could be separated from each other by chromatography on DEAE-cellulose and hydroxyapatite. The cinnamyl alcohol dehydrogenase isoenzymes were partially purified by (NH4)2SO4 fractionation, and column chromatography on DEAE-cellulose, Sephadex G-100, and hydroxyapatite. The molecular weight of the enzymes were estimated by the elution volumes from a Sephadex G-100 column and were found to be about 43,000 (isoenzyme 1) and 69,000 (isoenzyme 2). Maximum rates of reaction were observed in the case of coniferyl alcohol oxidation at pH 9.2 (Isoenzyme 1) and pH 8.8 (isoenzyme 2); in the reverse reaction pH 6.5 was optimal for isoenzyme 2. Whereas isoenzyme 1 is specific for coniferyl alcohol, isoenzyme 2 can also oxidize cinnamyl alcohol and a number of substituted cinnamyl alcohols, Km values for substituted cinnamaldehydes are 3-11 times lower than for the corresponding alcohols. Neither isoenzyme reacted with benzyl alcohol, anisic alcohol or ethanol. Substrate inhibition for the forward and reverse reaction was found with isoenzyme 2 but not with isoenzyme 1. The equilibrium constant was determined to be about 10(9) in favour of coniferaldehyde reduction. The possible role of the cinnamyl alcohol dehydrogenase in lignin biosynthesis is discussed. | Purification and properties of isoenzymes of cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures. Two isoenzymes of an NADP+ -dependent cinnamyl alcohol dehydrogenase and an NAD+ - dependent aliphatic alcohol dehydrogenase were extracted from cell suspension cultures of soybean (Glycine max L., var. Mandarin) which form lignin during growth. These enzymes could be separated from each other by chromatography on DEAE-cellulose and hydroxyapatite. The cinnamyl alcohol dehydrogenase isoenzymes were partially purified by (NH4)2SO4 fractionation, and column chromatography on DEAE-cellulose, Sephadex G-100, and hydroxyapatite. The molecular weight of the enzymes were estimated by the elution volumes from a Sephadex G-100 column and were found to be about 43,000 (isoenzyme 1) and 69,000 (isoenzyme 2). Maximum rates of reaction were observed in the case of coniferyl alcohol oxidation at pH 9.2 (Isoenzyme 1) and pH 8.8 (isoenzyme 2); in the reverse reaction pH 6.5 was optimal for isoenzyme 2. Whereas isoenzyme 1 is specific for coniferyl alcohol, isoenzyme 2 can also oxidize cinnamyl alcohol and a number of substituted cinnamyl alcohols, Km values for substituted cinnamaldehydes are 3-11 times lower than for the corresponding alcohols. Neither isoenzyme reacted with benzyl alcohol, anisic alcohol or ethanol. Substrate inhibition for the forward and reverse reaction was found with isoenzyme 2 but not with isoenzyme 1. The equilibrium constant was determined to be about 10(9) in favour of coniferaldehyde reduction. The possible role of the cinnamyl alcohol dehydrogenase in lignin biosynthesis is discussed. | 1,250 |
pubmed23n0001_603 | D-glucose-6-phosphate dehydrogenase (Entner-Doudoroff enzyme) from Pseudomonas fluorescens. Purification, properties and regulation. | 1. The existence of two different D-glucose-6-phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway. 2. A procedure is described for the isolation of that D-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300 mumol NADH formed per min per mg protein, was shown to be homogeneous. 3. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265000. 4. The pI of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal activity at pH 8.9. 5. The Entner-Doudoroff enzyme showed specificity for NAD+ as well as for NADP+ and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory. 6. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of beta-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase. | D-glucose-6-phosphate dehydrogenase (Entner-Doudoroff enzyme) from Pseudomonas fluorescens. Purification, properties and regulation. 1. The existence of two different D-glucose-6-phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway. 2. A procedure is described for the isolation of that D-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300 mumol NADH formed per min per mg protein, was shown to be homogeneous. 3. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265000. 4. The pI of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal activity at pH 8.9. 5. The Entner-Doudoroff enzyme showed specificity for NAD+ as well as for NADP+ and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory. 6. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of beta-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase. | 1,257 |
pubmed23n0001_605 | Factors affecting the molecular structure and the agglutinating ability of concanavalin A and other lectins. | Ultracentrifugation analyses were performed on lectins under varying conditions of pH, ionic strength and temperature. It has been demonstrated that the phytohemagglutinin from Phaseolus vulgaris, the wheat germ agglutinin and the soybean agglutinin are stable when these parameters are varied, whereas the concanavalin A molecule exhibits a striking reversible dimer-tetramer transition with variation in pH (from 6.0 to 7.2) and temperature (from 4 degrees up to 37 degrees C). It has also been demonstrated that, in agglutination experiments undertaken at different temperatures, cells do eventually aggregate with the first three lectins provided that incubation time is sufficient, whereas the concanavalin-A-induced agglutination was previously found to be temperature-sensitive. These results strongly suggest that the effect of temperature on agglutination by lectins may essentially be due to a structural transition of the lectin itself and nott only to modification of cell surface properties. | Factors affecting the molecular structure and the agglutinating ability of concanavalin A and other lectins. Ultracentrifugation analyses were performed on lectins under varying conditions of pH, ionic strength and temperature. It has been demonstrated that the phytohemagglutinin from Phaseolus vulgaris, the wheat germ agglutinin and the soybean agglutinin are stable when these parameters are varied, whereas the concanavalin A molecule exhibits a striking reversible dimer-tetramer transition with variation in pH (from 6.0 to 7.2) and temperature (from 4 degrees up to 37 degrees C). It has also been demonstrated that, in agglutination experiments undertaken at different temperatures, cells do eventually aggregate with the first three lectins provided that incubation time is sufficient, whereas the concanavalin-A-induced agglutination was previously found to be temperature-sensitive. These results strongly suggest that the effect of temperature on agglutination by lectins may essentially be due to a structural transition of the lectin itself and nott only to modification of cell surface properties. | 1,259 |
pubmed23n0001_608 | Multiple forms of human glutathione S-transferase and their affinity for bilirubin. | The initial enzymic step in mercapturic acid formation is catalyzed by glutathione S-transferase. Several species of this enzyme, designated as transferases alpha, beta, gamma, delta and epsilon on the basis of increasing isoelectric points, were isolated from human liver. Evidence is presented that each of the purified species is homogeneous with respect to sodium dodecylsulfate-gel electrophoresis. Transferases alpha, beta and epsilon each appear as a single band on gel electrofocusing; transferases gamma and delta are present as two and three bands, respectively, with each band catalytically active. Amino acid analysis indicated the five transferases to be either very closely related or identical in this respect. All enzyme species have a molecular weight of about 48500 and consist of two apparently identical subunits. The spectrum of substrates is the same for each although the enzymes differ slightly in specific activity. As is the case for the rat liver enzymes, each of the human transferases binds bilirubin although this compound is not a substrate. | Multiple forms of human glutathione S-transferase and their affinity for bilirubin. The initial enzymic step in mercapturic acid formation is catalyzed by glutathione S-transferase. Several species of this enzyme, designated as transferases alpha, beta, gamma, delta and epsilon on the basis of increasing isoelectric points, were isolated from human liver. Evidence is presented that each of the purified species is homogeneous with respect to sodium dodecylsulfate-gel electrophoresis. Transferases alpha, beta and epsilon each appear as a single band on gel electrofocusing; transferases gamma and delta are present as two and three bands, respectively, with each band catalytically active. Amino acid analysis indicated the five transferases to be either very closely related or identical in this respect. All enzyme species have a molecular weight of about 48500 and consist of two apparently identical subunits. The spectrum of substrates is the same for each although the enzymes differ slightly in specific activity. As is the case for the rat liver enzymes, each of the human transferases binds bilirubin although this compound is not a substrate. | 1,262 |
pubmed23n0001_610 | Spin-labelled phosphofructokinase. A simple and direct approach to the study of allosteric equilibria under near-physiological conditions. | Rabbit muscle phosphofructokinase, spin-labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors. The spectral changes have been interpreted in terms of a concerted allosteric transition between two conformational states with non-exclusive binding of effectors. On this basis MgATP, fructose 6-phosphate plus ATP, and NH+4ions behave as potent positive effectors, inorganic phosphate, sulphate, AMP, fructose 6-phosphate and fructose 1,6-bisphosphate are less potent activators, and free ATP and H+ions are negative effectors, in agreement with the kinetic behaviour, but citrate behaves anomalously. In addition, the allosteric equilibrium can be displaced towards the inhibited state by selectively modifying two further thiol groups. Strong positive cooperativity occurs under suitable conditions with ATP, metal-ATP and fructose 6-phosphate. Biphasic changes of conformation, attributed to binding at the catalytic and inhibitory sites, have been observed in titrations with ATP. The differentiation of the two ATP binding sites arises in the presence of fructose 6-phosphate because of a distinct concerted effect on conformation between the two substrates at the active site. A similar effect occurs between ATP and citrate. Other heterotropic effects are more consistent with simple models; phosphates favour the binding, and reduce the cooperativity, of fructose 6-phosphate and metal-ATP, whereas excess ATP and H+ ions antagonise the binding and increase the cooperativity of fructose 6-phosphate. The observations are related to existing kinetic and binding studies where possible. Anomalous features of the behaviour suggest that the model should be regarded only as a first approximation. | Spin-labelled phosphofructokinase. A simple and direct approach to the study of allosteric equilibria under near-physiological conditions. Rabbit muscle phosphofructokinase, spin-labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors. The spectral changes have been interpreted in terms of a concerted allosteric transition between two conformational states with non-exclusive binding of effectors. On this basis MgATP, fructose 6-phosphate plus ATP, and NH+4ions behave as potent positive effectors, inorganic phosphate, sulphate, AMP, fructose 6-phosphate and fructose 1,6-bisphosphate are less potent activators, and free ATP and H+ions are negative effectors, in agreement with the kinetic behaviour, but citrate behaves anomalously. In addition, the allosteric equilibrium can be displaced towards the inhibited state by selectively modifying two further thiol groups. Strong positive cooperativity occurs under suitable conditions with ATP, metal-ATP and fructose 6-phosphate. Biphasic changes of conformation, attributed to binding at the catalytic and inhibitory sites, have been observed in titrations with ATP. The differentiation of the two ATP binding sites arises in the presence of fructose 6-phosphate because of a distinct concerted effect on conformation between the two substrates at the active site. A similar effect occurs between ATP and citrate. Other heterotropic effects are more consistent with simple models; phosphates favour the binding, and reduce the cooperativity, of fructose 6-phosphate and metal-ATP, whereas excess ATP and H+ ions antagonise the binding and increase the cooperativity of fructose 6-phosphate. The observations are related to existing kinetic and binding studies where possible. Anomalous features of the behaviour suggest that the model should be regarded only as a first approximation. | 1,264 |
pubmed23n0001_613 | Investigations of the structure of 3-methylcrotonyl-CoA carboxylase from Achromobacter. | It was shown by gel electrophoresis in sodium dodecylsulphate solution that 3-methylcrotonyl-CoA carboxylase from Achromobacter IVS is composed of two different subunits with molecular weights of about 78000 and 96000, respectively. The biotin is bound to the heavier subunit. It was previously found that 3-methylcrotonyl-CoA carboxylase contains four biotin molecules per complex. A complex composed of four of each subunit would thus have a molecular weight of about 700000. This is compatible with the molecular weight of 760000 determined earlier by analytical ultracentrifugation. Both subunits were isolated preparatively. As the subunits, unlike the complex, are very sensitive to oxygen, special precautions had to be taken during isolation. The biotin-containing subunit was isolated by chromatography on DEAE-cellulose in 5 M urea. It no longer catalyzed the overall reaction, yet could still carboxylate free biotin. The biotin-free subunit was separated after dissociation of the enzyme by three-days' dialysis at pH 9.8 under nitrogen. On chromatography over a Sepharose-bound avidin column, the biotin-subunit was fixed and the biotin-free subunit was eluted unretarded. The latter subunit showed no enzymic activity. After the addition of the biotin-containing subunit, overall activity was regenerated. The speed of reassociation is very much enhanced by 3-methylcrotonyl-CoA. It was shown by reassociation experiments under different conditions that probably an initial complex, AxBy is formed, possessing a binding site for 3-methylcrotonyl-CoA. Upon the binding of this substrate the conformation may be changed to a form favourable for reconstitution. Finally, the structures of biotin enzymes from different sources are compared. In the course of evolution there is a tendency toward integration of the different constituent proteins into only one polypeptide chain. | Investigations of the structure of 3-methylcrotonyl-CoA carboxylase from Achromobacter. It was shown by gel electrophoresis in sodium dodecylsulphate solution that 3-methylcrotonyl-CoA carboxylase from Achromobacter IVS is composed of two different subunits with molecular weights of about 78000 and 96000, respectively. The biotin is bound to the heavier subunit. It was previously found that 3-methylcrotonyl-CoA carboxylase contains four biotin molecules per complex. A complex composed of four of each subunit would thus have a molecular weight of about 700000. This is compatible with the molecular weight of 760000 determined earlier by analytical ultracentrifugation. Both subunits were isolated preparatively. As the subunits, unlike the complex, are very sensitive to oxygen, special precautions had to be taken during isolation. The biotin-containing subunit was isolated by chromatography on DEAE-cellulose in 5 M urea. It no longer catalyzed the overall reaction, yet could still carboxylate free biotin. The biotin-free subunit was separated after dissociation of the enzyme by three-days' dialysis at pH 9.8 under nitrogen. On chromatography over a Sepharose-bound avidin column, the biotin-subunit was fixed and the biotin-free subunit was eluted unretarded. The latter subunit showed no enzymic activity. After the addition of the biotin-containing subunit, overall activity was regenerated. The speed of reassociation is very much enhanced by 3-methylcrotonyl-CoA. It was shown by reassociation experiments under different conditions that probably an initial complex, AxBy is formed, possessing a binding site for 3-methylcrotonyl-CoA. Upon the binding of this substrate the conformation may be changed to a form favourable for reconstitution. Finally, the structures of biotin enzymes from different sources are compared. In the course of evolution there is a tendency toward integration of the different constituent proteins into only one polypeptide chain. | 1,267 |
pubmed23n0001_617 | Purification and properties of a periplasmic aminoendopeptidase from Escherichia coli. | A periplasmic aminoendopeptidase from Escherichia coli has been purified to hemogeneity. It is a monomer of molecular weight 45000 and containing one -- SH group that is necessary for catalytic activity. The study of its substrate specificity indicated that the enzyme has both aminopeptidase and endopeptidase activity. The pH optimum for L-alanine p-nitroanilide hydrolysis is between 7 and 7.5 and that for 125I-labeled casein proteolysis between 7.3 and 7.6. The activation energy for the hydrolysis of L-anine p-nitroanilide was calculated to be 5.3 kcal X mol-1 (22.2 kJ X mol-1). | Purification and properties of a periplasmic aminoendopeptidase from Escherichia coli. A periplasmic aminoendopeptidase from Escherichia coli has been purified to hemogeneity. It is a monomer of molecular weight 45000 and containing one -- SH group that is necessary for catalytic activity. The study of its substrate specificity indicated that the enzyme has both aminopeptidase and endopeptidase activity. The pH optimum for L-alanine p-nitroanilide hydrolysis is between 7 and 7.5 and that for 125I-labeled casein proteolysis between 7.3 and 7.6. The activation energy for the hydrolysis of L-anine p-nitroanilide was calculated to be 5.3 kcal X mol-1 (22.2 kJ X mol-1). | 1,271 |
pubmed23n0001_619 | Hepatic nucleases. Extrahepatic origin and association of neutral liver ribonuclease with lysosomes. | In the large granule fraction of rat liver, the density distribution of inhibitor-sensitive neutral ribonuclease is similar to that for acid hydrolases and its density distribution is similarly modified by Triton WR-1339 accumulation in lysosomes. Particulate neutral ribonuclease is latent; the enzyme is unmasked by very low digitonin concentrations or hypoosmotic shock. These observations demonstrate that the bulk of liver neutral ribonuclease is associated with the lysosomal system. In view of the neutral pH optimum of the enzyme and of some particularities of its distribution in fractionation experiments, the possiblilty of an extrahepatic origin of neutral ribonuclease has been investigated. After partial pancreatectomy, a significant decrease is observed in both plasma and liver neutral ribonuclease. The effect is specific, for it does not occur for other lysosomal enzymes. Also, labelled bovine pancreatic ribonuclease, when injected intravenously, is taken up by the liver. The sedimentable labelled enzyme has a density distribution similar to the distribution of other foreign proteins, horseradish peroxidase or yeast invertase. These results are explained by the uptake of plasmatic neutral ribonuclease from pancreatic origin by the liver. | Hepatic nucleases. Extrahepatic origin and association of neutral liver ribonuclease with lysosomes. In the large granule fraction of rat liver, the density distribution of inhibitor-sensitive neutral ribonuclease is similar to that for acid hydrolases and its density distribution is similarly modified by Triton WR-1339 accumulation in lysosomes. Particulate neutral ribonuclease is latent; the enzyme is unmasked by very low digitonin concentrations or hypoosmotic shock. These observations demonstrate that the bulk of liver neutral ribonuclease is associated with the lysosomal system. In view of the neutral pH optimum of the enzyme and of some particularities of its distribution in fractionation experiments, the possiblilty of an extrahepatic origin of neutral ribonuclease has been investigated. After partial pancreatectomy, a significant decrease is observed in both plasma and liver neutral ribonuclease. The effect is specific, for it does not occur for other lysosomal enzymes. Also, labelled bovine pancreatic ribonuclease, when injected intravenously, is taken up by the liver. The sedimentable labelled enzyme has a density distribution similar to the distribution of other foreign proteins, horseradish peroxidase or yeast invertase. These results are explained by the uptake of plasmatic neutral ribonuclease from pancreatic origin by the liver. | 1,273 |
pubmed23n0001_629 | Analysis of skin grafts across the MSA-barrier in mice pretreated with sera from specifically or syngeeically grafted donors. | Prolonged survival of weakly incompatible skin allografts in mice (across the barrier presented by the MSA) can be induced by pretreating the recipients not only with a specific anti-MSA serum (obtained on day 5 after a single MSA-incompatible skin graft) but also be means of control serum obtained in a similar way from the recipients of fully compatible (syngeneic) skin grafts. Administration of serum from non-grafted mice had no effect on graft survival. The similar biological effect of both sera had a counterpart in their similar content and spectrum of glycosaminoglycans. Also in the skin grafts themselves, the course of both qualitative and quantitative changes of GAG in the early postgrafting period was in the allogeneic and syngeneic situation similar. The possible role of these substances in the serum and at the site of grafting and their effect on the outcome of the allograft response are discussed. | Analysis of skin grafts across the MSA-barrier in mice pretreated with sera from specifically or syngeeically grafted donors. Prolonged survival of weakly incompatible skin allografts in mice (across the barrier presented by the MSA) can be induced by pretreating the recipients not only with a specific anti-MSA serum (obtained on day 5 after a single MSA-incompatible skin graft) but also be means of control serum obtained in a similar way from the recipients of fully compatible (syngeneic) skin grafts. Administration of serum from non-grafted mice had no effect on graft survival. The similar biological effect of both sera had a counterpart in their similar content and spectrum of glycosaminoglycans. Also in the skin grafts themselves, the course of both qualitative and quantitative changes of GAG in the early postgrafting period was in the allogeneic and syngeneic situation similar. The possible role of these substances in the serum and at the site of grafting and their effect on the outcome of the allograft response are discussed. | 1,295 |
pubmed23n0001_634 | Comparison of amino acids bathing the oxyntic gland area in the stimulation of gastric secretion. | This study was undertaken to compare the ability of L- and D-isomers of amino acids bathing the oxyntic gland area to stimulate acid secretion in conscious dogs with Heidenhain pouch (HP), gastric fistula (GF) and pancreatic fistula (PF). Acid outputs from HP were determined by an intragastric titration method when amino acid solutions were perfused into HP at various concentrations, pH values, and distention pressures. Only L-isomers of all natural amino acids were found to stimulate acid secretion, whereas D-isomers of amino acids tested were completely inert in this respect. The comparison of the secretagogue activity of amino acids shows that L-histidine among essential amino acids and glycine among nonessential amino acids exhibited the strongest stimulation of acid outputs, reaching, respectively, 52 and 40% of the maximal response to histamine. Decreasing the pH of L-histidine solution perfused into HP in sequential order from 5.0 to 1.0 resulted in a stepwise reduction of acid output, falling at pH 1.0 to about 40% of the peak response achieved at pH 5.0. Local irrigation of HP by 2% xylocaine and intravenous infusion of atropine (100 mug per kg per hr) or metiamide (2.9 mg per kg per hr) reduced but did not abolish HP response to chemical stimulation and the pH dependency of this response. We conclude that only L- and not D-isomers of amino acids bathing the oxyntic gland area stimulate acid secretion by a local, gastrin-independent mechanism sensitive to distention pressure and pH. | Comparison of amino acids bathing the oxyntic gland area in the stimulation of gastric secretion. This study was undertaken to compare the ability of L- and D-isomers of amino acids bathing the oxyntic gland area to stimulate acid secretion in conscious dogs with Heidenhain pouch (HP), gastric fistula (GF) and pancreatic fistula (PF). Acid outputs from HP were determined by an intragastric titration method when amino acid solutions were perfused into HP at various concentrations, pH values, and distention pressures. Only L-isomers of all natural amino acids were found to stimulate acid secretion, whereas D-isomers of amino acids tested were completely inert in this respect. The comparison of the secretagogue activity of amino acids shows that L-histidine among essential amino acids and glycine among nonessential amino acids exhibited the strongest stimulation of acid outputs, reaching, respectively, 52 and 40% of the maximal response to histamine. Decreasing the pH of L-histidine solution perfused into HP in sequential order from 5.0 to 1.0 resulted in a stepwise reduction of acid output, falling at pH 1.0 to about 40% of the peak response achieved at pH 5.0. Local irrigation of HP by 2% xylocaine and intravenous infusion of atropine (100 mug per kg per hr) or metiamide (2.9 mg per kg per hr) reduced but did not abolish HP response to chemical stimulation and the pH dependency of this response. We conclude that only L- and not D-isomers of amino acids bathing the oxyntic gland area stimulate acid secretion by a local, gastrin-independent mechanism sensitive to distention pressure and pH. | 1,322 |
pubmed23n0001_637 | Genetic hybridization at the unlinked thy and str loci of Streptococcus. | The sanguis and pneumoniae species of Streptococcus were used as recipients in transformations from str+ to str-r and from thy- to thy+. The str-r mutations in the two species had been previously shown to be allelic. Homology of the thy- mutations in the two species was demonstrated in the similar phenotypic properties they conferred (death in the absence of thymidine, lack of thymidylate synthetase). The str and thy loci are unlinked in each species.--- When the two species are transformed by both homospecific and heterospecific DNA, the efficiency is always lower in the heterospecific cross. The efficiency of heterospecific transformation is considerably lower at the thy than at the str locus. DNA was extracted from recipients that had integrated markers of heterospecific origin. When such hybrid DNA is tested on the original recipient species, the heterospecific markers are usually as efficient as homospecific markers. When tested on the original donor species, however, the hybrid DNA is usually more efficient than heterospecific DNA. This is true for both thy and str transformation. -- -- Forty independent thy+ hybrids were obtained in the cross of sanguis thy- recipients with pneumoniae thy+ DNA. These hybrids fall into a number of classes based upon the relative efficiency with which their extracted DNA's are able to transfer the thy+ marker into pneumoniae thy- cells. The most efficient of these DNA's exhibits about 20% of the efficiency of homospecific pneumoniae thy+ DNA and three orders of magnitude greater efficiency than heterospecific sanguis thy+ DNA. Thus, very little of the inefficiency of heterospecific transformation of the thy locus is ascribable to a classic restriction mechanism. Rather, the wild-type thy+ loci in the two species appear to differ at multiple sites, and independent heterospecific transfers result in differential extents of integration of these sites. On this basis, the thy+ loci of the two species differ at a greater number of sites than do the respective str+ loci. | Genetic hybridization at the unlinked thy and str loci of Streptococcus. The sanguis and pneumoniae species of Streptococcus were used as recipients in transformations from str+ to str-r and from thy- to thy+. The str-r mutations in the two species had been previously shown to be allelic. Homology of the thy- mutations in the two species was demonstrated in the similar phenotypic properties they conferred (death in the absence of thymidine, lack of thymidylate synthetase). The str and thy loci are unlinked in each species.--- When the two species are transformed by both homospecific and heterospecific DNA, the efficiency is always lower in the heterospecific cross. The efficiency of heterospecific transformation is considerably lower at the thy than at the str locus. DNA was extracted from recipients that had integrated markers of heterospecific origin. When such hybrid DNA is tested on the original recipient species, the heterospecific markers are usually as efficient as homospecific markers. When tested on the original donor species, however, the hybrid DNA is usually more efficient than heterospecific DNA. This is true for both thy and str transformation. -- -- Forty independent thy+ hybrids were obtained in the cross of sanguis thy- recipients with pneumoniae thy+ DNA. These hybrids fall into a number of classes based upon the relative efficiency with which their extracted DNA's are able to transfer the thy+ marker into pneumoniae thy- cells. The most efficient of these DNA's exhibits about 20% of the efficiency of homospecific pneumoniae thy+ DNA and three orders of magnitude greater efficiency than heterospecific sanguis thy+ DNA. Thus, very little of the inefficiency of heterospecific transformation of the thy locus is ascribable to a classic restriction mechanism. Rather, the wild-type thy+ loci in the two species appear to differ at multiple sites, and independent heterospecific transfers result in differential extents of integration of these sites. On this basis, the thy+ loci of the two species differ at a greater number of sites than do the respective str+ loci. | 1,325 |
pubmed23n0001_638 | Fibrin plate method with reagents purified by affinity chromatography and its use for determination of fibrinolytic and other proteolytic activity in saliva, bile and plasma. | A modification of the fibrin plate method is presented. Plasminogen-free human fibrinogen and plasminogen purified by affinity chromatography have been used. Fibrin plates without and with a constant amount of plasminogen and with agarose as stabilizing medium were used for the estimation of plasmin and plasminogen activator activity. Activator activity could be demonstrated in sterile bile and saliva. When plasmin activity was present, estimations of plasminogen activator were approximate. The method is sensitive, small volumes of reagents and samples are needed. The error of the method is comparatively low and the reproducibility is good. | Fibrin plate method with reagents purified by affinity chromatography and its use for determination of fibrinolytic and other proteolytic activity in saliva, bile and plasma. A modification of the fibrin plate method is presented. Plasminogen-free human fibrinogen and plasminogen purified by affinity chromatography have been used. Fibrin plates without and with a constant amount of plasminogen and with agarose as stabilizing medium were used for the estimation of plasmin and plasminogen activator activity. Activator activity could be demonstrated in sterile bile and saliva. When plasmin activity was present, estimations of plasminogen activator were approximate. The method is sensitive, small volumes of reagents and samples are needed. The error of the method is comparatively low and the reproducibility is good. | 1,332 |
pubmed23n0001_642 | Human liver acid phosphatases. | Human liver contains three chromatographically distinct forms of non-specific acid phosphatase (EC 3.1.3.2). Acid phosphatases I, II and III have molecular weights of greater than 200 000, of 107 000, and of 13 400, respectively. Following partial purification, isoenzyme II was obtained as a single activity band, as assessed by activity staining with p-nitrophenyl phosphate and alpha-naphthyl phosphate on polyacrylamide gels run at several pH values. With 50mM p-nitrophenyl phosphate as a substrate, enzymes II and III exhibit plateaus of activity over the pH range 3 - 5 and 3.5 - 6, respectively. Acid phosphatase II is not significantly inhibited by 0.5% formaldehyde. The activity of human liver acid phosphatase II and of human prostatic acid phosphatase towards several substrates is compared. The liver enzyme, is marked contrast to the prostatic enzyme, does not hydrolyze O-phosphoryl choline. | Human liver acid phosphatases. Human liver contains three chromatographically distinct forms of non-specific acid phosphatase (EC 3.1.3.2). Acid phosphatases I, II and III have molecular weights of greater than 200 000, of 107 000, and of 13 400, respectively. Following partial purification, isoenzyme II was obtained as a single activity band, as assessed by activity staining with p-nitrophenyl phosphate and alpha-naphthyl phosphate on polyacrylamide gels run at several pH values. With 50mM p-nitrophenyl phosphate as a substrate, enzymes II and III exhibit plateaus of activity over the pH range 3 - 5 and 3.5 - 6, respectively. Acid phosphatase II is not significantly inhibited by 0.5% formaldehyde. The activity of human liver acid phosphatase II and of human prostatic acid phosphatase towards several substrates is compared. The liver enzyme, is marked contrast to the prostatic enzyme, does not hydrolyze O-phosphoryl choline. | 1,336 |
pubmed23n0001_648 | [Past and present aspects of diarrheal disease in childhood. Clinical study and treatment (author's transl)]. | The etiologic and pathophysiologic findings described in the first part of this paper have important consequences: The recognition of the specific etiology of diarrhea requires new laboratory methods: most of these, however, are technically easy to perform and do not require a large laboratory. A long-ranging consequence of this changed concept is a well-founded modification of therapy. The most important discovery was, that in a well balanced glucose electrolyte solution sodium and glucose enhance their absorption mutually and increase the absorption of water by solvent drag. Since in most acute diarrheas the mechanisms of absorption of glucose and electrolytes are retained this mechanism can be utilized for fast oral rehydration and reinstitution of normal intestinal homeostasis. Prompt institution of a diet consisting of the previously mentioned glucose-electrolyte solution usually prevents severe dehydration and the need for stationary treatment. The elimination of lactose and long chain fatty acids from the diet prevents continuation of the pathologic osmotic and chemical conditions in the intestine. Antibiotics are not indicated in acute diarrhea with the exception of diarrhea caused by enteroinvasive E. Coli or Shigella, in the case of Salmonella-gastroenteritis even contraindicated. Further research concentrates on the development of drugs for neutralisation of E. Coli enterotoxin and the prevention of diarrheas by development of effective vaccines. | [Past and present aspects of diarrheal disease in childhood. Clinical study and treatment (author's transl)]. The etiologic and pathophysiologic findings described in the first part of this paper have important consequences: The recognition of the specific etiology of diarrhea requires new laboratory methods: most of these, however, are technically easy to perform and do not require a large laboratory. A long-ranging consequence of this changed concept is a well-founded modification of therapy. The most important discovery was, that in a well balanced glucose electrolyte solution sodium and glucose enhance their absorption mutually and increase the absorption of water by solvent drag. Since in most acute diarrheas the mechanisms of absorption of glucose and electrolytes are retained this mechanism can be utilized for fast oral rehydration and reinstitution of normal intestinal homeostasis. Prompt institution of a diet consisting of the previously mentioned glucose-electrolyte solution usually prevents severe dehydration and the need for stationary treatment. The elimination of lactose and long chain fatty acids from the diet prevents continuation of the pathologic osmotic and chemical conditions in the intestine. Antibiotics are not indicated in acute diarrhea with the exception of diarrhea caused by enteroinvasive E. Coli or Shigella, in the case of Salmonella-gastroenteritis even contraindicated. Further research concentrates on the development of drugs for neutralisation of E. Coli enterotoxin and the prevention of diarrheas by development of effective vaccines. | 1,346 |
pubmed23n0001_654 | Induction of corneal graft rejection by passive cell transfer. | An experimental model is presented demonstrating that penetrating corneal grafts in the rabbit may be rejected by passive transfer into the anterior chamber of specifically sensitized lymphoid cells. Destruction of histo-incompatible corneal endothelium is always marked by the formation of focal pock-like areas of damage in this system, rather than by the typical moving line of rejecting endothelium usually seen in spontaneous graft rejection. Where the transferred lymphoid cells are compatible with the tissues of the graft recipient, the picture is one of a severely affected graft on a field of uninvolved recipient corneal endothelium. Where the lymphoid cells are compatible with the graft and not with the tissues of the recipient, one sees a clear corneal graft surviving on a field of endothelial destruction on the recipient bed. The specificity of these reactions is illustrated in terms of the histocompatibility relationships between corneal donor, graft recipient, and the donor of the sensitized lymphoid cells. | Induction of corneal graft rejection by passive cell transfer. An experimental model is presented demonstrating that penetrating corneal grafts in the rabbit may be rejected by passive transfer into the anterior chamber of specifically sensitized lymphoid cells. Destruction of histo-incompatible corneal endothelium is always marked by the formation of focal pock-like areas of damage in this system, rather than by the typical moving line of rejecting endothelium usually seen in spontaneous graft rejection. Where the transferred lymphoid cells are compatible with the tissues of the graft recipient, the picture is one of a severely affected graft on a field of uninvolved recipient corneal endothelium. Where the lymphoid cells are compatible with the graft and not with the tissues of the recipient, one sees a clear corneal graft surviving on a field of endothelial destruction on the recipient bed. The specificity of these reactions is illustrated in terms of the histocompatibility relationships between corneal donor, graft recipient, and the donor of the sensitized lymphoid cells. | 1,357 |
pubmed23n0001_662 | Purification and properties of polyol dehydrogenase from Cephalosporium chrysogenus. | A polyol dehydrogenase of broad specificity was purified 178-fold from extracts of the filamentous fungus Cephalosporium chrysogenum. The enzyme was found to act as an oxido-reductase in two substrate-coenzyme systems: D-sorbitol (or xylitol)-nicotinamide-adenine dinucleotide (NAD) and D-mannitol-nicotinamide adenine dinucleotide phosphate (NADP). The dehydrogenase was composed of five isozymes, which, as a mixture, exhibited these properties: Km to D-sorbitol and D-mannitol, 7.15 to 10(-2) M; PH optimum, 9 to 10; molecular weight, 300,000 subunit weight, 29,000; PI, 5.8 to 7.5. The NADP-linked activity was labile to treatment with heat or ethylenediaminetetraacetic acid. Mixed substrate assays support the hypothesis that both NAD-, and NADP-linked activities are associated with isozymes of a single dehydrogenase. | Purification and properties of polyol dehydrogenase from Cephalosporium chrysogenus. A polyol dehydrogenase of broad specificity was purified 178-fold from extracts of the filamentous fungus Cephalosporium chrysogenum. The enzyme was found to act as an oxido-reductase in two substrate-coenzyme systems: D-sorbitol (or xylitol)-nicotinamide-adenine dinucleotide (NAD) and D-mannitol-nicotinamide adenine dinucleotide phosphate (NADP). The dehydrogenase was composed of five isozymes, which, as a mixture, exhibited these properties: Km to D-sorbitol and D-mannitol, 7.15 to 10(-2) M; PH optimum, 9 to 10; molecular weight, 300,000 subunit weight, 29,000; PI, 5.8 to 7.5. The NADP-linked activity was labile to treatment with heat or ethylenediaminetetraacetic acid. Mixed substrate assays support the hypothesis that both NAD-, and NADP-linked activities are associated with isozymes of a single dehydrogenase. | 1,374 |
pubmed23n0001_665 | Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli. | beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase. | Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli. beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase. | 1,377 |
pubmed23n0001_666 | Acid protease activity during germination of microcysts of the cellular slime mold Polysphondylium pallidum. | Extracts of dormant microcysts of Polysphondylium pallidum demonstrate pH optima for the hydrolysis of casein at 3.5 and 6.0. During germination the intracellular pH 6.0 caseinolytic specific activity does not change significantly. The pH 6.0 protease is also active on azo-albumin, revealing the same developmental pattern with this substrate. Both acid protease activities are excreted during the germination process. Addition of purified nonspecific protease to cultures speeds up germination, suggesting that the excreted protease may play a role in removal of the microcyst wall. When cycloheximide is added to cultures, complete germination (emergence) is stopped whereas the pH 6.0 protease activity still accumulates to between 50 and 60% of the maximum control activity. Although this suggests that post-translational controls might mediate the accumulation of a portion of the pH 6.0 protease increase, mixing and dilution experiments with cell extracts do not reveal the differential presence of soluble activators or inhibitors of this activity at different developmental stages. The presence of tightly bound enzyme-inhibitor complexes for protease B in dormant microcysts has not been ruled out and is currently under study. | Acid protease activity during germination of microcysts of the cellular slime mold Polysphondylium pallidum. Extracts of dormant microcysts of Polysphondylium pallidum demonstrate pH optima for the hydrolysis of casein at 3.5 and 6.0. During germination the intracellular pH 6.0 caseinolytic specific activity does not change significantly. The pH 6.0 protease is also active on azo-albumin, revealing the same developmental pattern with this substrate. Both acid protease activities are excreted during the germination process. Addition of purified nonspecific protease to cultures speeds up germination, suggesting that the excreted protease may play a role in removal of the microcyst wall. When cycloheximide is added to cultures, complete germination (emergence) is stopped whereas the pH 6.0 protease activity still accumulates to between 50 and 60% of the maximum control activity. Although this suggests that post-translational controls might mediate the accumulation of a portion of the pH 6.0 protease increase, mixing and dilution experiments with cell extracts do not reveal the differential presence of soluble activators or inhibitors of this activity at different developmental stages. The presence of tightly bound enzyme-inhibitor complexes for protease B in dormant microcysts has not been ruled out and is currently under study. | 1,378 |
pubmed23n0001_667 | Altered nutritional requirements associated with mutations affecting the structures of ribonucleic acid polymerase in Lactobacillus casei. | Rifampin-resistant mutants were isolated from Lactobacillus casei S1 and examined for possible simultaneous alteration in nutritional properties. Among the 36 mutants obtained either spontaneously or after mutagenesis with 2-aminopurine, 22 were found to be altered with respect to the specific growth requirements. The majority (20 of 22) of the latter mutants were shown to require L-glutamine in addition to the nutrients required by the parental strain for maximal growth, whereas the remaining mutants had apparently lost the requirement for L-aspartate. Further studies with one of the glutamine-requiring mutants revealed that the rifampin resistance of this strain is due to the resistance of ribonucleic acid polymerase itself and that a single mutation is responsible for both rifampin resistance and the glutamine requirement. These results strongly indicate that a structural alteration of the ribonucleic acid polymerase caused by the rifampin resistance mutation somehow affected glutamine metabolism, possibly through change in selective transcription of the genes involved. | Altered nutritional requirements associated with mutations affecting the structures of ribonucleic acid polymerase in Lactobacillus casei. Rifampin-resistant mutants were isolated from Lactobacillus casei S1 and examined for possible simultaneous alteration in nutritional properties. Among the 36 mutants obtained either spontaneously or after mutagenesis with 2-aminopurine, 22 were found to be altered with respect to the specific growth requirements. The majority (20 of 22) of the latter mutants were shown to require L-glutamine in addition to the nutrients required by the parental strain for maximal growth, whereas the remaining mutants had apparently lost the requirement for L-aspartate. Further studies with one of the glutamine-requiring mutants revealed that the rifampin resistance of this strain is due to the resistance of ribonucleic acid polymerase itself and that a single mutation is responsible for both rifampin resistance and the glutamine requirement. These results strongly indicate that a structural alteration of the ribonucleic acid polymerase caused by the rifampin resistance mutation somehow affected glutamine metabolism, possibly through change in selective transcription of the genes involved. | 1,379 |
pubmed23n0001_671 | Derepression of certain aromatic amino acid biosynthetic enzymes of Escherichia coli K-12 by growth in Fe3+-deficient medium. | 3-Deoxy-arabino-heptulosonic acid 7-phosphate synthase, prephenate dehydratase, tryptophan synthase, and 2,3-dihydroxybenzoylserine synthase enzyme activities are derepressed in wild-type Escherichia coli K-12 cells grown on Fe3+-deficient medium. This derepression is reversed when FeSO4 is added to the growth medium. Addition of shikimic acid to the Fe3+-deficient growth medium caused repression of the first three enzyme activities but not of 2,3-dihydroxybenzoylserine synthase activity. Addition of 2,3-dihydroxybenzoic acid to the Fe3+-deficient growth medium has no effect on any of the above-mentioned enzyme activities. The Fe3+ deficiency-mediated derepression of 3-deoxyarabino-heptulosonic acid 7-phosphate synthase activity is due to an elevation of the tyrosine-sensitive isoenzyme; the phenylalanine-sensitive isoenzyme is not derepressed under these conditions. | Derepression of certain aromatic amino acid biosynthetic enzymes of Escherichia coli K-12 by growth in Fe3+-deficient medium. 3-Deoxy-arabino-heptulosonic acid 7-phosphate synthase, prephenate dehydratase, tryptophan synthase, and 2,3-dihydroxybenzoylserine synthase enzyme activities are derepressed in wild-type Escherichia coli K-12 cells grown on Fe3+-deficient medium. This derepression is reversed when FeSO4 is added to the growth medium. Addition of shikimic acid to the Fe3+-deficient growth medium caused repression of the first three enzyme activities but not of 2,3-dihydroxybenzoylserine synthase activity. Addition of 2,3-dihydroxybenzoic acid to the Fe3+-deficient growth medium has no effect on any of the above-mentioned enzyme activities. The Fe3+ deficiency-mediated derepression of 3-deoxyarabino-heptulosonic acid 7-phosphate synthase activity is due to an elevation of the tyrosine-sensitive isoenzyme; the phenylalanine-sensitive isoenzyme is not derepressed under these conditions. | 1,383 |
pubmed23n0001_672 | Control of differentiation in streptomycetes: involvement of extrachromosomal deoxyribonucleic acid and glucose repression in aerial mycelia development. | When Streptomyces alboniger spores were grown in Hickey-Tresner broth containing 5 muM ethidium bromide, a high frequency of permanently cured aerial mycelia-negative (am-) colonies was recovered. The appearance an am- colonies was time dependent: a very low frequency (0.3%) at zero time, a maximum (9 to 21%) after 2 to 5 days of growth, and a decline again to low frequencies later in the growth cycle. On agar, cured am- colonies of S. alboniger still produced puromycin. The development of aerial mycelia in S. alboniger, S. scabies, and S. coelicolor was also sensitive to glucose repression. Colonies grown on Hickey-Tresner agar containing 2% glucose remained phenotypically am- throughout the observation period. Adenine (2.5 mM or greater), and to a lesser extent adenosine and guanosine, specifically reversed the repression. The accumulation of undissociated organic acids appears to be involved in glucose repression of aerial mycelia formation. However, this does not appear to be the case with puromycin production in S. alboniger; glucose repression was observed over the pH range 5.0 to 7.5. | Control of differentiation in streptomycetes: involvement of extrachromosomal deoxyribonucleic acid and glucose repression in aerial mycelia development. When Streptomyces alboniger spores were grown in Hickey-Tresner broth containing 5 muM ethidium bromide, a high frequency of permanently cured aerial mycelia-negative (am-) colonies was recovered. The appearance an am- colonies was time dependent: a very low frequency (0.3%) at zero time, a maximum (9 to 21%) after 2 to 5 days of growth, and a decline again to low frequencies later in the growth cycle. On agar, cured am- colonies of S. alboniger still produced puromycin. The development of aerial mycelia in S. alboniger, S. scabies, and S. coelicolor was also sensitive to glucose repression. Colonies grown on Hickey-Tresner agar containing 2% glucose remained phenotypically am- throughout the observation period. Adenine (2.5 mM or greater), and to a lesser extent adenosine and guanosine, specifically reversed the repression. The accumulation of undissociated organic acids appears to be involved in glucose repression of aerial mycelia formation. However, this does not appear to be the case with puromycin production in S. alboniger; glucose repression was observed over the pH range 5.0 to 7.5. | 1,384 |
pubmed23n0001_674 | Isolation and characterization of crystalline methylglyoxal synthetase from Proteus vulgaris. | Methylglyoxal synthetase, which catalyzes the conversion of dihydroxyacetone phosphate to methylglyoxal and inorganic phosphate, has been isolated and crystalized in good yields from Proteus vulgaris. The enzyme was shown to be homogeneous by a variety of criteria and was found to be a dimer (Mr = 135,000; s20,w = 7.2 S) composed of two apparently identical catalytic and physical properties and their interconvertible nature suggest that they do not represent true isozymes. The enzyme is specific for dihydroxyacetone phosphate and does not form methylglyoxal from glyceraldehyde 3-phophate, glyceraldehyde, or dihydroxyacetone. Nonphosphorylated analogs are neither substrates nor competive inhibitors, but a variety of phosphorylated analogs are competitive with respect to dihydroxyacetone phosphate. The enzyme is inhibited by inorganic orthophosphate in a complex manner which is overcome by dihydroxyacetone phosphate in a signoidal manner | Isolation and characterization of crystalline methylglyoxal synthetase from Proteus vulgaris. Methylglyoxal synthetase, which catalyzes the conversion of dihydroxyacetone phosphate to methylglyoxal and inorganic phosphate, has been isolated and crystalized in good yields from Proteus vulgaris. The enzyme was shown to be homogeneous by a variety of criteria and was found to be a dimer (Mr = 135,000; s20,w = 7.2 S) composed of two apparently identical catalytic and physical properties and their interconvertible nature suggest that they do not represent true isozymes. The enzyme is specific for dihydroxyacetone phosphate and does not form methylglyoxal from glyceraldehyde 3-phophate, glyceraldehyde, or dihydroxyacetone. Nonphosphorylated analogs are neither substrates nor competive inhibitors, but a variety of phosphorylated analogs are competitive with respect to dihydroxyacetone phosphate. The enzyme is inhibited by inorganic orthophosphate in a complex manner which is overcome by dihydroxyacetone phosphate in a signoidal manner | 1,386 |
pubmed23n0001_677 | Rickettsial permeability. An ADP-ATP transport system. | The obligate intracellular parasitic bacterium, Rickettsia prowazeki, has a carrier-mediated transport system for ADP and ATP. The transport of nucleotides was measured by membrane filtration assays; the assay was shown not to harm the relatively labile rickettsiae. The nucleotide transport system was shown to reside in the rickettsiae, not in the contaminating yolk sac mitochondria of the preparation. The influx of nucleotide had an activation energy of 12 to 13 kcal above 22 deg-rees (an apparent transition temperature), and 30 kcal below this value. The uptake of nucleotide was independent of the Mg2+ concentration, but was markedly stimulated by the phosphate concentration. The pH optimum of the influx of nucleotide was pH 7. The specificity of the transport system was remarkable in that it required a specific moiety in each portion of the nucleotide, i.e. an adenine base, a ribose sugar, and two or three, but not one, phosphates. Of the wide variety of compounds tested, the system could transport only ADP, ATP, and (beta, gamma-methylene) adenosine 5'-triphosphate. The influx of nucleotide was a saturable process; half-maximum velocity was achieved at a nucleotide concentration of about 75 muM. ADP and ATP were competitive inhibitors of each other's transport. Although at least 95% of the labeled intracellular nucleotide was exchangeable, efflux of labeled nucleotide was observed only in the presence of unlabeled nucleotide in the medium. Half-maximum efflux was achieved at a concentration of about 75 muM. A large intracellular to extracellular concentration gradient of labeled nucleotide was maintained in the presence of metabolic inhibitors and uncouplers, which completely abolished rickettsial hemolysis. While having no effect on the steady state, KCN and DNP accelerated both influx and efflux. Measurements of the endogenous pool of adenine nucleotides in isolated rickettsiae show that is was large (5 mM), and that these unlabeled nucleotides exchanged, on approximately a 1/1 basis, with exogenously added nucleotide. These studies support the proposal that rickettsiae are not "leaky" to adenine nucleotides or to small molecules in general, and that they have a carrier-mediated transport system which allows an exchange of host and parasite ADP and ATP. | Rickettsial permeability. An ADP-ATP transport system. The obligate intracellular parasitic bacterium, Rickettsia prowazeki, has a carrier-mediated transport system for ADP and ATP. The transport of nucleotides was measured by membrane filtration assays; the assay was shown not to harm the relatively labile rickettsiae. The nucleotide transport system was shown to reside in the rickettsiae, not in the contaminating yolk sac mitochondria of the preparation. The influx of nucleotide had an activation energy of 12 to 13 kcal above 22 deg-rees (an apparent transition temperature), and 30 kcal below this value. The uptake of nucleotide was independent of the Mg2+ concentration, but was markedly stimulated by the phosphate concentration. The pH optimum of the influx of nucleotide was pH 7. The specificity of the transport system was remarkable in that it required a specific moiety in each portion of the nucleotide, i.e. an adenine base, a ribose sugar, and two or three, but not one, phosphates. Of the wide variety of compounds tested, the system could transport only ADP, ATP, and (beta, gamma-methylene) adenosine 5'-triphosphate. The influx of nucleotide was a saturable process; half-maximum velocity was achieved at a nucleotide concentration of about 75 muM. ADP and ATP were competitive inhibitors of each other's transport. Although at least 95% of the labeled intracellular nucleotide was exchangeable, efflux of labeled nucleotide was observed only in the presence of unlabeled nucleotide in the medium. Half-maximum efflux was achieved at a concentration of about 75 muM. A large intracellular to extracellular concentration gradient of labeled nucleotide was maintained in the presence of metabolic inhibitors and uncouplers, which completely abolished rickettsial hemolysis. While having no effect on the steady state, KCN and DNP accelerated both influx and efflux. Measurements of the endogenous pool of adenine nucleotides in isolated rickettsiae show that is was large (5 mM), and that these unlabeled nucleotides exchanged, on approximately a 1/1 basis, with exogenously added nucleotide. These studies support the proposal that rickettsiae are not "leaky" to adenine nucleotides or to small molecules in general, and that they have a carrier-mediated transport system which allows an exchange of host and parasite ADP and ATP. | 1,389 |
pubmed23n0001_678 | Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization. | Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme. | Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization. Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme. | 1,390 |
pubmed23n0001_682 | Molecular size of nerve growth factor in dilute solution. | Nerve growth factor (NGF) is a protein composed of two identical chains of mass 13,259. An analysis of the sedimentation equilibrium, sedimentation velocity, and gel filtration behavior of dilute solutions of NGF indicates the existence of a rapidly reversible monomer in equilibrium dimer equilibrium and that the association constant K for the reaction at neutral pH is 9.4 X 10(6)M-1. Reaction mixtures consist of equal concentrations of monomer and dimer at a total protein concentration as high as 1.4 mug/ml, and at 1 ng/ml, monomer accounts for greater than 99% of the total. The latter concentration is 20 to 30 times that required for the biological activity of NGF. Several lines of evidence suggest that the dimerization reaction is highly stereospecific, although its biological significance is not known. | Molecular size of nerve growth factor in dilute solution. Nerve growth factor (NGF) is a protein composed of two identical chains of mass 13,259. An analysis of the sedimentation equilibrium, sedimentation velocity, and gel filtration behavior of dilute solutions of NGF indicates the existence of a rapidly reversible monomer in equilibrium dimer equilibrium and that the association constant K for the reaction at neutral pH is 9.4 X 10(6)M-1. Reaction mixtures consist of equal concentrations of monomer and dimer at a total protein concentration as high as 1.4 mug/ml, and at 1 ng/ml, monomer accounts for greater than 99% of the total. The latter concentration is 20 to 30 times that required for the biological activity of NGF. Several lines of evidence suggest that the dimerization reaction is highly stereospecific, although its biological significance is not known. | 1,394 |
pubmed23n0001_683 | Carbon 13 resonances of 13CO2 carbamino adducts of alpha and beta chains in human adult hemoglobin. | The principal component of normal adult human hemoglobin Ao, was equilibrated under various conditions with 13CO2. In addition, derivatives containing specifically carbamylated NH2-terinal groups in alpha or beta chains, or both, were prepared by treatment with cyanate, and equilibrated likewise to allow the identification of specific resonances observed by 13C nuclear magnetic resonance. In deoxyhemoglobin, a resonanance at 29.2 ppm upfield of external CS2 was assigned to the alpha chain terminal adduct, and one at 29.8 ppm to the beta chain terminal adduct. In the liganded state as the CO derivative, the terminal adduct on both chains showed a common resonance position at 29.8 ppm. Small effects of pH on the resonance positions were observed. Under certain conditions, a resonance was observed at 33.4 ppm, probably not ascribable to a carbamino compound. A carbamino resonance that became prominent at higher pH was found at 28.4 ppm, and is tentatively ascribed to one or more adducts on epsilon amino groups. The beta chain resonances in particular are minimized by the presence of inositol hexaphosphate or 2,3-diphosphoglycerate. Quantitative analysis of the resonance intensities shows that the effects of conversion from the deoxy to the liganded state in reducing the degree of carbamino adduct is much more pronounced for the beta than for the alpha chains. | Carbon 13 resonances of 13CO2 carbamino adducts of alpha and beta chains in human adult hemoglobin. The principal component of normal adult human hemoglobin Ao, was equilibrated under various conditions with 13CO2. In addition, derivatives containing specifically carbamylated NH2-terinal groups in alpha or beta chains, or both, were prepared by treatment with cyanate, and equilibrated likewise to allow the identification of specific resonances observed by 13C nuclear magnetic resonance. In deoxyhemoglobin, a resonanance at 29.2 ppm upfield of external CS2 was assigned to the alpha chain terminal adduct, and one at 29.8 ppm to the beta chain terminal adduct. In the liganded state as the CO derivative, the terminal adduct on both chains showed a common resonance position at 29.8 ppm. Small effects of pH on the resonance positions were observed. Under certain conditions, a resonance was observed at 33.4 ppm, probably not ascribable to a carbamino compound. A carbamino resonance that became prominent at higher pH was found at 28.4 ppm, and is tentatively ascribed to one or more adducts on epsilon amino groups. The beta chain resonances in particular are minimized by the presence of inositol hexaphosphate or 2,3-diphosphoglycerate. Quantitative analysis of the resonance intensities shows that the effects of conversion from the deoxy to the liganded state in reducing the degree of carbamino adduct is much more pronounced for the beta than for the alpha chains. | 1,395 |
pubmed23n0001_686 | Serum stimulation of phosphate uptake into 3T3 cells. | The stimulation by calf serum of phosphate uptake into 3T3 cells results from a change in maximum velocity of the transport process with no change in the Michaelis constant. Only arsenate among a series of inorganic structural analogs of phosphate inhibited phosphate uptake indicating a high specificity for the process. The arsenate inhibition was competitive in nature. Papaverine, theophylline, and protaglandin E1, drugs known to maintain high intracellular levels of cAMP, had little effect on serum stimulated phosphate uptake. The phosphate uptake stimulating factor(s) in serum could be distinguised from the 3T3 cell survival and migration factors by stability characteristics, but this factor(s) could not be completely separated from a uridine uptake stimulation activity or growth promoting activity using a variety of serum fractionation procedures. Only partial stimulation of the uptake process was achieved with any one serum fraction indicating a multiplicity of serum components is probably involved in this process. Because of the rapidity of serum activation of phosphate uptake and its apparent independence of intracellular cyclic nucleotide levels, it is suggested that serum factors may stimulate phosphate uptake by inducing structural changes in the phosphate carrier system. | Serum stimulation of phosphate uptake into 3T3 cells. The stimulation by calf serum of phosphate uptake into 3T3 cells results from a change in maximum velocity of the transport process with no change in the Michaelis constant. Only arsenate among a series of inorganic structural analogs of phosphate inhibited phosphate uptake indicating a high specificity for the process. The arsenate inhibition was competitive in nature. Papaverine, theophylline, and protaglandin E1, drugs known to maintain high intracellular levels of cAMP, had little effect on serum stimulated phosphate uptake. The phosphate uptake stimulating factor(s) in serum could be distinguised from the 3T3 cell survival and migration factors by stability characteristics, but this factor(s) could not be completely separated from a uridine uptake stimulation activity or growth promoting activity using a variety of serum fractionation procedures. Only partial stimulation of the uptake process was achieved with any one serum fraction indicating a multiplicity of serum components is probably involved in this process. Because of the rapidity of serum activation of phosphate uptake and its apparent independence of intracellular cyclic nucleotide levels, it is suggested that serum factors may stimulate phosphate uptake by inducing structural changes in the phosphate carrier system. | 1,399 |
pubmed23n0001_690 | High-speed ion-pair partition chromatography in pharmaceutical analysis. | Ion-pair chromatography offers attractive possibilities in pharmaceutical analysis. The specificity of the separation systems can be varied over a wide range by appropriate selection of the stationary phase. The choice of a suitable counter-ion can also drastically improve the detection limit, permitting the determination of drug substances in low dosage and possibly of by-products or breakdown products. Ion-pair chromatography of tropane and ergot alkaloids has been investigated using picrate as counter-ion. Alumina, Kieselguhr and various grades of silica gel have been tested as supports. Partition properties studied in a batch procedure have been compared with the actual chromatographic conditions. Columns (10 cm) filled with silical gel (particle size, 5 mum; pore size, 1000 A) show the best performance in the separation of hyoscyamine, scopolamine and ergotamine as picrate ion-pairs. Close control of pH and temperature is essential for reproducible separations. Improvements in detection limits between 100 and 300 times have been observed with these systems. Ion-pair extractions of these alkaloids from dosage forms can be used for sample preparation prior to injection on the the column. This provides an added degree of selectivity and sensitivity. | High-speed ion-pair partition chromatography in pharmaceutical analysis. Ion-pair chromatography offers attractive possibilities in pharmaceutical analysis. The specificity of the separation systems can be varied over a wide range by appropriate selection of the stationary phase. The choice of a suitable counter-ion can also drastically improve the detection limit, permitting the determination of drug substances in low dosage and possibly of by-products or breakdown products. Ion-pair chromatography of tropane and ergot alkaloids has been investigated using picrate as counter-ion. Alumina, Kieselguhr and various grades of silica gel have been tested as supports. Partition properties studied in a batch procedure have been compared with the actual chromatographic conditions. Columns (10 cm) filled with silical gel (particle size, 5 mum; pore size, 1000 A) show the best performance in the separation of hyoscyamine, scopolamine and ergotamine as picrate ion-pairs. Close control of pH and temperature is essential for reproducible separations. Improvements in detection limits between 100 and 300 times have been observed with these systems. Ion-pair extractions of these alkaloids from dosage forms can be used for sample preparation prior to injection on the the column. This provides an added degree of selectivity and sensitivity. | 1,405 |
pubmed23n0001_698 | Specific ion-exchange chromatography and fluorimetric assay for urinary 3-O-methyldopamine. | A technique for the selective extraction of 3-O-methyldopamine, normetanephrine and metanephrine from a single urine sample has been investigated. After hydrolysis of the conjugates, the diluted mixture is passed through a Dowex 50W-X2 column and the methoxylated amines are eluted by means of concentrated ammonia. The eluate, containing metanephrine, normetanephrine and 3-O-methyldopamine is evaporated, and a solution of the residue in borate buffer is fractionated under strictly controlled conditions on an Amberlite CG-50 column. The three amines so separated are estimated by specific fluorimetric methods. The extraction recovery is 80 +/- 3% for pure solutions and 78 +/- 4% for 3-O-methyldopamine added to urine. The fluorimetric procedure, carried out under well-defined conditions, allows the estimation of 10 ng of 3-O-methethyldopamine. The spectral characteristics of the fluorescent derivative are similar to those obtained with dopamine, so that it can be assumed that iodine oxidation of 3-O-methyldopamine demethylates this compound and oxidises the resulting dopamine to the dopamine fluorophore (5,6-dihydroxy-indole). Of the compounds that might interfere in the fluorimetric procedure, dopamine, DOPA and alpha-methyl-DOPA are destroyed by the ammoniacal elution from the Dowex column and 3-O-methyl-DOPA is eliminated in the effluent from the Amberlite column. The elimination of interfering compounds and the improved separation on Amberlite ensure high specificity for this procedure. We have applied the method to normal urine and to pathological urines from patients with adrenergic tumours or untreated and treated parkinsonian subjects; vital information has been obtained on the prognosis of adrenergic tumours. The presence of large amounts of dopamine, normetanephrine and/or metanephrine does not affect the assay for 3-O-methyldopamine. The method is also applicable to rat and dog urine, and can be applied to tissue extracts with little modification. | Specific ion-exchange chromatography and fluorimetric assay for urinary 3-O-methyldopamine. A technique for the selective extraction of 3-O-methyldopamine, normetanephrine and metanephrine from a single urine sample has been investigated. After hydrolysis of the conjugates, the diluted mixture is passed through a Dowex 50W-X2 column and the methoxylated amines are eluted by means of concentrated ammonia. The eluate, containing metanephrine, normetanephrine and 3-O-methyldopamine is evaporated, and a solution of the residue in borate buffer is fractionated under strictly controlled conditions on an Amberlite CG-50 column. The three amines so separated are estimated by specific fluorimetric methods. The extraction recovery is 80 +/- 3% for pure solutions and 78 +/- 4% for 3-O-methyldopamine added to urine. The fluorimetric procedure, carried out under well-defined conditions, allows the estimation of 10 ng of 3-O-methethyldopamine. The spectral characteristics of the fluorescent derivative are similar to those obtained with dopamine, so that it can be assumed that iodine oxidation of 3-O-methyldopamine demethylates this compound and oxidises the resulting dopamine to the dopamine fluorophore (5,6-dihydroxy-indole). Of the compounds that might interfere in the fluorimetric procedure, dopamine, DOPA and alpha-methyl-DOPA are destroyed by the ammoniacal elution from the Dowex column and 3-O-methyl-DOPA is eliminated in the effluent from the Amberlite column. The elimination of interfering compounds and the improved separation on Amberlite ensure high specificity for this procedure. We have applied the method to normal urine and to pathological urines from patients with adrenergic tumours or untreated and treated parkinsonian subjects; vital information has been obtained on the prognosis of adrenergic tumours. The presence of large amounts of dopamine, normetanephrine and/or metanephrine does not affect the assay for 3-O-methyldopamine. The method is also applicable to rat and dog urine, and can be applied to tissue extracts with little modification. | 1,415 |
pubmed23n0001_699 | Analysis of the radioimmunoassay for gonadotropin-releasing hormone (GnRH): studies on the effect of radioiodinated GnRH. | When GnRH is radioiodinated by the chloramine-T method, two immunoreactive labeled species are formed at pH 6.5 with a chloramine-T: GnRH molar ratio of 11:1, whereas four bands (I, IIa, IIb, and III) are separated by polyacrylamide gel electrophoresis when the hormone is iodinated at pH 7.5 in a system containing a 97:1 molar ratio of chloramine-T:GnRH. Because they were more stable and were more immunoreactive than the other products, band I and band IIa from the latter system were used separately as tracers with Niswender antiserum R-42 in radioimmunoassays for GnRH. The standard curves of each tracer are distinct: when analyzed after log-logit transformation, the band I curve had a mean slope of -3.31 +/- 0.2 (SE) and a 50% B/Bt level of 9 +/- 0.8 pg (n=8) of synthetic GnRH, whereas the band IIa standard curve had a slope of -2.30 +/- 0.6 and a 50% B/Bt value of 20 +/- 0.9 pg (n=11). The sensitivity of both assays is approximately 2.0 pg. Gn RH concentrations in plasma and serum samples assayed with band I were consistently greater than those assayed with band IIa. Normal adult male plasmas assayed with band I measured 21 +/- 0.9 pg/ml, whereas band IIa values were 8 +/- 0.4 pg/ml. No difference between plasma and serum was detected, nor was there any difference among adult men, adult women, prepubertal children, hypogonadal patients, or hypopituitary patients with either assay. Plasma GnRH concentrations were also similar in jugular and vena cava samples from intact and castrated male rats. Because many of the samples were at or below the sensitivity of the band IIa assay, they were concentrated after extraction with either methanol or acid-ethanol. However, endogenous immunoreactive GnRH could not be concentrated by these extraction procedures. As measured in the band IIa assay, hypothalamic extracts from control adult male rats contained 3.1 +/- 0.4 ng while hypothalami from castrated rats contained 1.4 +/- 0.1 ng. Similar but slightly lower values were obtained with band I. In contrast, the GnRH content of pineal glands from intact and castrated male rats was similar (approximately 150 pg) when determined in either assay. These studies emphasize that: 1) the characteristics of the radioiodinated hormone can influence the quantitation of GnRH; and 2) endogenous plasma concentrations of GnRH are much lower than previously reported. | Analysis of the radioimmunoassay for gonadotropin-releasing hormone (GnRH): studies on the effect of radioiodinated GnRH. When GnRH is radioiodinated by the chloramine-T method, two immunoreactive labeled species are formed at pH 6.5 with a chloramine-T: GnRH molar ratio of 11:1, whereas four bands (I, IIa, IIb, and III) are separated by polyacrylamide gel electrophoresis when the hormone is iodinated at pH 7.5 in a system containing a 97:1 molar ratio of chloramine-T:GnRH. Because they were more stable and were more immunoreactive than the other products, band I and band IIa from the latter system were used separately as tracers with Niswender antiserum R-42 in radioimmunoassays for GnRH. The standard curves of each tracer are distinct: when analyzed after log-logit transformation, the band I curve had a mean slope of -3.31 +/- 0.2 (SE) and a 50% B/Bt level of 9 +/- 0.8 pg (n=8) of synthetic GnRH, whereas the band IIa standard curve had a slope of -2.30 +/- 0.6 and a 50% B/Bt value of 20 +/- 0.9 pg (n=11). The sensitivity of both assays is approximately 2.0 pg. Gn RH concentrations in plasma and serum samples assayed with band I were consistently greater than those assayed with band IIa. Normal adult male plasmas assayed with band I measured 21 +/- 0.9 pg/ml, whereas band IIa values were 8 +/- 0.4 pg/ml. No difference between plasma and serum was detected, nor was there any difference among adult men, adult women, prepubertal children, hypogonadal patients, or hypopituitary patients with either assay. Plasma GnRH concentrations were also similar in jugular and vena cava samples from intact and castrated male rats. Because many of the samples were at or below the sensitivity of the band IIa assay, they were concentrated after extraction with either methanol or acid-ethanol. However, endogenous immunoreactive GnRH could not be concentrated by these extraction procedures. As measured in the band IIa assay, hypothalamic extracts from control adult male rats contained 3.1 +/- 0.4 ng while hypothalami from castrated rats contained 1.4 +/- 0.1 ng. Similar but slightly lower values were obtained with band I. In contrast, the GnRH content of pineal glands from intact and castrated male rats was similar (approximately 150 pg) when determined in either assay. These studies emphasize that: 1) the characteristics of the radioiodinated hormone can influence the quantitation of GnRH; and 2) endogenous plasma concentrations of GnRH are much lower than previously reported. | 1,416 |
pubmed23n0001_700 | Counterimmunoelectrophoresis of pneumococcal antigens:improved sensitivity for the detection of types VII and XIV. | Rapid identification of Streptococcus pneumoniae has been reported using counterimmunoelectrophoresis for the detection of specific capsular antigens in serum, cerebrospinal fluid, and urine. Previous clinical studies have failed to detect type VII or XIV pneumococcal antigen. These two types, however, account for a significant portion of pneumococcal disease. The incorporation of a sulfonated derivative of phenylboronic acid in the buffer system provides a method for the sensitive detection of these types in artificial mixtures without greatly reducing the sensitivity for the detection of other pneumococcal types. A problem with false positives encountered using human serum and barbitalbuffer was reduced by the use of buffer containing sulfonated phenylboronic acid. | Counterimmunoelectrophoresis of pneumococcal antigens:improved sensitivity for the detection of types VII and XIV. Rapid identification of Streptococcus pneumoniae has been reported using counterimmunoelectrophoresis for the detection of specific capsular antigens in serum, cerebrospinal fluid, and urine. Previous clinical studies have failed to detect type VII or XIV pneumococcal antigen. These two types, however, account for a significant portion of pneumococcal disease. The incorporation of a sulfonated derivative of phenylboronic acid in the buffer system provides a method for the sensitive detection of these types in artificial mixtures without greatly reducing the sensitivity for the detection of other pneumococcal types. A problem with false positives encountered using human serum and barbitalbuffer was reduced by the use of buffer containing sulfonated phenylboronic acid. | 1,417 |
pubmed23n0001_705 | Demonstration of a nonadrenergic inhibitory nervous system in the trachea of the guinea pig. | A nonadrenergic inhibitory nervous system has been demonstrated in the guinea pig trachea. Electrical field stimulation of this system, in the presence of adrenergic and cholinergic blockade, resulted in relaxation of tracheal rings contracted by the mediators of immediate hypersensitivity or histamine. The relaxation was blocked by tetrodotoxin, which indicated that nerve stimulation was responsible for the relaxation. The gastrointestinal tract, which has a similar embryological origin to the respiratory tract, also has a nonadrenergic inhibitory system. In the gastrointestinal tract, this system is thought to be responsible for the relaxation phase of peristalsis, and absence of this system, in the colon and the rectum, is thought to be an explanation for the spastic bowel in Hirschsprung's disease. It is possible that an abnormality of the respiratory nonadrenergic inhibitory system may play a role in the pathogenesis of the hyperreactive airways in asthma. The airways, due to a lack of inhibition, may be either partially contracted or unable to relax, and thus appear hyperreactive to stimuli. | Demonstration of a nonadrenergic inhibitory nervous system in the trachea of the guinea pig. A nonadrenergic inhibitory nervous system has been demonstrated in the guinea pig trachea. Electrical field stimulation of this system, in the presence of adrenergic and cholinergic blockade, resulted in relaxation of tracheal rings contracted by the mediators of immediate hypersensitivity or histamine. The relaxation was blocked by tetrodotoxin, which indicated that nerve stimulation was responsible for the relaxation. The gastrointestinal tract, which has a similar embryological origin to the respiratory tract, also has a nonadrenergic inhibitory system. In the gastrointestinal tract, this system is thought to be responsible for the relaxation phase of peristalsis, and absence of this system, in the colon and the rectum, is thought to be an explanation for the spastic bowel in Hirschsprung's disease. It is possible that an abnormality of the respiratory nonadrenergic inhibitory system may play a role in the pathogenesis of the hyperreactive airways in asthma. The airways, due to a lack of inhibition, may be either partially contracted or unable to relax, and thus appear hyperreactive to stimuli. | 1,437 |
pubmed23n0001_706 | Non-specific alkaline phosphomonoesterases of eight species of digenetic trematodes. | Alkaline phosphatases from different trematodes occupying the same habitat have identical pH otima but different levels of enzyme activities. Isoparorchis hypselobagri, from the fish Wallago attu, shows four to six times more enzyme activity than Fasciolopsis buski, Gastrodiscoides hominis and Echinostoma malayanum, from the pig Sus scrofa, and Fasciola gigantica, Gigantocotyle explanatum, Cotylophoron cotylophorum and Gastrothylax crumenifer, from the buffalo Bubalus bubalis. At least two peaks of activity at different levels of pH were obtained for each trematode examined. Both Gastrodiscoides hominis and Isoparorchis hypselobagri enzymes had three peaks of alkaline phosphatase activity. The optimum temperature for maximum enzyme activity was 40 degrees C, above which rapid inactivation occurred. At temperatures below 40 degrees C, the enzymes of fish and mammalian trematodes did not behave similarly; I. hypselobagri enzyme being active over a wider range of temperature (20 degrees-40 degrees C. Various concentrations of KCN and arsenate proportionately inhibited enzyme activity. NaF Did not significantly influence enzyme activity, while Mg++ and Co++ acted as activators. The extent of inhibition or activation of enzyme activity of different trematodes varied, probably due to species differences. Both inhibition and activation of I. hypselobagri enzyme was higher than in the case of other trematodes. | Non-specific alkaline phosphomonoesterases of eight species of digenetic trematodes. Alkaline phosphatases from different trematodes occupying the same habitat have identical pH otima but different levels of enzyme activities. Isoparorchis hypselobagri, from the fish Wallago attu, shows four to six times more enzyme activity than Fasciolopsis buski, Gastrodiscoides hominis and Echinostoma malayanum, from the pig Sus scrofa, and Fasciola gigantica, Gigantocotyle explanatum, Cotylophoron cotylophorum and Gastrothylax crumenifer, from the buffalo Bubalus bubalis. At least two peaks of activity at different levels of pH were obtained for each trematode examined. Both Gastrodiscoides hominis and Isoparorchis hypselobagri enzymes had three peaks of alkaline phosphatase activity. The optimum temperature for maximum enzyme activity was 40 degrees C, above which rapid inactivation occurred. At temperatures below 40 degrees C, the enzymes of fish and mammalian trematodes did not behave similarly; I. hypselobagri enzyme being active over a wider range of temperature (20 degrees-40 degrees C. Various concentrations of KCN and arsenate proportionately inhibited enzyme activity. NaF Did not significantly influence enzyme activity, while Mg++ and Co++ acted as activators. The extent of inhibition or activation of enzyme activity of different trematodes varied, probably due to species differences. Both inhibition and activation of I. hypselobagri enzyme was higher than in the case of other trematodes. | 1,442 |
pubmed23n0001_708 | Kinetics of the antibody response to type III pneumococcal polysaccharide (SSS-III). I. Use of 125I-labeled SSS-III to study serum antibody levels, as well as the distribution and excretion of antigen after immunization. | A simple method was described for the preparation of 125I-labeled type III neumococcal polysaccharide (SSS-III) with a high specific radioactivity which retained the physical and immunologic properties of native SSS-III. SSS-III was used to study the serum and tissue levels of antigen, as well as its excretion, after i.p. injection. When an optimally immunogenic dose (0.5 mug) of antigen was given, greater than 90% of the injected antigen was excreted during the first 3 days after injection; however, after day 3, the SSS-III which remained in each mouse was firmly bound to various tissues, and less than 5 ng SSS-III was released into the circulation daily. SSS-III was also used in a Farr test to measure serum antibody levels; the kinetics for the appearance of PFC/spleen and serum antibody levels were measured at 24-hr intervals after immunization with 0.5 mug of antigen. Maximum PFC/spleen were observed on day 4 after immunization whereas the peak serum antibody level was seen on day 5. The decay of serum antibody level from its maximum value was much slower than that of the PFC/spleen. The data describing the distribution of SSS-III in vivo and the measurement of serum antibody levels indicated that treadmill neutralization was not a factor in determining the serum antibody levels after immunization with an optimally immunogenic dose of SSS-III. | Kinetics of the antibody response to type III pneumococcal polysaccharide (SSS-III). I. Use of 125I-labeled SSS-III to study serum antibody levels, as well as the distribution and excretion of antigen after immunization. A simple method was described for the preparation of 125I-labeled type III neumococcal polysaccharide (SSS-III) with a high specific radioactivity which retained the physical and immunologic properties of native SSS-III. SSS-III was used to study the serum and tissue levels of antigen, as well as its excretion, after i.p. injection. When an optimally immunogenic dose (0.5 mug) of antigen was given, greater than 90% of the injected antigen was excreted during the first 3 days after injection; however, after day 3, the SSS-III which remained in each mouse was firmly bound to various tissues, and less than 5 ng SSS-III was released into the circulation daily. SSS-III was also used in a Farr test to measure serum antibody levels; the kinetics for the appearance of PFC/spleen and serum antibody levels were measured at 24-hr intervals after immunization with 0.5 mug of antigen. Maximum PFC/spleen were observed on day 4 after immunization whereas the peak serum antibody level was seen on day 5. The decay of serum antibody level from its maximum value was much slower than that of the PFC/spleen. The data describing the distribution of SSS-III in vivo and the measurement of serum antibody levels indicated that treadmill neutralization was not a factor in determining the serum antibody levels after immunization with an optimally immunogenic dose of SSS-III. | 1,445 |
pubmed23n0001_714 | The polysaccharide capsule of Bacteroides fragilis subspecies fragilis: immunochemical and morphologic definition. | A large-molecular-weight capsular polysaccharide was isolated from strains of Bacteroides fragilis subspecies fragilis. By means of electron microscopy and staining with ruthenium red, the thick polysaccharide capsule was also visualized. With use of a radioactive antigen-binding assay, antibody to this capsular polysaccharide was demonstrated in antisera prepared in rabbits to each of eight strains of B. fragilis fragilis. Antibody of similar specificity was not found in antisera prepared to Bacteroides melaninogenicus or to strains of Bacteroides fragilis subspecies vulgatus and Bacteroides fragilis subspecies distasonis; such antibody was found in antisera to only one of two strains of Bacteroides fragilis subspecies thetaiotaomicron. The radioactive antigen-binding assay is a sensitive test for the detection of antibody to capsular polysaccharide. This polysaccharide antigen may form the basis of a serogrouping system for B. fragilis. | The polysaccharide capsule of Bacteroides fragilis subspecies fragilis: immunochemical and morphologic definition. A large-molecular-weight capsular polysaccharide was isolated from strains of Bacteroides fragilis subspecies fragilis. By means of electron microscopy and staining with ruthenium red, the thick polysaccharide capsule was also visualized. With use of a radioactive antigen-binding assay, antibody to this capsular polysaccharide was demonstrated in antisera prepared in rabbits to each of eight strains of B. fragilis fragilis. Antibody of similar specificity was not found in antisera prepared to Bacteroides melaninogenicus or to strains of Bacteroides fragilis subspecies vulgatus and Bacteroides fragilis subspecies distasonis; such antibody was found in antisera to only one of two strains of Bacteroides fragilis subspecies thetaiotaomicron. The radioactive antigen-binding assay is a sensitive test for the detection of antibody to capsular polysaccharide. This polysaccharide antigen may form the basis of a serogrouping system for B. fragilis. | 1,451 |
pubmed23n0001_718 | Peptide-binding macromolecules in the blood of seriously ill or mentally retarded patients. | This report describes macromolecules that bind (des-aspartic acid1)-angiotensin II, the des aspartic acid1 derivative of angiotensin I, and several biologically active and inactive analogues of these polypeptides. The macromolecules were found in the plasma of approximately 2 per cent of ambulatory adults and hospitalized children and 32 per cent of the patients at two institutions for the mentally retarded. The binding properties of these macromolecules were studied by incubating with peptides labeled with 125iodine, and separating bound from free labeled peptide using small gel filtration columns. The peptide-binding macromolecules from several patients were compared. They showed very similar specificity for a group of arginyl peptides of the des-aspartyl1-angiotensin sequence. The plasma binders differed from one another in their optimum pH and their mobility in electrophoretic fields. Those with more acid pH optima displayed more rapid electrophoretic mobility. The binders fell into two classes based on apparent molecular weight, approximately 140,000 and 250,000. Those with the higher apparent molecular weight contained a large proportion of binder that could be precipitated with antiserum to human IgA. Kinetic measurements showed that the plasma binders were somewhat heterogeneous with respect to affinity for (des-asp1)-angiotensin, with apparent association constants ranging from 10(7) to 10(8) M-1. Binding activity was labile to heat, and to treatment with pepsin or trypsin. It was inhibited by calcium, protamine, streptomycin, and some other cationic compounds. The plasma peptide binder differed in specificity and molecular weight from soluble angiotensin-binding molecules extracted from tissues, and from properties expected of a receptor for angiotensin. These macromolecules may be useful reagents for measuring (des-asp1)-angiotensins. Their presence in plasma samples may interfere with angiotensin assays in some circumstances. | Peptide-binding macromolecules in the blood of seriously ill or mentally retarded patients. This report describes macromolecules that bind (des-aspartic acid1)-angiotensin II, the des aspartic acid1 derivative of angiotensin I, and several biologically active and inactive analogues of these polypeptides. The macromolecules were found in the plasma of approximately 2 per cent of ambulatory adults and hospitalized children and 32 per cent of the patients at two institutions for the mentally retarded. The binding properties of these macromolecules were studied by incubating with peptides labeled with 125iodine, and separating bound from free labeled peptide using small gel filtration columns. The peptide-binding macromolecules from several patients were compared. They showed very similar specificity for a group of arginyl peptides of the des-aspartyl1-angiotensin sequence. The plasma binders differed from one another in their optimum pH and their mobility in electrophoretic fields. Those with more acid pH optima displayed more rapid electrophoretic mobility. The binders fell into two classes based on apparent molecular weight, approximately 140,000 and 250,000. Those with the higher apparent molecular weight contained a large proportion of binder that could be precipitated with antiserum to human IgA. Kinetic measurements showed that the plasma binders were somewhat heterogeneous with respect to affinity for (des-asp1)-angiotensin, with apparent association constants ranging from 10(7) to 10(8) M-1. Binding activity was labile to heat, and to treatment with pepsin or trypsin. It was inhibited by calcium, protamine, streptomycin, and some other cationic compounds. The plasma peptide binder differed in specificity and molecular weight from soluble angiotensin-binding molecules extracted from tissues, and from properties expected of a receptor for angiotensin. These macromolecules may be useful reagents for measuring (des-asp1)-angiotensins. Their presence in plasma samples may interfere with angiotensin assays in some circumstances. | 1,455 |
pubmed23n0001_725 | Properties of some bacteriocins produced by Rhizobium trifolii. | Bacteriocins produced by six strains of Rhizobium trifolii were found to be of the relatively low molecular weight, non-phage type. The molecular weights ranged from approximately 1-8 X 105 to 2-0 X 105. All were of protein composition, as indicated by buoyant density (1-32 to 1-34 g/cm3) in CsC1 and by sensitivity to proteolytic enzymes. They were resistant to RNAase but sensitive to DNAase. The six bacteriocins could further be separated into two subgroups on the basis of sensitivity to extremes of pH, binding to filter membranes, activity spectrum on sensitive strains of R. trifolii, and possibly mode of action on sensitive bacteria. Bacteriocin production occurred spontaneously during the early-to mid-exponential phase of bacterial growth in broth culture. | Properties of some bacteriocins produced by Rhizobium trifolii. Bacteriocins produced by six strains of Rhizobium trifolii were found to be of the relatively low molecular weight, non-phage type. The molecular weights ranged from approximately 1-8 X 105 to 2-0 X 105. All were of protein composition, as indicated by buoyant density (1-32 to 1-34 g/cm3) in CsC1 and by sensitivity to proteolytic enzymes. They were resistant to RNAase but sensitive to DNAase. The six bacteriocins could further be separated into two subgroups on the basis of sensitivity to extremes of pH, binding to filter membranes, activity spectrum on sensitive strains of R. trifolii, and possibly mode of action on sensitive bacteria. Bacteriocin production occurred spontaneously during the early-to mid-exponential phase of bacterial growth in broth culture. | 1,463 |
pubmed23n0001_726 | Production and purification of the gamma haemolysin of Staphylococcus aureus 'Smith 5R'. | The gamma haemolysin of Staphylococcus aureus 'Smith 5R' was produced on Dolman-Wilson agar overlain with cellophane. Maximal yields of crude lysin with titres of 2000 to 4000 haemolytic units/ml were obtained within 24 h at 37 degrees C in 10% (v/v) CO2 in air, on medium adjusted to pH 7-0. The crude lysin was purified 2700-fold (with 75% recovery) by ultrafiltration, gel filtration and ammonium sulphate fractionation. The specific activity of the lysin was 10(5) haemolytic units/mg protein after the dialysed active precipitate was extracted with NaCl and reprecipitated with ammonium sulphate. Purified gamma lysin was homogeneous by disc electrophoresis and immunoelectrophoresis. | Production and purification of the gamma haemolysin of Staphylococcus aureus 'Smith 5R'. The gamma haemolysin of Staphylococcus aureus 'Smith 5R' was produced on Dolman-Wilson agar overlain with cellophane. Maximal yields of crude lysin with titres of 2000 to 4000 haemolytic units/ml were obtained within 24 h at 37 degrees C in 10% (v/v) CO2 in air, on medium adjusted to pH 7-0. The crude lysin was purified 2700-fold (with 75% recovery) by ultrafiltration, gel filtration and ammonium sulphate fractionation. The specific activity of the lysin was 10(5) haemolytic units/mg protein after the dialysed active precipitate was extracted with NaCl and reprecipitated with ammonium sulphate. Purified gamma lysin was homogeneous by disc electrophoresis and immunoelectrophoresis. | 1,465 |
pubmed23n0001_727 | Basic and neutral amino acid transport in Aspergillus nidulans. | Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases. | Basic and neutral amino acid transport in Aspergillus nidulans. Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases. | 1,466 |
pubmed23n0001_729 | Disruption of Vi bacteriophage III and localization of its deacetylase activity. | It has been shown that particles of Vi bacteriophage III catalyse deacetylation of O-acetyl pectic (polygalacturonic) acid, a structural analogue of Vi polysaccharide (Vi antigen). Using this substrate, and determining the acetic acid liberated by gas-liquid chromatogrphy, a method for the estimation of Vi phage deacetylase activity has been developed. Purified particles of Vi phage III were exposed to a variety of mildly dissociative reagents and conditions, and then tested for plaque-forming and for deacetylase activity. They have also been inspected under the electron microscope. Osmotic shock, and incubation in the presence of ethylenediamine tetraacetic acid (greater than or equil 0-01 M), or of L-arginine (0-25 M), were found to cause disintegration of the virions into empty head capsids, deoxyribonucleic acid, and base plates still carrying the spikes. The mixtures of viral fragments exhibited an increased deacetylase activity. Using zonal sedimentation and ion exchange chromatography, the phage fragments obtained by treatment with ethylenediaminetetraacetic acid have been fractionated and the base plates isolated. Amongst the viral components, these structures showed the highest specific deacetylase activity. They had the shape of six-pointed stars (about 9-5 nm inner, and 14-5 nm outer diam.) with a central hole or plug (approximately 3 nm), carrying six spikes, roughly cylindrical organelles of approx. 11 X 4 nm, one at each of the points. Of the polypeptides of six sizes (P.1, about 153,000 daltons; P.2, 91,000; P.3, 71,000; P.4 56,500; P.6, 22,000), detected in whole Vi phage III virions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, only two, P.2 and P.3 were found in the base plates. | Disruption of Vi bacteriophage III and localization of its deacetylase activity. It has been shown that particles of Vi bacteriophage III catalyse deacetylation of O-acetyl pectic (polygalacturonic) acid, a structural analogue of Vi polysaccharide (Vi antigen). Using this substrate, and determining the acetic acid liberated by gas-liquid chromatogrphy, a method for the estimation of Vi phage deacetylase activity has been developed. Purified particles of Vi phage III were exposed to a variety of mildly dissociative reagents and conditions, and then tested for plaque-forming and for deacetylase activity. They have also been inspected under the electron microscope. Osmotic shock, and incubation in the presence of ethylenediamine tetraacetic acid (greater than or equil 0-01 M), or of L-arginine (0-25 M), were found to cause disintegration of the virions into empty head capsids, deoxyribonucleic acid, and base plates still carrying the spikes. The mixtures of viral fragments exhibited an increased deacetylase activity. Using zonal sedimentation and ion exchange chromatography, the phage fragments obtained by treatment with ethylenediaminetetraacetic acid have been fractionated and the base plates isolated. Amongst the viral components, these structures showed the highest specific deacetylase activity. They had the shape of six-pointed stars (about 9-5 nm inner, and 14-5 nm outer diam.) with a central hole or plug (approximately 3 nm), carrying six spikes, roughly cylindrical organelles of approx. 11 X 4 nm, one at each of the points. Of the polypeptides of six sizes (P.1, about 153,000 daltons; P.2, 91,000; P.3, 71,000; P.4 56,500; P.6, 22,000), detected in whole Vi phage III virions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, only two, P.2 and P.3 were found in the base plates. | 1,468 |
pubmed23n0001_733 | Studies of the effect of dietary cholesterol on hepatic protein synthesis, reduced glutathione levels and serine dehydratase activity in the rat. | A basal diet or a basal diet plus 1% of cholesterol and 0.33% cholic acid was fed to rats for varying lengths of time and (1) the activities of liver phosphoenolpyruvate-carboxykinase (PEP-CK), tyrosine transaminase (TT), and serine dehydratase (SD); (2) the rate of total hepatic protein synthesis and (3) the concentration of hepatic reduced glutathione (GSH) were quantitated. The specific activity of PEP-CK was significantly depressed by cholesterol plus cholic acid feeding, while the specific activity of TT was unchanged. No significant effect of dietary cholesterol plus cholic acid was found on the total liver activities. In contrast, SD specific activity was increased 3-fold. The rate of (U-14C)-L-leucine incorporation into total TCA precipitable protein following ingestion of cholesterol plus acid was significantly reduced when the data were expressed as dpm (U-14C)-L-leucine/mg protein. After correcting this expression for specific radioactivity of the liver tissue free leucine pool, no significant effect of dietary cholesterol plus cholic acid on hepatic protein synthesis existed. In fact, the amount of 14C-leucine incorporated into protein on a total liver basis was 50% greater for the cholesterol group. On a per gram of liver basis, the concentration of GSH in the liver of rats fed a cholesterol plus cholic acid diet was significantly decreased. Considering the liver enlargement in rats fed cholesterol plus cholic acid, total organ GSH was found to be significantly greater than for rats fed a basal diet. | Studies of the effect of dietary cholesterol on hepatic protein synthesis, reduced glutathione levels and serine dehydratase activity in the rat. A basal diet or a basal diet plus 1% of cholesterol and 0.33% cholic acid was fed to rats for varying lengths of time and (1) the activities of liver phosphoenolpyruvate-carboxykinase (PEP-CK), tyrosine transaminase (TT), and serine dehydratase (SD); (2) the rate of total hepatic protein synthesis and (3) the concentration of hepatic reduced glutathione (GSH) were quantitated. The specific activity of PEP-CK was significantly depressed by cholesterol plus cholic acid feeding, while the specific activity of TT was unchanged. No significant effect of dietary cholesterol plus cholic acid was found on the total liver activities. In contrast, SD specific activity was increased 3-fold. The rate of (U-14C)-L-leucine incorporation into total TCA precipitable protein following ingestion of cholesterol plus acid was significantly reduced when the data were expressed as dpm (U-14C)-L-leucine/mg protein. After correcting this expression for specific radioactivity of the liver tissue free leucine pool, no significant effect of dietary cholesterol plus cholic acid on hepatic protein synthesis existed. In fact, the amount of 14C-leucine incorporated into protein on a total liver basis was 50% greater for the cholesterol group. On a per gram of liver basis, the concentration of GSH in the liver of rats fed a cholesterol plus cholic acid diet was significantly decreased. Considering the liver enlargement in rats fed cholesterol plus cholic acid, total organ GSH was found to be significantly greater than for rats fed a basal diet. | 1,480 |
pubmed23n0001_744 | Determination of low cencentrations of the quaternary ammonium compound thiazinamium methylsulphate in plasma and urine. | A sensitive and selective method for the quantitative determination of the quaternary ammonium antiacetylcholine-compound thiazinamium methylsulphate (Multergan) in plasma and urine is described. The procedure is based on ion pair extraction of the compound with iodide as the counter ion. This is followed by gas chromatography using an alkali flame ionization detector. The detection limit is 2 ng ml-1 with a recovery of 88-0 +/- 6-2% from plasma, 91-4 +/- 4-6% from urine. The described method can also be applied to other quaternary ammonium compounds. | Determination of low cencentrations of the quaternary ammonium compound thiazinamium methylsulphate in plasma and urine. A sensitive and selective method for the quantitative determination of the quaternary ammonium antiacetylcholine-compound thiazinamium methylsulphate (Multergan) in plasma and urine is described. The procedure is based on ion pair extraction of the compound with iodide as the counter ion. This is followed by gas chromatography using an alkali flame ionization detector. The detection limit is 2 ng ml-1 with a recovery of 88-0 +/- 6-2% from plasma, 91-4 +/- 4-6% from urine. The described method can also be applied to other quaternary ammonium compounds. | 1,494 |
pubmed23n0001_747 | New and expedient determination of atenolol in biological samples. | A method is described for the GLC determination of atenolol BP in plasma and urine. Extraction is accomplished under dehydrating conditions, and interfering impurities are removed by using an acidified cyclohexane-isopropanol mixture (2:1) and charcoal-treated paper disks. The drug thus isolated appears to react more efficiently with heptafluorobutyric anhydride, increasing the sensitivity of GLC electron-capture analysis. Concentrations as low as 0.02 mug/ml were measured using 0.5-ml aliquots of plasma or 0.1 ml of urine. Amino alcohols such as atenolol may form hydrates or alcoholates, precluding complete derivatization with heptafluorobutyric anhydride. | New and expedient determination of atenolol in biological samples. A method is described for the GLC determination of atenolol BP in plasma and urine. Extraction is accomplished under dehydrating conditions, and interfering impurities are removed by using an acidified cyclohexane-isopropanol mixture (2:1) and charcoal-treated paper disks. The drug thus isolated appears to react more efficiently with heptafluorobutyric anhydride, increasing the sensitivity of GLC electron-capture analysis. Concentrations as low as 0.02 mug/ml were measured using 0.5-ml aliquots of plasma or 0.1 ml of urine. Amino alcohols such as atenolol may form hydrates or alcoholates, precluding complete derivatization with heptafluorobutyric anhydride. | 1,511 |
pubmed23n0001_749 | Quantitative analysis of tribromsalan in blood and urine. | Tribromsalan can be quantitatively measured in whole blood and urine by a technique involving extraction with ethyl acetate, treatment with silica gel, separation by TLC, and quantitative measurement by fluorescent spectrophotometry. This method has a sensitivity down to 125 ng (25 ppb in 5.0 ml of sample) of free tribromsalan and shows an average 90% recovery of tribromsalan in blood and urine with standard deviations of 9.7 and 7.4%, respectively. | Quantitative analysis of tribromsalan in blood and urine. Tribromsalan can be quantitatively measured in whole blood and urine by a technique involving extraction with ethyl acetate, treatment with silica gel, separation by TLC, and quantitative measurement by fluorescent spectrophotometry. This method has a sensitivity down to 125 ng (25 ppb in 5.0 ml of sample) of free tribromsalan and shows an average 90% recovery of tribromsalan in blood and urine with standard deviations of 9.7 and 7.4%, respectively. | 1,513 |
pubmed23n0001_757 | Micro-electrode measurement of the internal pH of crab muscle fibres. | The internal pH of crab muscle fibres was measured using recessed-tip pH-sensitive micro-electrodes. Immediately following electrode penetration the mean internal pH was 7-21 +/- 0-02 (S.E. of mean) and the mean membrane potential was -64-9 +/- 0-6 mV (S.E. of mean). If H+ ions were passively distributed across the fibre membrane the internal pH would have been 6-39. 2. The internal pH tended to rise before stabilizing at a mean value of 7-27 +/- 0-02 (S.E. of mean). The difference between immediate and stabilized values is highly significant and suggests acid injury on electrode penetration. 3. Changing the membrane potential or external pH had only small, slow effects on internal pH. 4. External CO2 caused a large and rapid decrease in internal pH. With low concentrations of CO2, the effect was dependent on the initial pH as predicted by the Law of Mass Action. During a long exposure to 2-65% CO2 at pH 7-5, the internal pH returned slowly to its previous value, suggesting active transport of H+ (or OH- or HCO3-) ions across the fibre membrane. 5. The internal buffering power calculated from the response to 2-65% CO2 was 47-3 +/- 2-8 slykes (m-equiv H+/pH unit per l.) (S.E. of mean). | Micro-electrode measurement of the internal pH of crab muscle fibres. The internal pH of crab muscle fibres was measured using recessed-tip pH-sensitive micro-electrodes. Immediately following electrode penetration the mean internal pH was 7-21 +/- 0-02 (S.E. of mean) and the mean membrane potential was -64-9 +/- 0-6 mV (S.E. of mean). If H+ ions were passively distributed across the fibre membrane the internal pH would have been 6-39. 2. The internal pH tended to rise before stabilizing at a mean value of 7-27 +/- 0-02 (S.E. of mean). The difference between immediate and stabilized values is highly significant and suggests acid injury on electrode penetration. 3. Changing the membrane potential or external pH had only small, slow effects on internal pH. 4. External CO2 caused a large and rapid decrease in internal pH. With low concentrations of CO2, the effect was dependent on the initial pH as predicted by the Law of Mass Action. During a long exposure to 2-65% CO2 at pH 7-5, the internal pH returned slowly to its previous value, suggesting active transport of H+ (or OH- or HCO3-) ions across the fibre membrane. 5. The internal buffering power calculated from the response to 2-65% CO2 was 47-3 +/- 2-8 slykes (m-equiv H+/pH unit per l.) (S.E. of mean). | 1,523 |
pubmed23n0001_758 | [Adrenergic and cholinergic control of oxytocin release evoked by vaginal, vagal and mammary stimulation in lactating rats (author's transl)]. | 1. The amounts of oxytocin released during Ferguson and vago-pituitary reflexes are estimated by measurements of intramammary pressure. For the milk-ejection reflex, the gain in weight of the young over a period of 30 minutes is taken as an indirect index of the release of oxytocin. 2. Antagonists of specific cholinoceptors and adrenoceptors were injected into the third ventricle in order to delineate the role of the mediators and receptors in the control of oxytocin release. 3. The results suggest that three reflexes have a specific chemical transmission since: a) The Ferguson reflex is inhibited by the drugs that only block alpha and beta adrenoceptors. b) The vago-pituitary reflex is inhibited by the drugs that block alpha and beta adrenoceptors and muscarinic cholinoceptors. c) The milk-ejection reflex is inhibited by the drugs that block alpha adrenoceptors and muscarinic and nicotinic cholinoceptors. | [Adrenergic and cholinergic control of oxytocin release evoked by vaginal, vagal and mammary stimulation in lactating rats (author's transl)]. 1. The amounts of oxytocin released during Ferguson and vago-pituitary reflexes are estimated by measurements of intramammary pressure. For the milk-ejection reflex, the gain in weight of the young over a period of 30 minutes is taken as an indirect index of the release of oxytocin. 2. Antagonists of specific cholinoceptors and adrenoceptors were injected into the third ventricle in order to delineate the role of the mediators and receptors in the control of oxytocin release. 3. The results suggest that three reflexes have a specific chemical transmission since: a) The Ferguson reflex is inhibited by the drugs that only block alpha and beta adrenoceptors. b) The vago-pituitary reflex is inhibited by the drugs that block alpha and beta adrenoceptors and muscarinic cholinoceptors. c) The milk-ejection reflex is inhibited by the drugs that block alpha adrenoceptors and muscarinic and nicotinic cholinoceptors. | 1,524 |
pubmed23n0001_781 | Enzymic deacetylation of carcinogenic arylacetamides by tissue microsomes of the dog and other species. | The relative ability of arylacetamide deacetylase enzyme systems of dog liver to carry out the deacetylation of the carcinogens, 4-acetylaminobiphenyl, 2-acetylaminofluorene, and 2-acetylaminaphthalene, was examined. The arylacetamides were incubated with unfortified dog liver microsomes, and enzyme activity (nmol arylamine/mg protein/hr) was estimated by colorimetric quantitation of the resulting arylamines. The dog liver enzyme system displayed characteristics similar to those described for the rodent liver enzyme system in that enzyme activity was greatest in liver tissue, was localized in the microsomal subcellular fraction, required no cofactors, and was inhibited by heat, sodium fluoride, and thiol reagents. In five replicate assays, the relative rates of deacetylation were about 10, 6, and 1 with 4-acetylaminobiphenyl (84.8 +/- 12.4), 2-acetylaminofluorene (52.5 +/- 5.1), and 2-acetylaminonaphthalene (8.8 +/- 3.3), respectively. As a canine urinary bladder carcinogen, 4-acetylaminobiphenyl is considered more potent than 2-acetylaminofluroene, while 2-acetylaminonaphthalene is devoid of detectable carcinogenic activity, despite the fact that 2-aminoaphthalene is a well-established canine urinary bladder carcinogen. Removal of the acetyl group may be a requirement for urinary bladder carcinogenesis; accordingly, the present studies demonstrate the appearance of a direct relationship between dog liver deacetylase enzyme specificity and urinary bladder susceptibility to these carcinogenic arylacetamides. | Enzymic deacetylation of carcinogenic arylacetamides by tissue microsomes of the dog and other species. The relative ability of arylacetamide deacetylase enzyme systems of dog liver to carry out the deacetylation of the carcinogens, 4-acetylaminobiphenyl, 2-acetylaminofluorene, and 2-acetylaminaphthalene, was examined. The arylacetamides were incubated with unfortified dog liver microsomes, and enzyme activity (nmol arylamine/mg protein/hr) was estimated by colorimetric quantitation of the resulting arylamines. The dog liver enzyme system displayed characteristics similar to those described for the rodent liver enzyme system in that enzyme activity was greatest in liver tissue, was localized in the microsomal subcellular fraction, required no cofactors, and was inhibited by heat, sodium fluoride, and thiol reagents. In five replicate assays, the relative rates of deacetylation were about 10, 6, and 1 with 4-acetylaminobiphenyl (84.8 +/- 12.4), 2-acetylaminofluorene (52.5 +/- 5.1), and 2-acetylaminonaphthalene (8.8 +/- 3.3), respectively. As a canine urinary bladder carcinogen, 4-acetylaminobiphenyl is considered more potent than 2-acetylaminofluroene, while 2-acetylaminonaphthalene is devoid of detectable carcinogenic activity, despite the fact that 2-aminoaphthalene is a well-established canine urinary bladder carcinogen. Removal of the acetyl group may be a requirement for urinary bladder carcinogenesis; accordingly, the present studies demonstrate the appearance of a direct relationship between dog liver deacetylase enzyme specificity and urinary bladder susceptibility to these carcinogenic arylacetamides. | 1,550 |
pubmed23n0001_786 | Sarcolemmal slow conductance increase of frog sartorius fibers during hyperpolarization. | The properties of the hyperpolarization-activated sarcolemmal slow conductance increase in frog sartorius muscle fibers have been investigated using ethylenediaminetetraacetic acid disodium salt (EDTA) and propionate Ringer solution. More than 1 sec was required for maximum activation of the sarcolemmal slwo conductance increase. It is suggested that, although the sarcolemmal slow conductance increase was affected by deterioration, the conductance increase is not a direct product of deterioration but it represents a property of the sarcolemma which is encountered in physiological range. The sarcolemmal conductance increase was rather insensitive to the change in pH of Ringer solution. It is inferred that the absence of bellying in newly penetrated intact fibers at neutral and alkaline pHs is caused mainly by the shunting effect of large parallel conductance. Apparent augmentation with EDTA of the sarcolemmal conductance increase infers that Ca ions affect the conductance increase. The conductance increase occurred also in the EDTA-containing Cl-deficient solution. The sarcolemmal slow conductance increase has been compared with the change in Cl conductance reported by Hutter and Warner, and Warner. | Sarcolemmal slow conductance increase of frog sartorius fibers during hyperpolarization. The properties of the hyperpolarization-activated sarcolemmal slow conductance increase in frog sartorius muscle fibers have been investigated using ethylenediaminetetraacetic acid disodium salt (EDTA) and propionate Ringer solution. More than 1 sec was required for maximum activation of the sarcolemmal slwo conductance increase. It is suggested that, although the sarcolemmal slow conductance increase was affected by deterioration, the conductance increase is not a direct product of deterioration but it represents a property of the sarcolemma which is encountered in physiological range. The sarcolemmal conductance increase was rather insensitive to the change in pH of Ringer solution. It is inferred that the absence of bellying in newly penetrated intact fibers at neutral and alkaline pHs is caused mainly by the shunting effect of large parallel conductance. Apparent augmentation with EDTA of the sarcolemmal conductance increase infers that Ca ions affect the conductance increase. The conductance increase occurred also in the EDTA-containing Cl-deficient solution. The sarcolemmal slow conductance increase has been compared with the change in Cl conductance reported by Hutter and Warner, and Warner. | 1,560 |
pubmed23n0001_825 | Fatty acid synthesis from 2-14C-acetate in rat testis mitochondrial and cytosol fractions in vitro. | An in vitro system for acetate incorporation into fatty acids by the mitochondrial and the cytosol fractions of rat testis is described. The rate of incorporation of acetate into fatty acids was twice as fast with the mitochondrial as with the cytosol fraction; both systems were stimulated in the presence of adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate, coenzyme A, and MgC1(2). The optimum pH was between 7.0-7.5 for the mitochondrial fraction and between 6.5-8.0 for the cytosol fraction. Radio gas chromatography showed that palmitic acid was the most highly labeled acid, followed by stearic acid, in the mitochondrial fraction in accord with the pathway of de novo fatty acid synthesis. Some of the labeled acetate was also incorporated into the 16:1 and 18:1 fatty acids of this fraction. Distribution of radioactivity among the mitochondrial lipid classes was highest in the phospholipids and monoglycerides, followed by diglycerides and cholesterol; little radioactivity was present in the triglyceride fraction. These observations are in accord with studies of the incorporation of labeled metabolites into testicular lipids following intratesticular injection and indicate the validity of the in vitro system for studies of specific reactions occurring in vivo. | Fatty acid synthesis from 2-14C-acetate in rat testis mitochondrial and cytosol fractions in vitro. An in vitro system for acetate incorporation into fatty acids by the mitochondrial and the cytosol fractions of rat testis is described. The rate of incorporation of acetate into fatty acids was twice as fast with the mitochondrial as with the cytosol fraction; both systems were stimulated in the presence of adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate, coenzyme A, and MgC1(2). The optimum pH was between 7.0-7.5 for the mitochondrial fraction and between 6.5-8.0 for the cytosol fraction. Radio gas chromatography showed that palmitic acid was the most highly labeled acid, followed by stearic acid, in the mitochondrial fraction in accord with the pathway of de novo fatty acid synthesis. Some of the labeled acetate was also incorporated into the 16:1 and 18:1 fatty acids of this fraction. Distribution of radioactivity among the mitochondrial lipid classes was highest in the phospholipids and monoglycerides, followed by diglycerides and cholesterol; little radioactivity was present in the triglyceride fraction. These observations are in accord with studies of the incorporation of labeled metabolites into testicular lipids following intratesticular injection and indicate the validity of the in vitro system for studies of specific reactions occurring in vivo. | 1,622 |
pubmed23n0001_827 | Stearoyl-CoA desaturase activity in mammary adenocarcinomas carried by C3H mice. | Transplantable mammary adenocarcinomas and livers of C3H mice fed a stock diet or a linoleate rich diet (15% corn oil) contain similar amounts of oleate (ca 3 mg/gm tissue). On feeding either a high carbohydrate, fat free or a high carbohydrate, saturated fat-containing (15% hydrogenated coconut or cottonseed oil) diet for 6 weeks, oleate levels increased 2-fold in tumor and 5-fold in liver. The specific activity of stearoyl-CoA desaturase in liver microsomes was similar to that in the corresponding fractions of mammary glands of lactating mice. In liver, this activity was enhanced 2- to 3-fold by feeding a high carbohydrate, fat free or a high carbohydrate, saturated fat diet. The desaturase activity in mammary tumor microsomes, while only 10% of that in hepatic microsomes, remained unaltered regardless of the type of diet fed. These observations suggest that (a) a major portion of the oleate in the mammary tumor is not produced within the tissue, (b) dietary adaptation is not a general characteristic of stearoyl-CoA desaturase in neoplastic tissues, and (c) enhanced desaturase activity in liver is directly related to the absence of linoleate or oleate, or to a large decrease in oleate in the diet. | Stearoyl-CoA desaturase activity in mammary adenocarcinomas carried by C3H mice. Transplantable mammary adenocarcinomas and livers of C3H mice fed a stock diet or a linoleate rich diet (15% corn oil) contain similar amounts of oleate (ca 3 mg/gm tissue). On feeding either a high carbohydrate, fat free or a high carbohydrate, saturated fat-containing (15% hydrogenated coconut or cottonseed oil) diet for 6 weeks, oleate levels increased 2-fold in tumor and 5-fold in liver. The specific activity of stearoyl-CoA desaturase in liver microsomes was similar to that in the corresponding fractions of mammary glands of lactating mice. In liver, this activity was enhanced 2- to 3-fold by feeding a high carbohydrate, fat free or a high carbohydrate, saturated fat diet. The desaturase activity in mammary tumor microsomes, while only 10% of that in hepatic microsomes, remained unaltered regardless of the type of diet fed. These observations suggest that (a) a major portion of the oleate in the mammary tumor is not produced within the tissue, (b) dietary adaptation is not a general characteristic of stearoyl-CoA desaturase in neoplastic tissues, and (c) enhanced desaturase activity in liver is directly related to the absence of linoleate or oleate, or to a large decrease in oleate in the diet. | 1,624 |
pubmed23n0001_828 | Iowa wrestling study: changes in the urinary profiles of wrestlers prior to and after competition. | During the 1973 and 1974 state high school wrestling championships, urine samples were obtained from wrestlers prior to the weigh-in, immediately before they wrestled, and immediately after the subjects had completed their match. Specific gravity, osmolarity, pH, sodium and potassium determinations, as well as qualitative tests for protein and ketones, indicated that the wrestlers were in a dehydrated state at the time of weigh-in. After the five hour interim between the weigh-in and the first match, all but the pH measure remained essentially unchanged. This absence of significant changes in the urinary profile suggests that the wrestlers were unable to rehydrate during the five hour time period between the weigh-in and the first match and that they were competing in a dehydrated state. Urine samples collected after competition were significantly lower in specific gravity, osmolarity and potassium concentration than samples obtained before the match. The urinary potassium levels were of interest because at the three conditions (weigh-in, before the first match, after competition) they were 73-182% higher than values reported for high school students who were nonwrestlers. | Iowa wrestling study: changes in the urinary profiles of wrestlers prior to and after competition. During the 1973 and 1974 state high school wrestling championships, urine samples were obtained from wrestlers prior to the weigh-in, immediately before they wrestled, and immediately after the subjects had completed their match. Specific gravity, osmolarity, pH, sodium and potassium determinations, as well as qualitative tests for protein and ketones, indicated that the wrestlers were in a dehydrated state at the time of weigh-in. After the five hour interim between the weigh-in and the first match, all but the pH measure remained essentially unchanged. This absence of significant changes in the urinary profile suggests that the wrestlers were unable to rehydrate during the five hour time period between the weigh-in and the first match and that they were competing in a dehydrated state. Urine samples collected after competition were significantly lower in specific gravity, osmolarity and potassium concentration than samples obtained before the match. The urinary potassium levels were of interest because at the three conditions (weigh-in, before the first match, after competition) they were 73-182% higher than values reported for high school students who were nonwrestlers. | 1,625 |
pubmed23n0001_832 | [Alcohol dehydrogenase activity of nonsulfur purple bacteria]. | Rhodopseudomonas palustris, Rh. viridis, Rh. acidophila, and Rhodomicrobium vanniellii grow on media containing ethanol, n-propanol, and n-butanol. The highest amount of lower alcohols is utilized by the strains of Rh. palustris. Only Rh. acidophila accumulates methanol. Alcohol dehydrogenase of Rh. palustris, Rh. viridis, and Rhodospirillum rubrum requires for its activity NAD, that of Rhodomicrobium vanniellii--NADP, and the enzyme of Rh. acidophila is active in the presence of phenazine metasulphate (PMS) and ammonium ions. Aldehyde dehydrogenase from two strains of Rh. palustris also requires NAD; the Nakamura strain is active in the presence of PMS. Aldehyde dehydrogenase of Rh. acidophila is active in the presence of PMS and ammonium ions. Different bacterial species vary in the substrate specificity of their alcohol dehydrogenases. | [Alcohol dehydrogenase activity of nonsulfur purple bacteria]. Rhodopseudomonas palustris, Rh. viridis, Rh. acidophila, and Rhodomicrobium vanniellii grow on media containing ethanol, n-propanol, and n-butanol. The highest amount of lower alcohols is utilized by the strains of Rh. palustris. Only Rh. acidophila accumulates methanol. Alcohol dehydrogenase of Rh. palustris, Rh. viridis, and Rhodospirillum rubrum requires for its activity NAD, that of Rhodomicrobium vanniellii--NADP, and the enzyme of Rh. acidophila is active in the presence of phenazine metasulphate (PMS) and ammonium ions. Aldehyde dehydrogenase from two strains of Rh. palustris also requires NAD; the Nakamura strain is active in the presence of PMS. Aldehyde dehydrogenase of Rh. acidophila is active in the presence of PMS and ammonium ions. Different bacterial species vary in the substrate specificity of their alcohol dehydrogenases. | 1,631 |
pubmed23n0001_842 | [Regulation of lipid synthesis in animal organs]. | Studies were made of the mechanisms regulating the quantity and catalytic efficiency of hepatic acetyl coenzyme A carboxylase, which plays a critical role in the control of fatty acid biosynthesis. The microsomal enzyme system responsible for the formation of phosphatidic acid, the initial step in glycerolipid biosynthesis, was resolved into two component enzymes. The acyl-donor specificities of these and other acyltransferases account for the asymmetric fatty acid distribution in naturally occurring glycerolipids. | [Regulation of lipid synthesis in animal organs]. Studies were made of the mechanisms regulating the quantity and catalytic efficiency of hepatic acetyl coenzyme A carboxylase, which plays a critical role in the control of fatty acid biosynthesis. The microsomal enzyme system responsible for the formation of phosphatidic acid, the initial step in glycerolipid biosynthesis, was resolved into two component enzymes. The acyl-donor specificities of these and other acyltransferases account for the asymmetric fatty acid distribution in naturally occurring glycerolipids. | 1,682 |
pubmed23n0001_847 | The preparation and properties of nerve growth factor protein at alkaline pH. | The nerve growth factor (NGF) subunit of 7S NGF was isolated by chromatography at high pH on QAE-Sephadex. It has the same specific NGF activity as betaNGF isolated at acid pH, showing that this activity is an intrinsic property of the subunit and is independent of the pathway of dissociation. Continued exposure of the NGF subunit to high pH resulted in an increase in the amount of the minor species beta2NGF and the formation of a new species, beta3NGF, of even lower isoelectric point. These two species and the original major species of the preparation, beta1, were isolated by isoelectric focusing. All three species had the same specific NGF activity, but differed in their ability to reform 7S NGF. The beta2 species was one-fifth as competent as beta1, while beta3 was unable to regenerate 7S NGF. Addition of alpha- and gamma-subunits to beta1NGF decreased the amount of NGF protein required to produce one Biological Unit of activity in the bioassay, but had no effect when added to beta3NGF. The interactions between the subunits in 7S NGF therefore determine, in part, the specific activity of the NGF subunit. | The preparation and properties of nerve growth factor protein at alkaline pH. The nerve growth factor (NGF) subunit of 7S NGF was isolated by chromatography at high pH on QAE-Sephadex. It has the same specific NGF activity as betaNGF isolated at acid pH, showing that this activity is an intrinsic property of the subunit and is independent of the pathway of dissociation. Continued exposure of the NGF subunit to high pH resulted in an increase in the amount of the minor species beta2NGF and the formation of a new species, beta3NGF, of even lower isoelectric point. These two species and the original major species of the preparation, beta1, were isolated by isoelectric focusing. All three species had the same specific NGF activity, but differed in their ability to reform 7S NGF. The beta2 species was one-fifth as competent as beta1, while beta3 was unable to regenerate 7S NGF. Addition of alpha- and gamma-subunits to beta1NGF decreased the amount of NGF protein required to produce one Biological Unit of activity in the bioassay, but had no effect when added to beta3NGF. The interactions between the subunits in 7S NGF therefore determine, in part, the specific activity of the NGF subunit. | 1,689 |
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