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pubmed23n0001_1300 | Electrodecantation of serum proteins. | The sedimentation of albumin under the action of the electric and gravitational fields was determined as a function of time in discontinuous experiments in a rectangular cell, using serum with the albumin fraction stained blue. It was shown that even under the influence of strong electric fields, the upper boundary of the albumin layer fell no further than the mid-point of the cell. In continuous single-stage separation of gamma-globulin from other serum proteins, only about half the gamma-globulins can be obtained from the solution because it remains homogeneously distributed throughout the solution and is only free from albumin and other proteins in the upper half of the cell. In experiments with continuously operated triangular cells, the process was optimized to give gamma-globulin of 97.5% purity in a yield of 80%, at serum flow-through rates of up to 0.5 l/h in a block composed of 40 cells. | Electrodecantation of serum proteins. The sedimentation of albumin under the action of the electric and gravitational fields was determined as a function of time in discontinuous experiments in a rectangular cell, using serum with the albumin fraction stained blue. It was shown that even under the influence of strong electric fields, the upper boundary of the albumin layer fell no further than the mid-point of the cell. In continuous single-stage separation of gamma-globulin from other serum proteins, only about half the gamma-globulins can be obtained from the solution because it remains homogeneously distributed throughout the solution and is only free from albumin and other proteins in the upper half of the cell. In experiments with continuously operated triangular cells, the process was optimized to give gamma-globulin of 97.5% purity in a yield of 80%, at serum flow-through rates of up to 0.5 l/h in a block composed of 40 cells. | 2,529 |
pubmed23n0001_1301 | Recent studies on cytochrome P-450-linked functions in isolated rat liver cells. | In rat liver cells isolated by perfusion in the perfusion in the presence of collagenase, the major portion of cytochrome P-450 is present in the oxidized, nonsubstrate-bound, low spin state. Drug addition to a suspension of liver cells results in the rapid formation of the cytochrome P-450 (Fe3+)-substrate complex which in turn is followed by the appearance of other species with different spectral characteristics before steady state drug monooxygenation is achieved. Cytochrome P-450-linked metabolism of various tested drugs and carcinogenic polycyclic hydrocarbons by isolated rat liver cells is as fast, or faster, as with rat liver microsomes supplemented with a NADPH generating system. Both experimental models respond similarily to phenobarbital or 3-methylcholanthrene pretreatment of the animals and to various of the wellknown inhibitors of drug metabolism. Except with liver cells isolated from fasted, phenobarbital-treated rats, generation of cytosolic NADPH seems sufficient to support optimal drug metabolism even in the absence of added substrates of intermediary metabolism. In isolated liver cells oxidized drug metabolites undergo subsequent metabolic conversion, most often to form the corresponding glucuronides and sulphates. These are readily excreted, whereas non-conjugated products, e.g. free phenols, tend to accumulate intracellularly. Cellular glucuronide formation is strongly inhibited by ethanol-presumably due to an unfavorable effect of the increased NADH/NAD+ ratio on the synthesis of uridine-5'-diphosphoglucuronic acid (UDPGA). In contrast, low concentrations of ethanol have no, or only a slight stimulatory effect on the cytochrome P-450-linked step of drug metabolism and there are indications that the oxidation of low concentrations of ethanol is in fact stimulated by a facilitated reoxidation of cytosolic NADH occuring during drug monooxygenation. | Recent studies on cytochrome P-450-linked functions in isolated rat liver cells. In rat liver cells isolated by perfusion in the perfusion in the presence of collagenase, the major portion of cytochrome P-450 is present in the oxidized, nonsubstrate-bound, low spin state. Drug addition to a suspension of liver cells results in the rapid formation of the cytochrome P-450 (Fe3+)-substrate complex which in turn is followed by the appearance of other species with different spectral characteristics before steady state drug monooxygenation is achieved. Cytochrome P-450-linked metabolism of various tested drugs and carcinogenic polycyclic hydrocarbons by isolated rat liver cells is as fast, or faster, as with rat liver microsomes supplemented with a NADPH generating system. Both experimental models respond similarily to phenobarbital or 3-methylcholanthrene pretreatment of the animals and to various of the wellknown inhibitors of drug metabolism. Except with liver cells isolated from fasted, phenobarbital-treated rats, generation of cytosolic NADPH seems sufficient to support optimal drug metabolism even in the absence of added substrates of intermediary metabolism. In isolated liver cells oxidized drug metabolites undergo subsequent metabolic conversion, most often to form the corresponding glucuronides and sulphates. These are readily excreted, whereas non-conjugated products, e.g. free phenols, tend to accumulate intracellularly. Cellular glucuronide formation is strongly inhibited by ethanol-presumably due to an unfavorable effect of the increased NADH/NAD+ ratio on the synthesis of uridine-5'-diphosphoglucuronic acid (UDPGA). In contrast, low concentrations of ethanol have no, or only a slight stimulatory effect on the cytochrome P-450-linked step of drug metabolism and there are indications that the oxidation of low concentrations of ethanol is in fact stimulated by a facilitated reoxidation of cytosolic NADH occuring during drug monooxygenation. | 2,526 |
pubmed23n0001_1302 | D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure. | 1) Glucose dehydrogenase from Bacillus megaterium has been purified to a specific activity of 550 U per mg protein. The homogeneity of the purified enzyme was demonstrated by gel electrophoresis and isoelectric focusing. 2) The amino acid composition has been determined. 3) The molecular weight of the native enzyme was found to be 116000 by gel permeation chromatography, in good agreement with the values of 120000 and 118000, which were ascertained electrophoretically according to the method of Hedrick and Smith and by density gradient centrifugation, respectively. 4) In the presence of 0.1% sodium dodecylsulfate and 8M urea, the enzyme dissociates into subunits with a molecular weight of 30000 as determined by dodecylsulfate gel electrophoresis. These values indicate that the native enzyme is composed of four polypeptide chains, each probably possessing one coenzyme binding site, which can be concluded from fluorescent titration of the NADH binding sites. 5) In polyacrylamide disc electrophoresis, samples of the purified enzyme exhibit three bands of activity, which present the native (tetrameric) form of glucose dehydrogenase and two monomeric forms (molecular weight 30000), arising under the conditions of pH and ionic strength of this method. 6) The enzyme shows a sharp pH optimum at pH 8.0 in Tris/HCl buffer, and a shift of the pH optimum to pH 9.0 in acetate/borate buffer. The limiting Michaelis constant at pH 9.0 for NAD is 4.5 mM and 47.5 mM for glucose. The dissociation constant for NAD is 0.69 mM. 7) D-Glucose dehydrogenase is highly specific for beta-D-glucose and is capable of using either NAD or NADP. The enzyme is insensitive to sulfhydryl group inhibitors, heavy metal ions and chelating agents. | D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure. 1) Glucose dehydrogenase from Bacillus megaterium has been purified to a specific activity of 550 U per mg protein. The homogeneity of the purified enzyme was demonstrated by gel electrophoresis and isoelectric focusing. 2) The amino acid composition has been determined. 3) The molecular weight of the native enzyme was found to be 116000 by gel permeation chromatography, in good agreement with the values of 120000 and 118000, which were ascertained electrophoretically according to the method of Hedrick and Smith and by density gradient centrifugation, respectively. 4) In the presence of 0.1% sodium dodecylsulfate and 8M urea, the enzyme dissociates into subunits with a molecular weight of 30000 as determined by dodecylsulfate gel electrophoresis. These values indicate that the native enzyme is composed of four polypeptide chains, each probably possessing one coenzyme binding site, which can be concluded from fluorescent titration of the NADH binding sites. 5) In polyacrylamide disc electrophoresis, samples of the purified enzyme exhibit three bands of activity, which present the native (tetrameric) form of glucose dehydrogenase and two monomeric forms (molecular weight 30000), arising under the conditions of pH and ionic strength of this method. 6) The enzyme shows a sharp pH optimum at pH 8.0 in Tris/HCl buffer, and a shift of the pH optimum to pH 9.0 in acetate/borate buffer. The limiting Michaelis constant at pH 9.0 for NAD is 4.5 mM and 47.5 mM for glucose. The dissociation constant for NAD is 0.69 mM. 7) D-Glucose dehydrogenase is highly specific for beta-D-glucose and is capable of using either NAD or NADP. The enzyme is insensitive to sulfhydryl group inhibitors, heavy metal ions and chelating agents. | 2,530 |
pubmed23n0001_1303 | [Enzyme induction in Streptomyces hydrogenans. V. Characterization of testosterone-17 beta-dehydrogenase and its induction by steroids]. | Testosterone 17beta-dehydrogenase can be enriched from Streptomyces hydrogenans. The enzyme dehydrogenizes testosterone with Km=13muM and estradiol-17beta with Km=21muM to the corresponding 17-ketoderivatives. NAD forms NADH with Km=125muM. The enzyme is strongly inhibited by androstandione and 17alpha-methyltestosterone. The Ki for 17alpha-methyltestosterone is 18muM. The enzyme activity increases with increasing pH up to alkali-mediated denaturation at about pH 10. The optimum temperature is at 45 degrees C. If Streptomyces hydrogenans is cultivated in the absence of steroids, the specific activity of testosterone 17beta-dehydrogenase in the cytosol of the microorganisms amounts to 10 mU/mg protein, and increases up to 10-fold if the cells are cultivated in the presence of certain steroids. Testosterone, alpha-dihydrotestosterone, beta-dihydrotestosterone, estradiol-17beta, and 17alpha-methyltestosterone are very effective inducers. Thus, for the first time, the ability of estradiol-17beta to induce an enzyme synthesis in a microorganism is shown. The steroid-dependent induction is inhibited by testosterone acetate and rifamycin SV. Cyproterone, however, does not decrease the testosterone-dependent enzyme induction of testosterone 17beta-dehydrogenase. | [Enzyme induction in Streptomyces hydrogenans. V. Characterization of testosterone-17 beta-dehydrogenase and its induction by steroids]. Testosterone 17beta-dehydrogenase can be enriched from Streptomyces hydrogenans. The enzyme dehydrogenizes testosterone with Km=13muM and estradiol-17beta with Km=21muM to the corresponding 17-ketoderivatives. NAD forms NADH with Km=125muM. The enzyme is strongly inhibited by androstandione and 17alpha-methyltestosterone. The Ki for 17alpha-methyltestosterone is 18muM. The enzyme activity increases with increasing pH up to alkali-mediated denaturation at about pH 10. The optimum temperature is at 45 degrees C. If Streptomyces hydrogenans is cultivated in the absence of steroids, the specific activity of testosterone 17beta-dehydrogenase in the cytosol of the microorganisms amounts to 10 mU/mg protein, and increases up to 10-fold if the cells are cultivated in the presence of certain steroids. Testosterone, alpha-dihydrotestosterone, beta-dihydrotestosterone, estradiol-17beta, and 17alpha-methyltestosterone are very effective inducers. Thus, for the first time, the ability of estradiol-17beta to induce an enzyme synthesis in a microorganism is shown. The steroid-dependent induction is inhibited by testosterone acetate and rifamycin SV. Cyproterone, however, does not decrease the testosterone-dependent enzyme induction of testosterone 17beta-dehydrogenase. | 2,532 |
pubmed23n0001_1304 | Boar acrosin, II: Amino acid composition, amino terminal residue and molecular weight estimations by ultracentrifugation. | The molecular weight of boar acrosin in neutral solution was estimated to be 41000 +/- 1000 by high-speed sedimentation equilibrium analysis. This result is in good agreement with the value found earlier[1] by sodium dodecylsulfate polyacrylamide gel electrophoresis. The sedimentation coefficeint of acrosin obtained by active enzyme centrifugation of partly purified preparations is in accordance with the sedimentation coefficient of the pure preparation estimated by conventional sedimentation velocity analysis. The sedimentation coefficient of acrosin is considerably decreased in slightly acidic solution (pH 4), indicating that changes in the tertiary structure occur upon acidification. The amino acid composition of the acrosin preparation homogeneous by electrophoretic and chromatographic criteria and in sedimentation studies was determined. Valine was found as the unique N-terminal amino acid. However, in microheterogeneous forms of acrosin, alanine and methionine were also detected in end group analysis. | Boar acrosin, II: Amino acid composition, amino terminal residue and molecular weight estimations by ultracentrifugation. The molecular weight of boar acrosin in neutral solution was estimated to be 41000 +/- 1000 by high-speed sedimentation equilibrium analysis. This result is in good agreement with the value found earlier[1] by sodium dodecylsulfate polyacrylamide gel electrophoresis. The sedimentation coefficeint of acrosin obtained by active enzyme centrifugation of partly purified preparations is in accordance with the sedimentation coefficient of the pure preparation estimated by conventional sedimentation velocity analysis. The sedimentation coefficient of acrosin is considerably decreased in slightly acidic solution (pH 4), indicating that changes in the tertiary structure occur upon acidification. The amino acid composition of the acrosin preparation homogeneous by electrophoretic and chromatographic criteria and in sedimentation studies was determined. Valine was found as the unique N-terminal amino acid. However, in microheterogeneous forms of acrosin, alanine and methionine were also detected in end group analysis. | 2,533 |
pubmed23n0001_1305 | Characterization of protein kinases from Blepharisma intermedium. | Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated. | Characterization of protein kinases from Blepharisma intermedium. Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated. | 2,534 |
pubmed23n0001_1306 | [Solubilization and purification of a 3alpha-hydroxysteroid dehydrogenase in rat liver microsomes (author's transl)]. | The microsomal 3-hydroxysteroid dehydrogenases were solubilized with lubrol, a non-ionic detergent. A 3alpha-hydroxysteroid dehydrogenase is purified about 100-fold by double affinity chromatography on 5alpha-dihydrotestosterone-Sepharose. This enzyme can use both NADH and NADPH as coenzymes. | [Solubilization and purification of a 3alpha-hydroxysteroid dehydrogenase in rat liver microsomes (author's transl)]. The microsomal 3-hydroxysteroid dehydrogenases were solubilized with lubrol, a non-ionic detergent. A 3alpha-hydroxysteroid dehydrogenase is purified about 100-fold by double affinity chromatography on 5alpha-dihydrotestosterone-Sepharose. This enzyme can use both NADH and NADPH as coenzymes. | 2,535 |
pubmed23n0001_1307 | The dissociation of insect embryos for cell culture. | Procedures and solutions were developed for dissociating embryos of Blattella germanica in preparation for primary cell culture. Trypsin solutions were maximally effective at 0.01% for germ bands but higher concentrations, 0.05 to 0.1% were needed for embryos in later stages. | The dissociation of insect embryos for cell culture. Procedures and solutions were developed for dissociating embryos of Blattella germanica in preparation for primary cell culture. Trypsin solutions were maximally effective at 0.01% for germ bands but higher concentrations, 0.05 to 0.1% were needed for embryos in later stages. | 2,549 |
pubmed23n0001_1308 | Effect of sodium butyrate on mammalian cells in culture: a review. | Sodium butyrate produces reversible changes in morphology, growth rate, and enzyme activities of several mammalian cell types in culture. Some of these changes are similar to those produced by agents which increase the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP) or by analogs of cAMP. Sodium butyrate increases the intracellular level of cAMP by about two fold in neuroblastoma cells; therefore, some of the effects of sodium butyrate on these cells may in part be mediated by cAMP. Sodium butyrate appears to have properties of a good chemotherapeutic agent for neuroblastoma tumors because the treatment of neuroblastoma cells in culture causes cell death and "differentiation"; however, it is either innocuous or produces reversible morphological and biochemical alterations in other cell types. | Effect of sodium butyrate on mammalian cells in culture: a review. Sodium butyrate produces reversible changes in morphology, growth rate, and enzyme activities of several mammalian cell types in culture. Some of these changes are similar to those produced by agents which increase the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP) or by analogs of cAMP. Sodium butyrate increases the intracellular level of cAMP by about two fold in neuroblastoma cells; therefore, some of the effects of sodium butyrate on these cells may in part be mediated by cAMP. Sodium butyrate appears to have properties of a good chemotherapeutic agent for neuroblastoma tumors because the treatment of neuroblastoma cells in culture causes cell death and "differentiation"; however, it is either innocuous or produces reversible morphological and biochemical alterations in other cell types. | 2,548 |
pubmed23n0001_1309 | On the nature of the presumed receptor for IgE on mast cells. III. Kinetics of the blocking of the PCA reaction by cell-free particulate preparations from rat peritoneal mast cells and effect of pH and calcium concentration on the reaction. | The 'binding' of IgE to particulate preparations derived from sonicated purified rat mast cells was measured by the blocking of PCA titrations of the supernatant solutions from incubations with such preparations. It was found that the PCA blocking reaction was inhibited by the addition of calcium ion to the incubations. The blocking reaction was strongly dependent on the pH of the incubations, being maximal at pH values lower than 5-0. The blocking reaction proceeded in a linear manner for at least 3 h provided that no more than 70 percent of the amount of IgE initially supplied had been removed by the particulate fraction. Only mast cell-derived preparations were capable of effecting PCA blocking. | On the nature of the presumed receptor for IgE on mast cells. III. Kinetics of the blocking of the PCA reaction by cell-free particulate preparations from rat peritoneal mast cells and effect of pH and calcium concentration on the reaction. The 'binding' of IgE to particulate preparations derived from sonicated purified rat mast cells was measured by the blocking of PCA titrations of the supernatant solutions from incubations with such preparations. It was found that the PCA blocking reaction was inhibited by the addition of calcium ion to the incubations. The blocking reaction was strongly dependent on the pH of the incubations, being maximal at pH values lower than 5-0. The blocking reaction proceeded in a linear manner for at least 3 h provided that no more than 70 percent of the amount of IgE initially supplied had been removed by the particulate fraction. Only mast cell-derived preparations were capable of effecting PCA blocking. | 2,550 |
pubmed23n0001_1310 | Effect of glucose and sucrose on survival in batch culture of Streptococcus mutans C67-1 and a noncariogenic mutant, C67-25. | The growth and survival of two strains of Streptococcus mutans in 5% (wt/vol) glucose or sucrose broth was investigated. S. mutans strain C67-1 showed little loss of viability after 30 h of incubation in batch culture in the presence of either sugar. S. mutans strain C67-25, a noncariogenic mutant of C67-1 that has lost the ability of the latter to produce sticky, insoluble extracellular polysaccharide when grown in sucrose broth, showed a dramatic loss of viability after 30 h of incubation in either glucose or sucrose broth, the effect being most marked in the presence of glucose. The loss of viability was shown to be due to acid production. Insoluble extracellular polysaccharide production appears to be a phenomenon favoring the survival of organisms subjected to high sucrose levels. Other factors must be involved, however, since there are differences between the two strains as regards their survival in glucose broth. | Effect of glucose and sucrose on survival in batch culture of Streptococcus mutans C67-1 and a noncariogenic mutant, C67-25. The growth and survival of two strains of Streptococcus mutans in 5% (wt/vol) glucose or sucrose broth was investigated. S. mutans strain C67-1 showed little loss of viability after 30 h of incubation in batch culture in the presence of either sugar. S. mutans strain C67-25, a noncariogenic mutant of C67-1 that has lost the ability of the latter to produce sticky, insoluble extracellular polysaccharide when grown in sucrose broth, showed a dramatic loss of viability after 30 h of incubation in either glucose or sucrose broth, the effect being most marked in the presence of glucose. The loss of viability was shown to be due to acid production. Insoluble extracellular polysaccharide production appears to be a phenomenon favoring the survival of organisms subjected to high sucrose levels. Other factors must be involved, however, since there are differences between the two strains as regards their survival in glucose broth. | 2,556 |
pubmed23n0001_1311 | Microcapsule of type III strains of group B Streptococcus: production and morphology. | The yield of purified type III polysaccharide of group B Streptococcus was significantly improved by modification of the growth medium. Culture of organisms in standard Todd-Hewitt broth resulted in acid accumulation during the exponential phase of growth and poor yield of type III polysaccharide when extracted from cells by washing with neutral buffer solution. By increasing the buffering capacity of the broth medium, acid accumulation was prevented, and the number of viable cells was increased at the stationary phase of growth. Further, by increasing the concentration of glucose in the buffered medium, the yield of type III polysaccharide was increased two to three times. Electron microscopic investigations of cells grown in the modified broth medium demonstrated a thicker microcapsule than was found in organisms grown in standard broth. | Microcapsule of type III strains of group B Streptococcus: production and morphology. The yield of purified type III polysaccharide of group B Streptococcus was significantly improved by modification of the growth medium. Culture of organisms in standard Todd-Hewitt broth resulted in acid accumulation during the exponential phase of growth and poor yield of type III polysaccharide when extracted from cells by washing with neutral buffer solution. By increasing the buffering capacity of the broth medium, acid accumulation was prevented, and the number of viable cells was increased at the stationary phase of growth. Further, by increasing the concentration of glucose in the buffered medium, the yield of type III polysaccharide was increased two to three times. Electron microscopic investigations of cells grown in the modified broth medium demonstrated a thicker microcapsule than was found in organisms grown in standard broth. | 2,557 |
pubmed23n0001_1312 | Central regulation of blood eosinophilia by the beta-adrenergic system in rats. | The effect of isoprenaline and three different beta-adrenergic blocking agents on blood eosinophilia was evaluated in the rat. Eosinopenic effect of isoprenaline was antagonized by intraperitoneal pretreatment with propranolol and by high but not low doses of practolol. Sotalol, a beta-blocker which in contrast to propranolol does not penetrate the blood-brain barrier, did not affect the eosinophil count when injected intraperitoneally, but produced a marked increase in number of circulating eosinophils following intracerebroventricular injection. Intraperitoneal administration of isoprenaline and propranolol as well as intracerebroventricular injection of sotalol did not affect eosinophil count in adrenalectomized or hypophysectomized rats. These results suggest that beta-adrenergic agents regulate the number of circulating eosinophils through a central mechanism and that presence of both hypophysis and adrenal glands is required for this action. | Central regulation of blood eosinophilia by the beta-adrenergic system in rats. The effect of isoprenaline and three different beta-adrenergic blocking agents on blood eosinophilia was evaluated in the rat. Eosinopenic effect of isoprenaline was antagonized by intraperitoneal pretreatment with propranolol and by high but not low doses of practolol. Sotalol, a beta-blocker which in contrast to propranolol does not penetrate the blood-brain barrier, did not affect the eosinophil count when injected intraperitoneally, but produced a marked increase in number of circulating eosinophils following intracerebroventricular injection. Intraperitoneal administration of isoprenaline and propranolol as well as intracerebroventricular injection of sotalol did not affect eosinophil count in adrenalectomized or hypophysectomized rats. These results suggest that beta-adrenergic agents regulate the number of circulating eosinophils through a central mechanism and that presence of both hypophysis and adrenal glands is required for this action. | 2,558 |
pubmed23n0001_1313 | Comparison between two antibody populations in the EBV system: anti-MA versus neutralizing antibody activity. | EBV-neutralizing antibody titers were determined in 11 sera derived from African Burkitt lymphoma or nasopharyngeal carcinoma patients and in the corresponding serum fractions retained above XM 100 Diaflo membranes after low pH treatment, and after recombination of the retained and passed fractions by neutralization of the acidified samples. They were compared with the corresponding antimembrane antigen (MA) titers in seven of the same sera. While all sera tested showed substantial increase of the anti-MA activity in the retained fraction, resulting in a significantly increased mean titer, EBV neutralizing activity did not change at all after identical treatment or changed only in a random fashion, resulting in stable mean titers. It is suggested that anti-MA and neutralizing antibodies are directed against at least partly different antigens on the virus. | Comparison between two antibody populations in the EBV system: anti-MA versus neutralizing antibody activity. EBV-neutralizing antibody titers were determined in 11 sera derived from African Burkitt lymphoma or nasopharyngeal carcinoma patients and in the corresponding serum fractions retained above XM 100 Diaflo membranes after low pH treatment, and after recombination of the retained and passed fractions by neutralization of the acidified samples. They were compared with the corresponding antimembrane antigen (MA) titers in seven of the same sera. While all sera tested showed substantial increase of the anti-MA activity in the retained fraction, resulting in a significantly increased mean titer, EBV neutralizing activity did not change at all after identical treatment or changed only in a random fashion, resulting in stable mean titers. It is suggested that anti-MA and neutralizing antibodies are directed against at least partly different antigens on the virus. | 2,559 |
pubmed23n0001_1314 | Gastric acid secretion, serum-gastrin levels and psychomotor function under the influence of placebo, insulin-hypoglycemia, and/or bromazepam. | Gastric acid output, blood-glucose, serum-gastrin and psychomotor-performance were measured in four healthy subjects one hour before and two hours after the intravenous injection of (a) 2ml saline, (b) 0.2 U/kg b.w. insulin, (c) 0.1 mg/kg b.w. bromazepam. Each subject underwent one experiment of each type. The study was layed out as a Latin-square and analysed accordingly. Gastric acid secretion was measured by means of intragastric titration and a telemetering capsule; blood-glucose and serum-gastrin levels as well as psychomotor performance as a measure of vigilance were determined in 15-minute-intervals. In the saline series (a), none of the four parameters showed any systematic variation. In series (b), a bimodal response of acid output to insulin, initial inhibition and subsequent stimulation was observed in all subjects. Serum-gastrin levels showed only a slight and transient increase in the first thirty minutes. Psychomotor performance decreased markedly with progressing hypoglycemia, and increased when glucose levels rose again. In the bromazepan series (c), acid output and psychomotor performance decreased and, after the first hour, increased almost parallely, while glucose and gastrin levels remained unchanged. In series (d), an additive effect of insulin and bromazepam occurred: acid output and psychomotor performance were lower than after insulin alone; peak acid secretion, maximal hypoglycemia and peak of serum-gastrin were shifted to the right. It is concluded that the lowered basal as well as insulin-stimulated acid secretion after bromazepam is due to the central effect of the drug, and that this effect is mediated to the gastric glands directly via autonomic nervous pathways without involving a release of endogenous gastrin. | Gastric acid secretion, serum-gastrin levels and psychomotor function under the influence of placebo, insulin-hypoglycemia, and/or bromazepam. Gastric acid output, blood-glucose, serum-gastrin and psychomotor-performance were measured in four healthy subjects one hour before and two hours after the intravenous injection of (a) 2ml saline, (b) 0.2 U/kg b.w. insulin, (c) 0.1 mg/kg b.w. bromazepam. Each subject underwent one experiment of each type. The study was layed out as a Latin-square and analysed accordingly. Gastric acid secretion was measured by means of intragastric titration and a telemetering capsule; blood-glucose and serum-gastrin levels as well as psychomotor performance as a measure of vigilance were determined in 15-minute-intervals. In the saline series (a), none of the four parameters showed any systematic variation. In series (b), a bimodal response of acid output to insulin, initial inhibition and subsequent stimulation was observed in all subjects. Serum-gastrin levels showed only a slight and transient increase in the first thirty minutes. Psychomotor performance decreased markedly with progressing hypoglycemia, and increased when glucose levels rose again. In the bromazepan series (c), acid output and psychomotor performance decreased and, after the first hour, increased almost parallely, while glucose and gastrin levels remained unchanged. In series (d), an additive effect of insulin and bromazepam occurred: acid output and psychomotor performance were lower than after insulin alone; peak acid secretion, maximal hypoglycemia and peak of serum-gastrin were shifted to the right. It is concluded that the lowered basal as well as insulin-stimulated acid secretion after bromazepam is due to the central effect of the drug, and that this effect is mediated to the gastric glands directly via autonomic nervous pathways without involving a release of endogenous gastrin. | 2,560 |
pubmed23n0001_1315 | [Correlation between plasma concentration and clinical effect of neuroleptics and antidepressants]. | Results of investigations concerning the correlation between plasma concentrations and clinical effect of neuroleptics and antidepressives are summarised. In the case of neuroleptics there is in general no relationship between clinical parameters of activity and plasma concentration. For antidepressives there is, in most cases, a correlation between side-effects and plasma concentrations. Whether the therapeutic effect is dependent on the plasma concentration or not, is however open to some doubt. The practical value of plasma concentration estimations during therapy with the above mentioned drugs is therefore limited. Inadequate efficacy and/or poor tolerance can occasionally be explained by estimating plasma concentrations which alone, however, scarcely allow one to make prophecies. In determination of the optimal individual dosage must continue to be an empirical process based on clinical observations and experience. | [Correlation between plasma concentration and clinical effect of neuroleptics and antidepressants]. Results of investigations concerning the correlation between plasma concentrations and clinical effect of neuroleptics and antidepressives are summarised. In the case of neuroleptics there is in general no relationship between clinical parameters of activity and plasma concentration. For antidepressives there is, in most cases, a correlation between side-effects and plasma concentrations. Whether the therapeutic effect is dependent on the plasma concentration or not, is however open to some doubt. The practical value of plasma concentration estimations during therapy with the above mentioned drugs is therefore limited. Inadequate efficacy and/or poor tolerance can occasionally be explained by estimating plasma concentrations which alone, however, scarcely allow one to make prophecies. In determination of the optimal individual dosage must continue to be an empirical process based on clinical observations and experience. | 2,561 |
pubmed23n0001_1316 | EEG sleep studies of insomniacs under flunitrazepam treatment. | This study investigates the effect of flunitrazepam, a new benzodiazepine, on the sleep of insomniac patients under chronic treatment. Polygraphic recordings have shown that this drug decreases not only the activity of the wakefulness system, but also the activity of the synchronizing system of slow-wave sleep. The subjective feeling of improved and sounder sleep seems to be related to a decrease of wakefulness pressure as well as to a decrease of body motoricity, but not with the modification of sleep stages themselves. Flunitrazepam appears to possess some regulatory properties on REM sleep, since this stage is enhanced in patients with an initial low amount of REM sleep and decreased in those having a higher initial REM sleep. Flunitrazepam possesses potent and useful hypnogenic properties in man but does not induce physiological sleep. | EEG sleep studies of insomniacs under flunitrazepam treatment. This study investigates the effect of flunitrazepam, a new benzodiazepine, on the sleep of insomniac patients under chronic treatment. Polygraphic recordings have shown that this drug decreases not only the activity of the wakefulness system, but also the activity of the synchronizing system of slow-wave sleep. The subjective feeling of improved and sounder sleep seems to be related to a decrease of wakefulness pressure as well as to a decrease of body motoricity, but not with the modification of sleep stages themselves. Flunitrazepam appears to possess some regulatory properties on REM sleep, since this stage is enhanced in patients with an initial low amount of REM sleep and decreased in those having a higher initial REM sleep. Flunitrazepam possesses potent and useful hypnogenic properties in man but does not induce physiological sleep. | 2,563 |
pubmed23n0001_1317 | A histochemical study of the apparent deamination of proteins by sodium hypochlorite. | The possible chemical mechanisms by which neutral solutions of sodium hypochlorite containing a high concentration of sodium chloride abolish the acidophilia of proteins in sections of fixed tissue are reviewed. The most probable one is the chlorination of the protein terminal amino groups, followed by the breakdown of the N-chloramine so formed into alpha-ketocarboxylic acid, nitrile or aldehyde groups. Hypochlorite solutions certainly do not deaminate tissue sections as was previously thought. Experimental evidence for the formation of relatively stable N-chloramine groups in situ and their limited conversion to aldehydes is reported. For example, the acidophilia of hypochlorite-treated sections was found to be restored after flooding them with hydriodic acid followed by the extraction of the liberated iodine with an alcohol. The significance of these experimental findings is discussed. | A histochemical study of the apparent deamination of proteins by sodium hypochlorite. The possible chemical mechanisms by which neutral solutions of sodium hypochlorite containing a high concentration of sodium chloride abolish the acidophilia of proteins in sections of fixed tissue are reviewed. The most probable one is the chlorination of the protein terminal amino groups, followed by the breakdown of the N-chloramine so formed into alpha-ketocarboxylic acid, nitrile or aldehyde groups. Hypochlorite solutions certainly do not deaminate tissue sections as was previously thought. Experimental evidence for the formation of relatively stable N-chloramine groups in situ and their limited conversion to aldehydes is reported. For example, the acidophilia of hypochlorite-treated sections was found to be restored after flooding them with hydriodic acid followed by the extraction of the liberated iodine with an alcohol. The significance of these experimental findings is discussed. | 2,567 |
pubmed23n0001_1318 | Effects of dbcAMP and theophylline on rat adrenal medulla grown in tissue culture. | Explants of rat adrenal medulla were grown in tissue culture. The effects of various doses of dbcAMP ranging from 0.001 mM up to 1 mM and equimolar amounts of theophylline were recorded by phase contrast optics and catecholamine histochemistry (glyoxylic acid method) over six days. There was a dose-dependent inhibition of the normally occurring outgrowth of Schwann cells, "chromaffin" cells and axons from the explants. Maintenance of glyoxylic acid-induced fluorescence in "chromaffin" cells was dose-dependent, too. Since theophylline is known to enhance intracellular levels of cAMP only, these effects are probably due to the action of cAMP. cAMP obviously maintains the degree of differentiation of chromaffin cells. Thus it could be argued that a certain degree of dedifferentiation is a prerequisite for the formation of axons from these cells. | Effects of dbcAMP and theophylline on rat adrenal medulla grown in tissue culture. Explants of rat adrenal medulla were grown in tissue culture. The effects of various doses of dbcAMP ranging from 0.001 mM up to 1 mM and equimolar amounts of theophylline were recorded by phase contrast optics and catecholamine histochemistry (glyoxylic acid method) over six days. There was a dose-dependent inhibition of the normally occurring outgrowth of Schwann cells, "chromaffin" cells and axons from the explants. Maintenance of glyoxylic acid-induced fluorescence in "chromaffin" cells was dose-dependent, too. Since theophylline is known to enhance intracellular levels of cAMP only, these effects are probably due to the action of cAMP. cAMP obviously maintains the degree of differentiation of chromaffin cells. Thus it could be argued that a certain degree of dedifferentiation is a prerequisite for the formation of axons from these cells. | 2,568 |
pubmed23n0001_1319 | The possibilities and limitations of membrane methods for the histochemical demonstration of cholinesterases. | The thiocholine method for the histochemical detection of cholinesterases according to Karnovsky-Roots was adapted for unfixed cryostat sections by addition of the agar solution to the incubation mixture and by using the semipermeable membrane interposed between the section and the incubation medium. The procedure prevents the leakage of the enzyme activity of the section and is suitable for tissues where the cholinesterase activity is low. | The possibilities and limitations of membrane methods for the histochemical demonstration of cholinesterases. The thiocholine method for the histochemical detection of cholinesterases according to Karnovsky-Roots was adapted for unfixed cryostat sections by addition of the agar solution to the incubation mixture and by using the semipermeable membrane interposed between the section and the incubation medium. The procedure prevents the leakage of the enzyme activity of the section and is suitable for tissues where the cholinesterase activity is low. | 2,569 |
pubmed23n0001_1320 | Copper catalyzed alkaline autoxidation of selenocystamine. | In alkaline medium and in the presence of cupric ions selenocystamine undergoes autoxidation and is entirely transformed into selenohypotaurine. Among the different metal ions tested, Fe, Co, Ni, Cu, Ag, Mg, Mn, only cupric ions are effective in catalyzing the reaction. The reaction shows an optimum around pH 13. In most respects the autoxidation of selenocystamine is similar to the alkaline autoxidation of cystamine. Some data on the paper and ion exchange chromatographic behaviour of selenohypotaurine and selenotaurine are reported, as also details for the synthesis of selenotaurine. | Copper catalyzed alkaline autoxidation of selenocystamine. In alkaline medium and in the presence of cupric ions selenocystamine undergoes autoxidation and is entirely transformed into selenohypotaurine. Among the different metal ions tested, Fe, Co, Ni, Cu, Ag, Mg, Mn, only cupric ions are effective in catalyzing the reaction. The reaction shows an optimum around pH 13. In most respects the autoxidation of selenocystamine is similar to the alkaline autoxidation of cystamine. Some data on the paper and ion exchange chromatographic behaviour of selenohypotaurine and selenotaurine are reported, as also details for the synthesis of selenotaurine. | 2,565 |
pubmed23n0001_1321 | The binding of nucleotides to 3'-nucleotidase from wheat germ. | The 3'-mononucleotidase (3'-ribonucleotide phosphohydrolase, EC 3.1.3.6) from wheat germ has been purified 2,000 fold. The enzyme has a molecular weight of approximatley 32,000, as judged by the use of G-100 gel filtration, and does not attack 2'- or 5'- nucleotides. In order to obtain some indications on the structural requirements for binding and reactivity, the purified enzyme has been subjected to kinetic analyses, including initial velocities with several 3'-ribomononucleotides, inhibition by 5'- nucleotides and nucleotide-analogues, and effect of pH and sulphydryl compounds. The data indicate one base binding site at the active site of the enzyme. This site appears to be the same involved in the binding of both substrates and inhibitors, with higher affinity for purine nucleotides than for pyrimidine compounds, in the order guanosine greater than adenosine greater than inosine greater than uridine greater than cytidine nucleotides. | The binding of nucleotides to 3'-nucleotidase from wheat germ. The 3'-mononucleotidase (3'-ribonucleotide phosphohydrolase, EC 3.1.3.6) from wheat germ has been purified 2,000 fold. The enzyme has a molecular weight of approximatley 32,000, as judged by the use of G-100 gel filtration, and does not attack 2'- or 5'- nucleotides. In order to obtain some indications on the structural requirements for binding and reactivity, the purified enzyme has been subjected to kinetic analyses, including initial velocities with several 3'-ribomononucleotides, inhibition by 5'- nucleotides and nucleotide-analogues, and effect of pH and sulphydryl compounds. The data indicate one base binding site at the active site of the enzyme. This site appears to be the same involved in the binding of both substrates and inhibitors, with higher affinity for purine nucleotides than for pyrimidine compounds, in the order guanosine greater than adenosine greater than inosine greater than uridine greater than cytidine nucleotides. | 2,566 |
pubmed23n0001_1322 | Improved histological localization of GABA-transaminase activity in rat cerebellar cortex after aldehyde fixation. | A method for the chemical fixation of the enzyme GABA-transaminase in nervous tissue is described. It is shown that after perfusion with a formaldehyde/glutaraldehyde fixative, activity of the enzyme in cerebellar cortex is demonstrable whilst cellular morphology is preserved. Results from the improved technique have shown new sites of GABA-transaminase activity in cerebellar cortex. In view of these results a special function for glial cells in this area of brain has been suggested. | Improved histological localization of GABA-transaminase activity in rat cerebellar cortex after aldehyde fixation. A method for the chemical fixation of the enzyme GABA-transaminase in nervous tissue is described. It is shown that after perfusion with a formaldehyde/glutaraldehyde fixative, activity of the enzyme in cerebellar cortex is demonstrable whilst cellular morphology is preserved. Results from the improved technique have shown new sites of GABA-transaminase activity in cerebellar cortex. In view of these results a special function for glial cells in this area of brain has been suggested. | 2,570 |
pubmed23n0001_1323 | Effect of plasma [K+] on the DC potential and on ion distributions between CSF and blood. | Keeping the arterial pH at 7.4 and PaCO2 at 40 mmHg in eight anesthetized dogs, we acutely raised plasma potassium concentration from 3.4 to 8.2 meq/1, then allowed it to decay back to control levels. The cerebrospinal fluid (CSF)-blood electrical potential difference (pd) increased 13.2 mV per 10-fold increase in plasma [K+]. Again keeping arterial pH at 7.4 and PaCO2 at 40 mmHg, we elevated plasma [K+] in four dogs from 3.3 to 8.0 meq/1 and maintained this level for 6 h. We found 1) that the PD increased from a control value of +1.3 to +8.9mV, showing no tendency to decay over the 6 h; and 2) that the change in PD did not affect the distribution of Na+, K+, H+, Cl-, or HCO3- between blood and CSF over the 6 h. These results suggest that under these conditions the PD between CSF and blood may play no effective role in determining the distributions of these charged species by 6 h. These results are contrasted with recent findings which suggest that H+ and HCO3- are distributed according to passive forces between CSF and blood. | Effect of plasma [K+] on the DC potential and on ion distributions between CSF and blood. Keeping the arterial pH at 7.4 and PaCO2 at 40 mmHg in eight anesthetized dogs, we acutely raised plasma potassium concentration from 3.4 to 8.2 meq/1, then allowed it to decay back to control levels. The cerebrospinal fluid (CSF)-blood electrical potential difference (pd) increased 13.2 mV per 10-fold increase in plasma [K+]. Again keeping arterial pH at 7.4 and PaCO2 at 40 mmHg, we elevated plasma [K+] in four dogs from 3.3 to 8.0 meq/1 and maintained this level for 6 h. We found 1) that the PD increased from a control value of +1.3 to +8.9mV, showing no tendency to decay over the 6 h; and 2) that the change in PD did not affect the distribution of Na+, K+, H+, Cl-, or HCO3- between blood and CSF over the 6 h. These results suggest that under these conditions the PD between CSF and blood may play no effective role in determining the distributions of these charged species by 6 h. These results are contrasted with recent findings which suggest that H+ and HCO3- are distributed according to passive forces between CSF and blood. | 2,572 |
pubmed23n0001_1324 | Ontogeny of tracheal fluid, pulmonary surfactant, and plasma corticoids in the fetal lamb. | We examined fetal plasma corticoids and flow rate, electrolyte composition, and surfactant content of tracheal fluid in chronic experiments with eight fetal lambs. From 120 to 148 days of gestation the rate of fluid production was 4.5 ml/kg per h, and there was no change in mean fluid sodium (147.8 meq/1), chloride (153.1 meq/1), calcium (2.2 mg/100 ml), and pH (6.23). Tracheal fluid potassium increased from 4.3 meq/1 at 120-130 days to 8.9 meq/1 at term, while plasma sodium, chloride, calcium, pH, and potassium were constant at 146.1 meq/1, 110.0 meq/1, 12.1 mg/100 ml, 7.39, and 4.0 meq/1, respectively. Plasma corticoids were less than 1.5 mug/100 ml total (0.3 mug/100 ml free) until 130 days, when they increased rapidly to 10.5 total (3.2 free) at 148 days. Surfactant was first detected in tracheal fluid between 124 and 133 days and its secretion increased rapidly after 135 days to a value of 125 mug/kg per h at 148 days. A sudden increase in fetal plasma corticoids does not seem to be the stimulus for appearance of surfactant in the lamb, although these hormones may induce the rapid accumulation of surfactant prior to delivery. | Ontogeny of tracheal fluid, pulmonary surfactant, and plasma corticoids in the fetal lamb. We examined fetal plasma corticoids and flow rate, electrolyte composition, and surfactant content of tracheal fluid in chronic experiments with eight fetal lambs. From 120 to 148 days of gestation the rate of fluid production was 4.5 ml/kg per h, and there was no change in mean fluid sodium (147.8 meq/1), chloride (153.1 meq/1), calcium (2.2 mg/100 ml), and pH (6.23). Tracheal fluid potassium increased from 4.3 meq/1 at 120-130 days to 8.9 meq/1 at term, while plasma sodium, chloride, calcium, pH, and potassium were constant at 146.1 meq/1, 110.0 meq/1, 12.1 mg/100 ml, 7.39, and 4.0 meq/1, respectively. Plasma corticoids were less than 1.5 mug/100 ml total (0.3 mug/100 ml free) until 130 days, when they increased rapidly to 10.5 total (3.2 free) at 148 days. Surfactant was first detected in tracheal fluid between 124 and 133 days and its secretion increased rapidly after 135 days to a value of 125 mug/kg per h at 148 days. A sudden increase in fetal plasma corticoids does not seem to be the stimulus for appearance of surfactant in the lamb, although these hormones may induce the rapid accumulation of surfactant prior to delivery. | 2,573 |
pubmed23n0001_1325 | Hydrogen ion concentration and oxygen uptake in an isolated canine hindlimb. | Oxygen utilization (VO2) and lactate production by an isolated perfused canine hindlimb was evaluated at various hydrogen ion concentrations. A membrane lung perfusion system was established such that blood flow and temperature could be fixed at normal levels. Oxygen, nitrogen, and carbon dioxide (CO2) gas flows to the membrane lung were independently regulated to provide a fixed arterial oxygen content (CaO2). By changing CO2 flow, the pH of the arterial blood was varied between 6.9 and 7.6 at 10-min intervals. The mean O2 delivery (CaO2 X blood flow) was between 16.3 ML O2/min and 20.5 ml O2/min. Standard error of the mean in each dog, however, was less than 0.4 ml O2/min. VO2 was linearly related to the pH of the perfusing blood: VO2% = 100.1 pH - 643 (r = 0.866). Oxygen consumption was inversely related to PCO2: VO2% = -0.62 PCO2 + 124, but the correlation was less good (r = 0.729). Lactate production was linearly related to the pH of the perfusing blood (above a pH of 7.4): lactate produced = 22.5 pH - 162.5 (r = 0.75). At a pH below 7.4, lactate was not produced. Oxygen consumption of skeletal muscle appears critically dependent on extracellular fluid pH. A change in pH of 0.1 alters VO2 almost exactly 10%. Alkalosis is a potent stimulus to lactic acid production by skeletal muscle. | Hydrogen ion concentration and oxygen uptake in an isolated canine hindlimb. Oxygen utilization (VO2) and lactate production by an isolated perfused canine hindlimb was evaluated at various hydrogen ion concentrations. A membrane lung perfusion system was established such that blood flow and temperature could be fixed at normal levels. Oxygen, nitrogen, and carbon dioxide (CO2) gas flows to the membrane lung were independently regulated to provide a fixed arterial oxygen content (CaO2). By changing CO2 flow, the pH of the arterial blood was varied between 6.9 and 7.6 at 10-min intervals. The mean O2 delivery (CaO2 X blood flow) was between 16.3 ML O2/min and 20.5 ml O2/min. Standard error of the mean in each dog, however, was less than 0.4 ml O2/min. VO2 was linearly related to the pH of the perfusing blood: VO2% = 100.1 pH - 643 (r = 0.866). Oxygen consumption was inversely related to PCO2: VO2% = -0.62 PCO2 + 124, but the correlation was less good (r = 0.729). Lactate production was linearly related to the pH of the perfusing blood (above a pH of 7.4): lactate produced = 22.5 pH - 162.5 (r = 0.75). At a pH below 7.4, lactate was not produced. Oxygen consumption of skeletal muscle appears critically dependent on extracellular fluid pH. A change in pH of 0.1 alters VO2 almost exactly 10%. Alkalosis is a potent stimulus to lactic acid production by skeletal muscle. | 2,574 |
pubmed23n0001_1326 | Cerebrospinal fluid sampling technique and Astrup pH and PCO2 values. | The pH and PCO2 values measured by the Astrup technique were compared in cerebrospinal fluid (CSF) obtained using two different sampling techniques: 1) a direct or in vivo technique and 2) the widely accepted syringe sampling technique. In 65 pairs of measurements in 9 dogs it was found that the pH was always overestimated and the PCO2 always underestimated in the syringe sample when compared to the in vivo sample. The equations describing the relationships are as follows: 1) pH (syringe = 0.995 pH (in vivo) + 0.084 and 2) PCO2 (syringe) = 0.873 PCO2 (in vivo) + 0.2. The amount by which the syringe sample underestimated the true PCO2 value increased with the absolute PCO2 value, consistent with the possibility of there being a diffusional loss of CO2 during the transfer of CSF from the syringe to the pH electrode (PCO2 (in vivo)- PCO2 (syringe) = 2.4, 4.9, 7.5, and 10.0 mmHg at in vivo PCO2's of 20, 40, 60, and 80 mmHg). This study indicates that the technique used for sampling CSF is crucial to the expected accuracy of the results and that the number of transfers of CSF during the sampling and measurement procedures should be minimized in order to obtain reliable results. | Cerebrospinal fluid sampling technique and Astrup pH and PCO2 values. The pH and PCO2 values measured by the Astrup technique were compared in cerebrospinal fluid (CSF) obtained using two different sampling techniques: 1) a direct or in vivo technique and 2) the widely accepted syringe sampling technique. In 65 pairs of measurements in 9 dogs it was found that the pH was always overestimated and the PCO2 always underestimated in the syringe sample when compared to the in vivo sample. The equations describing the relationships are as follows: 1) pH (syringe = 0.995 pH (in vivo) + 0.084 and 2) PCO2 (syringe) = 0.873 PCO2 (in vivo) + 0.2. The amount by which the syringe sample underestimated the true PCO2 value increased with the absolute PCO2 value, consistent with the possibility of there being a diffusional loss of CO2 during the transfer of CSF from the syringe to the pH electrode (PCO2 (in vivo)- PCO2 (syringe) = 2.4, 4.9, 7.5, and 10.0 mmHg at in vivo PCO2's of 20, 40, 60, and 80 mmHg). This study indicates that the technique used for sampling CSF is crucial to the expected accuracy of the results and that the number of transfers of CSF during the sampling and measurement procedures should be minimized in order to obtain reliable results. | 2,575 |
pubmed23n0001_1327 | Hypoventilation in ponies after carotid body denervation. | Seven ponies were subjected to carotid body denervation (CD) and two ponies were sham operated (S). Measurement of arterial blood gases and arterial blood and cerebrospinal fluid (CSF) acid-base balance were made prior to and 1,2,4,9, and 17 wks after surgery in unanesthetized animals. Resting ventilation and ventilatory responsiveness to hypoxia and NaCN infusion were assessed prior to and 2,9, and 17 wks after surgery. Alveolar hypoventilation in the CD ponies was marked 1-2 wk after surgery when VE and VA were reduced 40% and 10%, respectively, from control and PaCO2 was 12-15 mmHg above control. However, the effect was not nearly as great 4, 9, and 17 wk after surgery when the PaCO2 stabilized at approximately 6 mmHg above control PaCO2. Arterial blood pH was normal in the hypercapnic CD ponies, but CSF pH remained acid relative to normal throughout the 17-wk period. Changes in ventilatory responsiveness to hypoxia and NaCN tended to parallel changes in resting ventilation. These findings suggest: 1) the carotid bodies are essential in ponies to maintain normal ventilation: 2) in CD ponies peripheral chemosensitivity is partially regained at some unestablished locus; and 3) pH compensating mechanisms in chronically hypercapnic ponies function relatively better in blood than in CSF. | Hypoventilation in ponies after carotid body denervation. Seven ponies were subjected to carotid body denervation (CD) and two ponies were sham operated (S). Measurement of arterial blood gases and arterial blood and cerebrospinal fluid (CSF) acid-base balance were made prior to and 1,2,4,9, and 17 wks after surgery in unanesthetized animals. Resting ventilation and ventilatory responsiveness to hypoxia and NaCN infusion were assessed prior to and 2,9, and 17 wks after surgery. Alveolar hypoventilation in the CD ponies was marked 1-2 wk after surgery when VE and VA were reduced 40% and 10%, respectively, from control and PaCO2 was 12-15 mmHg above control. However, the effect was not nearly as great 4, 9, and 17 wk after surgery when the PaCO2 stabilized at approximately 6 mmHg above control PaCO2. Arterial blood pH was normal in the hypercapnic CD ponies, but CSF pH remained acid relative to normal throughout the 17-wk period. Changes in ventilatory responsiveness to hypoxia and NaCN tended to parallel changes in resting ventilation. These findings suggest: 1) the carotid bodies are essential in ponies to maintain normal ventilation: 2) in CD ponies peripheral chemosensitivity is partially regained at some unestablished locus; and 3) pH compensating mechanisms in chronically hypercapnic ponies function relatively better in blood than in CSF. | 2,576 |
pubmed23n0001_1328 | Total and regional cerebral blood flow during moderate and severe exercise in miniature swine. | To determine the influence of exercise on cerebral blood flow, we ran 14 swine at 3-6 mph and at 0-10% grades on a treadmill for 30 min at moderate and severe levels of exercise. Measuring heart rate, cardiac output, and aortic pressure via implanted probes, we injected 15-mum radiolabeled microspheres via the left atrium before and during exercise. We measured their distribution by gamma spectrometry, determining total cerebral blood flow, regional blood flow, and ratio of flow to gray and white matter. Heart rate, cardiac output, and aortic pressure rose progressively with increasing exercise. Total cerebral flow resembled that reported in humans, i.e., it did not change significantly with exercise. Regional flow distribution also failed to change significantly with exercise. The ratio of gray to white matter flow did not change except to the cerebellum where it rose significantly from resting values at both moderate and severe exercise. Gray matter received more flow than white matter during all three conditions of observation. Cerebral blood flow was remarkably constant during even severe exercise. | Total and regional cerebral blood flow during moderate and severe exercise in miniature swine. To determine the influence of exercise on cerebral blood flow, we ran 14 swine at 3-6 mph and at 0-10% grades on a treadmill for 30 min at moderate and severe levels of exercise. Measuring heart rate, cardiac output, and aortic pressure via implanted probes, we injected 15-mum radiolabeled microspheres via the left atrium before and during exercise. We measured their distribution by gamma spectrometry, determining total cerebral blood flow, regional blood flow, and ratio of flow to gray and white matter. Heart rate, cardiac output, and aortic pressure rose progressively with increasing exercise. Total cerebral flow resembled that reported in humans, i.e., it did not change significantly with exercise. Regional flow distribution also failed to change significantly with exercise. The ratio of gray to white matter flow did not change except to the cerebellum where it rose significantly from resting values at both moderate and severe exercise. Gray matter received more flow than white matter during all three conditions of observation. Cerebral blood flow was remarkably constant during even severe exercise. | 2,577 |
pubmed23n0001_1329 | Method for measuring hepatic uptake of oxygen or other blood-borne substances in situ. | A preparation is described by which hepatic arterial blood flow and portal venous blood flow can be accurately and continuously measured while simultaneously providing a method by which multiple blood samples can be taken from the hepatic artery, portal vein, and hepatic vein without disrupting hepatic hemodynamics or causing hemodilution. By this means hepatic uptake or release of blood-borne substances can be measured in situ and correlated with hemodynamic parameters. In 13 splenectomized cats, oxygen uptake by the denervated liver was 4.5 +/- 0.3 ml . min-1. 100 g-1 of tissue, representing 54% of total oxygen removed by the splanchnic bed. The hepatic hemodynamics determined by this method are similar to those reported by others in vivo and the metabolic state of the liver remained stable for at least 2 h during which an average of 29 blood samples were taken. Advantages of this preparation over other methods of obtaining similar data are discussed. | Method for measuring hepatic uptake of oxygen or other blood-borne substances in situ. A preparation is described by which hepatic arterial blood flow and portal venous blood flow can be accurately and continuously measured while simultaneously providing a method by which multiple blood samples can be taken from the hepatic artery, portal vein, and hepatic vein without disrupting hepatic hemodynamics or causing hemodilution. By this means hepatic uptake or release of blood-borne substances can be measured in situ and correlated with hemodynamic parameters. In 13 splenectomized cats, oxygen uptake by the denervated liver was 4.5 +/- 0.3 ml . min-1. 100 g-1 of tissue, representing 54% of total oxygen removed by the splanchnic bed. The hepatic hemodynamics determined by this method are similar to those reported by others in vivo and the metabolic state of the liver remained stable for at least 2 h during which an average of 29 blood samples were taken. Advantages of this preparation over other methods of obtaining similar data are discussed. | 2,578 |
pubmed23n0001_1330 | Analysis of pesticide residues by chemical derivatization. II. N-methylcarbamates in natural water and soils. | A method for the quantitative determination of several N-methylcarbamates in natural waters and the applicability of the derivative to soil samples using a previously published extraction procedure are described. After extraction of the carbamates from the substrate, the carbamates are hydrolyzed in a 10% methanol-potassium hydroxide solution to form the phenolic hydrolysis products, which are isolated and derivatized with pentafluorobenzyl (PFB) bromide to produce the PFB ether derivatives. The PFB derivatives are cleaned up and fractionated on a silica gel microcolumn and determined by electron capture gas-liquid chromatography (GLC). Eight organophosphate pesticides and 2 phthalate acid esters that hydrolyze to phenols or phthalic acid were evaluated as potential interferences and were found not to interfere with any of the carbamates tested. Quantitative determinations of 0.1 mug carbofuran and 3-ketocarbofuran and 0.5 mug carbaryl, metmercapturon, and Mobam in a 1 L water sample are possible. Propoxur was not determined at levels less than 1 mug/L due to the short GLC retention time of the derivative and interferences from the reagents at the lower levels. | Analysis of pesticide residues by chemical derivatization. II. N-methylcarbamates in natural water and soils. A method for the quantitative determination of several N-methylcarbamates in natural waters and the applicability of the derivative to soil samples using a previously published extraction procedure are described. After extraction of the carbamates from the substrate, the carbamates are hydrolyzed in a 10% methanol-potassium hydroxide solution to form the phenolic hydrolysis products, which are isolated and derivatized with pentafluorobenzyl (PFB) bromide to produce the PFB ether derivatives. The PFB derivatives are cleaned up and fractionated on a silica gel microcolumn and determined by electron capture gas-liquid chromatography (GLC). Eight organophosphate pesticides and 2 phthalate acid esters that hydrolyze to phenols or phthalic acid were evaluated as potential interferences and were found not to interfere with any of the carbamates tested. Quantitative determinations of 0.1 mug carbofuran and 3-ketocarbofuran and 0.5 mug carbaryl, metmercapturon, and Mobam in a 1 L water sample are possible. Propoxur was not determined at levels less than 1 mug/L due to the short GLC retention time of the derivative and interferences from the reagents at the lower levels. | 2,579 |
pubmed23n0001_1331 | Separation of pesticide residues from lipids prior to gas-liquid chromatographic analysis. | Lipids are separated from dieldrin, endrin, and p,p'-DDE residues by saponification in ethanolic sodium hydroxide, acidification with dilute sulfuric acid, and adsorption chromatography on deactivated alumina, using petroleum ether as the eluant. Dieldrin, endrin, and p,p'-DDE are efficiently recovered (95-102%), and p,p'-DDT is converted to p,p'-DDE, which is then recovered with high yield (90-96%). Extremely low lipid carryover (less than 0.3-0.5%) is observed for 0.5-1.0 g samples of chicken fat. | Separation of pesticide residues from lipids prior to gas-liquid chromatographic analysis. Lipids are separated from dieldrin, endrin, and p,p'-DDE residues by saponification in ethanolic sodium hydroxide, acidification with dilute sulfuric acid, and adsorption chromatography on deactivated alumina, using petroleum ether as the eluant. Dieldrin, endrin, and p,p'-DDE are efficiently recovered (95-102%), and p,p'-DDT is converted to p,p'-DDE, which is then recovered with high yield (90-96%). Extremely low lipid carryover (less than 0.3-0.5%) is observed for 0.5-1.0 g samples of chicken fat. | 2,580 |
pubmed23n0001_1332 | Collaborative study of the Food Chemicals Codex method for the determination of the neutralizing value of sodium aluminum phosphate. | Fifteen laboratories participated in a collaborative study to evaluate the Food Chemicals Codex method for the determination of the neutralizing value of sodium aluminum phosphate. The AOAC method for determining the neutralizing value of sodium acid pyrophosphate, sec. 8.010, was also included in the study. The precisions of the Food chemicals Codex method, based on the between-replicate standard deviation and on one collaborator making one determination, are 1.16 and 3.66, respectively. The Food Chemicals Codex method for the determination of the neutralizing value of sodium aluminum phosphate has been adopted as official first action. | Collaborative study of the Food Chemicals Codex method for the determination of the neutralizing value of sodium aluminum phosphate. Fifteen laboratories participated in a collaborative study to evaluate the Food Chemicals Codex method for the determination of the neutralizing value of sodium aluminum phosphate. The AOAC method for determining the neutralizing value of sodium acid pyrophosphate, sec. 8.010, was also included in the study. The precisions of the Food chemicals Codex method, based on the between-replicate standard deviation and on one collaborator making one determination, are 1.16 and 3.66, respectively. The Food Chemicals Codex method for the determination of the neutralizing value of sodium aluminum phosphate has been adopted as official first action. | 2,581 |
pubmed23n0001_1333 | Affinity chromatography of trypsin and related enzymes. I. Preparation and characteristics of an affinity adsorbent containing tryptic peptides from protamine as ligands. | An absorbent for the affinity chromatography of trypsin [EC 3.4.21.4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di- and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was absorbed at relatively low pH (7-4), but was not absorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not absorbed. The absorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful. | Affinity chromatography of trypsin and related enzymes. I. Preparation and characteristics of an affinity adsorbent containing tryptic peptides from protamine as ligands. An absorbent for the affinity chromatography of trypsin [EC 3.4.21.4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di- and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was absorbed at relatively low pH (7-4), but was not absorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not absorbed. The absorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful. | 2,582 |
pubmed23n0001_1334 | Effects on tryptophyl absorption of the ionization of the catalytic carboxyls in hen and turkey lysozymes. | The difference spectra of hen and turkey egg-white lysozymes [EC 3.2.1.17] produced by acidification were measured. The difference spectra of both lysozymes had peaks at 295 and 301 nm which are characteristic of tryptophyl residues. The pH dependence curves of the extinction differences (delta eplision) at 301 nm and 295 nm for hen lysozyme were identical with the corresponding curves for turkey lysozyme. The pH dependence of delta eplision at 301 nm was analyzed assuming that the extinction at 301 nm is due to Trp 108 only, which interacts with the catalytic carboxyls, Glu 35 and Asp 52. The macroscopic pK values of Glu 35 and Asp 52 in both lysozymes thus determined were 6.0 and 3.3, respectively. These values were in excellent agreement with those determined by measuring the pH dependence of the circular dichroic band at 305 nm (Kuramitsu et al. (1974) J. Biochem, 76, 671-683; (1975) ibid. 77, 291-301). The pH dependence of delta eplision at 295 nm could not be completely explained in terms of the electrostatic effects of the catalytic groups on Trp 108. | Effects on tryptophyl absorption of the ionization of the catalytic carboxyls in hen and turkey lysozymes. The difference spectra of hen and turkey egg-white lysozymes [EC 3.2.1.17] produced by acidification were measured. The difference spectra of both lysozymes had peaks at 295 and 301 nm which are characteristic of tryptophyl residues. The pH dependence curves of the extinction differences (delta eplision) at 301 nm and 295 nm for hen lysozyme were identical with the corresponding curves for turkey lysozyme. The pH dependence of delta eplision at 301 nm was analyzed assuming that the extinction at 301 nm is due to Trp 108 only, which interacts with the catalytic carboxyls, Glu 35 and Asp 52. The macroscopic pK values of Glu 35 and Asp 52 in both lysozymes thus determined were 6.0 and 3.3, respectively. These values were in excellent agreement with those determined by measuring the pH dependence of the circular dichroic band at 305 nm (Kuramitsu et al. (1974) J. Biochem, 76, 671-683; (1975) ibid. 77, 291-301). The pH dependence of delta eplision at 295 nm could not be completely explained in terms of the electrostatic effects of the catalytic groups on Trp 108. | 2,583 |
pubmed23n0001_1335 | Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes. | Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme. | Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes. Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme. | 2,584 |
pubmed23n0001_1336 | Studies on the microsomal electron-transport system of anaerobically grown yeast. III. Spectral characterization of cytochrome P-450. | A carbon monoxide-binding pigment which shows an absorption peak at about 450 nm in the reduced carbon monoxide difference spectrum was purified from the microsomal fraction of yeast grown anaerobically. The spectral characteristics of the pigment were practically identical with those of cytochrome P-450 of hepatic microsomes, especially from polycyclic hydrocarbon-induced animals. The pigment was denatured to P-420, and bound with ethyl isocyanide in the reduced state. Although Type I spectral change was not evident, the pigment showed Type II and modified Type II spectral changes upon binding with some organic compounds, as in the case of hepatic cytochrome P-450. These observations clearly indicate that the carbon monoxide-binding pigment of yeast microsomes may be designated as cytochrome P-450 of yeast. | Studies on the microsomal electron-transport system of anaerobically grown yeast. III. Spectral characterization of cytochrome P-450. A carbon monoxide-binding pigment which shows an absorption peak at about 450 nm in the reduced carbon monoxide difference spectrum was purified from the microsomal fraction of yeast grown anaerobically. The spectral characteristics of the pigment were practically identical with those of cytochrome P-450 of hepatic microsomes, especially from polycyclic hydrocarbon-induced animals. The pigment was denatured to P-420, and bound with ethyl isocyanide in the reduced state. Although Type I spectral change was not evident, the pigment showed Type II and modified Type II spectral changes upon binding with some organic compounds, as in the case of hepatic cytochrome P-450. These observations clearly indicate that the carbon monoxide-binding pigment of yeast microsomes may be designated as cytochrome P-450 of yeast. | 2,585 |
pubmed23n0001_1337 | Polarographic studies on ubiquinone-10 and rhodoquinone bound with chromatophores from Rhodospirillum rubrum. | Redox components bound with chromatophores of Rhodospirillum rubrum, and pure samples of ubiquinone-10 and rhodoquinone were studied polarographically at 24 degrees. In a mixture of ethanol and water (4 : 1, v/v) at pH 7, ubiquinone-10 and rhodoquinone had half-wave potentials (E1/2) OF +43 MV and -63 mV, respectively. For both quinones, values of the electron transfer number (n) were 2 , and plots of E1/2 versus pH formed straight lines with slopes of -30 mV/pH in the neutral pH range; thus, values of the proton transfer number (n-a) were estimated to be 1 for both quinones. When bound with chromatophores, ubiquinone-10 and rhodoquinone had E1/2 values of +50 mV (n=2) and -30 mV (n=2), respectively, at pH 7. Values of (n-a) were estimated to be 1 for ubiquinone-10 and 2 for rhodoquinone. A component (POC-170) thought to be one of the active center bacteriochlorophylls (Liac-890) was characterized; it has E1/2 value of -170 mV at pH 7 and its oxidation-reduction is possibly brought about by dehydrogenation-hydrogenation. Conceivably, the oxidation-reduction sites of ubiquinone-10, rhodoquinone and POC-170 partly, if not all, exist on the surface of chromatophore membrane or project outside the membrane, because of their accessibility to the polarographic electrode. | Polarographic studies on ubiquinone-10 and rhodoquinone bound with chromatophores from Rhodospirillum rubrum. Redox components bound with chromatophores of Rhodospirillum rubrum, and pure samples of ubiquinone-10 and rhodoquinone were studied polarographically at 24 degrees. In a mixture of ethanol and water (4 : 1, v/v) at pH 7, ubiquinone-10 and rhodoquinone had half-wave potentials (E1/2) OF +43 MV and -63 mV, respectively. For both quinones, values of the electron transfer number (n) were 2 , and plots of E1/2 versus pH formed straight lines with slopes of -30 mV/pH in the neutral pH range; thus, values of the proton transfer number (n-a) were estimated to be 1 for both quinones. When bound with chromatophores, ubiquinone-10 and rhodoquinone had E1/2 values of +50 mV (n=2) and -30 mV (n=2), respectively, at pH 7. Values of (n-a) were estimated to be 1 for ubiquinone-10 and 2 for rhodoquinone. A component (POC-170) thought to be one of the active center bacteriochlorophylls (Liac-890) was characterized; it has E1/2 value of -170 mV at pH 7 and its oxidation-reduction is possibly brought about by dehydrogenation-hydrogenation. Conceivably, the oxidation-reduction sites of ubiquinone-10, rhodoquinone and POC-170 partly, if not all, exist on the surface of chromatophore membrane or project outside the membrane, because of their accessibility to the polarographic electrode. | 2,586 |
pubmed23n0001_1338 | Effect of metal ions in the culture medium on the stearoyl-coenzyme A desaturase activity of Mycobacterium phlei. | A particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome C reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system. | Effect of metal ions in the culture medium on the stearoyl-coenzyme A desaturase activity of Mycobacterium phlei. A particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome C reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system. | 2,587 |
pubmed23n0001_1339 | Photoaffinity labeling of concanavalin A. Preparation of a concanavalin A derivative with reduced valence. | Concanavalin A (Con A) was labeled with p-azidophenyl alpha-D-mannopyranoside under ultraviolet irradiation and the reaction products were separated by affinity chromatography on Sephadex G-100 at pH 5. One of the Con A derivatives thus obtained was characterized as a monovalent dimer at pH 5 and a divalent tetramer at pH 7 by sedimentation equilibrium and equilibrium dialysis, indicating that this photoaffinity labeling did not alter the quaternary structure of Con A. In agreement with these results, the labeled Con A did not show the capacity to precipitate glycogen at pH 5, but it formed precipitates with glycogen at pH 7. Although its hemagglutinating activity was found to be weaker than that of the native Con A, the dose-response cure of the labeled Con A in the mitogenic stimulation of human peripheral lymphocytes was almost identical to that of the native con A. | Photoaffinity labeling of concanavalin A. Preparation of a concanavalin A derivative with reduced valence. Concanavalin A (Con A) was labeled with p-azidophenyl alpha-D-mannopyranoside under ultraviolet irradiation and the reaction products were separated by affinity chromatography on Sephadex G-100 at pH 5. One of the Con A derivatives thus obtained was characterized as a monovalent dimer at pH 5 and a divalent tetramer at pH 7 by sedimentation equilibrium and equilibrium dialysis, indicating that this photoaffinity labeling did not alter the quaternary structure of Con A. In agreement with these results, the labeled Con A did not show the capacity to precipitate glycogen at pH 5, but it formed precipitates with glycogen at pH 7. Although its hemagglutinating activity was found to be weaker than that of the native Con A, the dose-response cure of the labeled Con A in the mitogenic stimulation of human peripheral lymphocytes was almost identical to that of the native con A. | 2,589 |
pubmed23n0001_1340 | Chemical modification of carboxyl groups of fibrinogen and its effect on the binding of cationic detergent. | Carboxyl groups of native human fibrinogen were modified with glycine methyl ester and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. It seemed likely that the modification occurred stepwise. Approximately 26% of the carboxyl groups of fibrinogen was modified finally. The modified fibrinogen had no interaction with cationic detergent, and did not form any complex with the detergent. In dilute acid, fibrinogen was observed to show only a slight interaction with cationic detergent. It is probable that the exposed and ionized carboxyl groups are essential for the formation of a complex between fibrinogen and cationic detergent. | Chemical modification of carboxyl groups of fibrinogen and its effect on the binding of cationic detergent. Carboxyl groups of native human fibrinogen were modified with glycine methyl ester and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. It seemed likely that the modification occurred stepwise. Approximately 26% of the carboxyl groups of fibrinogen was modified finally. The modified fibrinogen had no interaction with cationic detergent, and did not form any complex with the detergent. In dilute acid, fibrinogen was observed to show only a slight interaction with cationic detergent. It is probable that the exposed and ionized carboxyl groups are essential for the formation of a complex between fibrinogen and cationic detergent. | 2,590 |
pubmed23n0001_1341 | Purification and characterization of proteinase inhibitors from adzuki beans (Phaseolus angularis). | Two proteinase inhibitors, designated as inhibitors I and II, were purified from adzuki beans (Phaseolus angularis) by chromatographies on DEAE- and CM-cellulose, and gel filtration on a Sephadex G-100 column. Each inhibitor shows unique inhibitory activities. Inhibitor I was a powerful inhibitor of trypsin [EC 3.4.21.4], but essentially not of chymotrypsin ]EC 3.4.21.1]. On the other hand, inhibitor II inhibited chymotrypsin more strongly than trypsin. The molecular weights estimated from the enzyme inhibition were 3,750 and 9,700 for inhibitors I and II, respectively, assuming that the inhibitions were stoichiometric and in 1 : 1 molar ratio. The amino acid compositions of both inhibitors closely resemble those of low molecular weight inhibitors of other leguminous seeds: they contain large amounts of half-cystine, aspartic acid and serine, and little or no hydrophobic and aromatic amino acids. Inhibitor I lacks both tyrosine and tryptophan residues. The molecular weights were calculated to be 7,894 and 8,620 for inhibitors I and II, respectively. The reliability of these molecular weights was confirmed by the sedimentation equilibrium and 6 M guanidine gel filtration methods. On comparison with the values obtained from enzyme inhibition, it was concluded that inhibitor I and two trypsin inhibitory sites on the molecule, whereas inhibitor II had one chymotrypsin and one trypsin inhibitory sites on the molecule. | Purification and characterization of proteinase inhibitors from adzuki beans (Phaseolus angularis). Two proteinase inhibitors, designated as inhibitors I and II, were purified from adzuki beans (Phaseolus angularis) by chromatographies on DEAE- and CM-cellulose, and gel filtration on a Sephadex G-100 column. Each inhibitor shows unique inhibitory activities. Inhibitor I was a powerful inhibitor of trypsin [EC 3.4.21.4], but essentially not of chymotrypsin ]EC 3.4.21.1]. On the other hand, inhibitor II inhibited chymotrypsin more strongly than trypsin. The molecular weights estimated from the enzyme inhibition were 3,750 and 9,700 for inhibitors I and II, respectively, assuming that the inhibitions were stoichiometric and in 1 : 1 molar ratio. The amino acid compositions of both inhibitors closely resemble those of low molecular weight inhibitors of other leguminous seeds: they contain large amounts of half-cystine, aspartic acid and serine, and little or no hydrophobic and aromatic amino acids. Inhibitor I lacks both tyrosine and tryptophan residues. The molecular weights were calculated to be 7,894 and 8,620 for inhibitors I and II, respectively. The reliability of these molecular weights was confirmed by the sedimentation equilibrium and 6 M guanidine gel filtration methods. On comparison with the values obtained from enzyme inhibition, it was concluded that inhibitor I and two trypsin inhibitory sites on the molecule, whereas inhibitor II had one chymotrypsin and one trypsin inhibitory sites on the molecule. | 2,591 |
pubmed23n0001_1342 | Mitochondrial malate dehydrogenase of bovine cerebrum. Characterization and mechanisms of inhibition by silver ions. | Attempts were made to characterize mitochondrial malate dehydrogenase [L-malate: NAD+ oxidoreductase, EC 1.1.1.37] (M-MDH) purified from bovine cerebrum and to elucidate the mechanisms responsible for inhibition of the enzymic activity by Ag+. The molecular weights of the native enzyme and its subunits were 54,000-55,000 and 30,000-32,000, respectively. In general, the physiochemical and catalytic properties of bovine cerebral M-MDH was not very different from those of other corresponding mammalian enzymes. Incubation of the enzyme with Ag+ caused the loss of equivalent amounts of sulfhydryls with a parallel decrease of the enzymic activity. When the enzyme was exposed to 2-, 3.5-, and 5-fold molar excesses of Ag+, the enzymic activity showed an initial rapid fall and a subsequent slow restoration to a partially inactivated level (60-70, 45-50, and 15-20% of an untreated control, respectively), while the alpha-helical content of the enzyme fell exponentially with time. A 7-fold molar excess of Ag+ reduced both the enzymic activity and the alpha-helical content to a much greater degree and no restoration of the enzymic activity was observed. The Km values of Ag+-inactivated enzyme for NADH and oxaloacetate were the same as those of the native enzyme. The data suggest that Ag+ could inhibit enzymic activity both by reducing the structural regularity of the enzyme molecule and by attacking sulfhydryl groups necessary for the catalytic activity of bovine cerebral M-MDH. | Mitochondrial malate dehydrogenase of bovine cerebrum. Characterization and mechanisms of inhibition by silver ions. Attempts were made to characterize mitochondrial malate dehydrogenase [L-malate: NAD+ oxidoreductase, EC 1.1.1.37] (M-MDH) purified from bovine cerebrum and to elucidate the mechanisms responsible for inhibition of the enzymic activity by Ag+. The molecular weights of the native enzyme and its subunits were 54,000-55,000 and 30,000-32,000, respectively. In general, the physiochemical and catalytic properties of bovine cerebral M-MDH was not very different from those of other corresponding mammalian enzymes. Incubation of the enzyme with Ag+ caused the loss of equivalent amounts of sulfhydryls with a parallel decrease of the enzymic activity. When the enzyme was exposed to 2-, 3.5-, and 5-fold molar excesses of Ag+, the enzymic activity showed an initial rapid fall and a subsequent slow restoration to a partially inactivated level (60-70, 45-50, and 15-20% of an untreated control, respectively), while the alpha-helical content of the enzyme fell exponentially with time. A 7-fold molar excess of Ag+ reduced both the enzymic activity and the alpha-helical content to a much greater degree and no restoration of the enzymic activity was observed. The Km values of Ag+-inactivated enzyme for NADH and oxaloacetate were the same as those of the native enzyme. The data suggest that Ag+ could inhibit enzymic activity both by reducing the structural regularity of the enzyme molecule and by attacking sulfhydryl groups necessary for the catalytic activity of bovine cerebral M-MDH. | 2,592 |
pubmed23n0001_1343 | Double-headed protease inhibitors from black-eyed peas. II. Structural studies by optical absorption and circular dichroism. | Two new double-headed protease inhibitors from black-eyed peas have amino acid compositions typical of the low molecular weight protease inhibitors from legume seeds. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) contains no tryptophan, 1 tyrosine, and 14 half-cystines out of 83 amino acid residues per monomer. Black-eyed pea trypsin inhibitor (BEPTI) contains no tryptophan, 1 tyrosine, and 14 half-cystines out of 75 residues per monomer. The molar extinctions at 280 nm are 2770 for BEPCI and 3440 for BEPTI. The single tyrosyl residue is very inaccessible to solvent in native BEPCI and BEPTI at neutral pH and titrates anomalously with an apparent pK = 12. Ionization of tyrosine is complete in 13 hours above pH 12. No heterogeneity of the local environment of the tyrosyl residues in different subunits can be detected spectrophotometrically. The large number of cystine residues leads to an intense and complex near-ultraviolet CD spectrum with cystine contributions in the regions of 248 and 280 nm and tyrosine contributions at 233 and 280 nm. An intact disulfide structure is required for appearance of the tyrosyl CD bands. The inhibitors are unusually resistant to denaturation when compared with similar low molecular weight proteins of high disulfide content. All observations are consistent with a far more rigid structure for BEPCI and BEPTI than for a typical protein. | Double-headed protease inhibitors from black-eyed peas. II. Structural studies by optical absorption and circular dichroism. Two new double-headed protease inhibitors from black-eyed peas have amino acid compositions typical of the low molecular weight protease inhibitors from legume seeds. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) contains no tryptophan, 1 tyrosine, and 14 half-cystines out of 83 amino acid residues per monomer. Black-eyed pea trypsin inhibitor (BEPTI) contains no tryptophan, 1 tyrosine, and 14 half-cystines out of 75 residues per monomer. The molar extinctions at 280 nm are 2770 for BEPCI and 3440 for BEPTI. The single tyrosyl residue is very inaccessible to solvent in native BEPCI and BEPTI at neutral pH and titrates anomalously with an apparent pK = 12. Ionization of tyrosine is complete in 13 hours above pH 12. No heterogeneity of the local environment of the tyrosyl residues in different subunits can be detected spectrophotometrically. The large number of cystine residues leads to an intense and complex near-ultraviolet CD spectrum with cystine contributions in the regions of 248 and 280 nm and tyrosine contributions at 233 and 280 nm. An intact disulfide structure is required for appearance of the tyrosyl CD bands. The inhibitors are unusually resistant to denaturation when compared with similar low molecular weight proteins of high disulfide content. All observations are consistent with a far more rigid structure for BEPCI and BEPTI than for a typical protein. | 2,593 |
pubmed23n0001_1344 | Double-headed protease inhibitors from black-eyed peas. III. Subunit interactions of the native and half-site chemically modified proteins. | The chemical modification of two new double-headed-protease inhibitors from black-eyed peas, a trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI) with dansyl chloride was investigated under various conditions. The NH2-terminal serine of both BEPCI and BEPTI, the 4 lysyl residues of BEPCI, and 4 of the 5 lysyl residues of BEPTI, could not be dansylated in the absence of urea. The single tyrosine per subunit of BEPCI and BEPTI was unreactive even in the presence of urea but could be labeled with half-site reactivity by the Celite method. Lysine, NH2-terminal serine, and tyrosine were reactive in fully reduced, carbamidomethylated BEPCI and BEPTI. Gel filtration was used to study the subunit interactions of BEPCI and BEPTI. At pH 8 or pH 3.0 there is a complex set of multiple equilibria with widely differing rates of attainment. We have found evidence for a rapid dimer-tetramer equilibrium, a distinct moderate rate dimer-tetramer equilibrium, a very slow monomer-dimer equilibrium, and postulate slow isomerization of the two forms of dimer and the two forms of tetramer. The monomer-dimer equilibrium is quite unusual in that the dimer is stabilized by chaotropic ions and even slightly by guanidine HC1. In contrast to the complex pattern seen in native BEPCI, the half-site, dansylated BEPCI exists at similar concentration exclusively as a tetramer at neutral pH. | Double-headed protease inhibitors from black-eyed peas. III. Subunit interactions of the native and half-site chemically modified proteins. The chemical modification of two new double-headed-protease inhibitors from black-eyed peas, a trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI) with dansyl chloride was investigated under various conditions. The NH2-terminal serine of both BEPCI and BEPTI, the 4 lysyl residues of BEPCI, and 4 of the 5 lysyl residues of BEPTI, could not be dansylated in the absence of urea. The single tyrosine per subunit of BEPCI and BEPTI was unreactive even in the presence of urea but could be labeled with half-site reactivity by the Celite method. Lysine, NH2-terminal serine, and tyrosine were reactive in fully reduced, carbamidomethylated BEPCI and BEPTI. Gel filtration was used to study the subunit interactions of BEPCI and BEPTI. At pH 8 or pH 3.0 there is a complex set of multiple equilibria with widely differing rates of attainment. We have found evidence for a rapid dimer-tetramer equilibrium, a distinct moderate rate dimer-tetramer equilibrium, a very slow monomer-dimer equilibrium, and postulate slow isomerization of the two forms of dimer and the two forms of tetramer. The monomer-dimer equilibrium is quite unusual in that the dimer is stabilized by chaotropic ions and even slightly by guanidine HC1. In contrast to the complex pattern seen in native BEPCI, the half-site, dansylated BEPCI exists at similar concentration exclusively as a tetramer at neutral pH. | 2,594 |
pubmed23n0001_1345 | Affinity labeling of the primary bilirubin binding site of human serum albumin. | A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site. | Affinity labeling of the primary bilirubin binding site of human serum albumin. A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site. | 2,595 |
pubmed23n0001_1346 | Acylation of subtilisin Carlsberg by phenyl esters. | Approximate Hammett reaction constants rho calculated from k2/K8 values of several phenyl esters of N-acetyl-L-phenylalanine, hippuric acid, and beta-phenylpropionic acid are 0.0, 0.4, and 1.0 respectively. To determine whether the lack of substituent effect of k2/K8 with the N-acetyl-L-phenylalanine esters is a result of substituent-insensitive k2 or rate-limiting association of enzyme and substrate, pH-k2/K8 deependences and solvent deuterium isotope effects were determined for certain of the substrates and compared with those found with the corresponding hippurates and beta-phenylpropionates. In the pH range 5 to 8, k2/K8 of the phenyl and 4-nitrophenyl esters of each series is dependent upon the unprotonated form of an enzymatic base of apparent pKa approximately 7.4, identical with the pKa found for the free enzyme. With the phenyl esters of each substrate class, k2/K8 decreased by 2 to 3 times in deuterium oxide compared with water. The results suggest that a step involving a general base-catalyzed proton transfer, almost certainly k2, is rate-limiting with the N-acetyl-L-phenylalaninates, as well as the hippurates and beta-phenylpropionates. Attack by the protein on the latter substrates is prediminantly nucleophilic, judged by the similarity of rho in the enzymatic and reference hydroxide ion-catalyzed hydrolyses. The power rho values for the N-acetyl-L-phenylalaninates and hippurates could result from an electrophilic component in their hydrolytic mechanisms. | Acylation of subtilisin Carlsberg by phenyl esters. Approximate Hammett reaction constants rho calculated from k2/K8 values of several phenyl esters of N-acetyl-L-phenylalanine, hippuric acid, and beta-phenylpropionic acid are 0.0, 0.4, and 1.0 respectively. To determine whether the lack of substituent effect of k2/K8 with the N-acetyl-L-phenylalanine esters is a result of substituent-insensitive k2 or rate-limiting association of enzyme and substrate, pH-k2/K8 deependences and solvent deuterium isotope effects were determined for certain of the substrates and compared with those found with the corresponding hippurates and beta-phenylpropionates. In the pH range 5 to 8, k2/K8 of the phenyl and 4-nitrophenyl esters of each series is dependent upon the unprotonated form of an enzymatic base of apparent pKa approximately 7.4, identical with the pKa found for the free enzyme. With the phenyl esters of each substrate class, k2/K8 decreased by 2 to 3 times in deuterium oxide compared with water. The results suggest that a step involving a general base-catalyzed proton transfer, almost certainly k2, is rate-limiting with the N-acetyl-L-phenylalaninates, as well as the hippurates and beta-phenylpropionates. Attack by the protein on the latter substrates is prediminantly nucleophilic, judged by the similarity of rho in the enzymatic and reference hydroxide ion-catalyzed hydrolyses. The power rho values for the N-acetyl-L-phenylalaninates and hippurates could result from an electrophilic component in their hydrolytic mechanisms. | 2,596 |
pubmed23n0001_1347 | Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin. | The formation of methemoglobin from oxyhemoglobin in a solution containing photoreduced riboflavin and oxygen was inhibited by superoxide dismutase. The rate of the reaction was pH-dependent in the range of 6.8 to 7.8, increasing as the pH was reduced. Inhibition by superoxide dismutase was enhanced as the EDTA concentration increased and was dependent on enzymatic activity. Under conditions in which superoxide dismutase inhibition was incomplete, catalase inhibited the reaction but mannitol had no effect. The data support the mediation of methemoglobin formation by superoxide. The hypothesis is offered that superoxide anion reduced the heme-bound oxygen in oxygemoglobin by one electron, permitting the subsequent dissociation of ferrihemoglobin and peroxide. The ability of superoxide dismutase to inhibit the formation of methemoglobin may represent one of its functions in the mature erythrocyte. | Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin. The formation of methemoglobin from oxyhemoglobin in a solution containing photoreduced riboflavin and oxygen was inhibited by superoxide dismutase. The rate of the reaction was pH-dependent in the range of 6.8 to 7.8, increasing as the pH was reduced. Inhibition by superoxide dismutase was enhanced as the EDTA concentration increased and was dependent on enzymatic activity. Under conditions in which superoxide dismutase inhibition was incomplete, catalase inhibited the reaction but mannitol had no effect. The data support the mediation of methemoglobin formation by superoxide. The hypothesis is offered that superoxide anion reduced the heme-bound oxygen in oxygemoglobin by one electron, permitting the subsequent dissociation of ferrihemoglobin and peroxide. The ability of superoxide dismutase to inhibit the formation of methemoglobin may represent one of its functions in the mature erythrocyte. | 2,597 |
pubmed23n0001_1348 | Transport and metabolism of vitamin B6 in the yeast Saccharomyces carlsbergensis 4228. | Active transport of pyridoxine, pyridoxal, and pyridoxamine occurs in resting cells of Saccharomyces carlsbergensis 4228 and can lead to intracellular concentrations of free vitamin much higher than those supplied externally. The initial Km for pyridoxine uptake is 3.6 x 10(-7) M at 30 degrees and pH 4.5, which are optimum for growth. Transport is inhibited by many unphosphorylated vitamin analogs, the most effective being 5'-deoxypyridoxine, 5'-deoxypridoxal, toxopyrimidine, 4'-deoxypyridoxine, and 3-amino-3-deoxypyridoxine. Two distinct uptake systems that differ in structural specificity and ionic requirements are present. One, with optimum pH of 3.5, transports pyridoxal effectively, but not pyridoxamine; the other (optimum pH 6.0) transports pyridoxamine effectively, but not pyridoxal. Both systems transport pyridoxine, while neither transports pyridoxal 5'-phosphate. Other properties of these systems are similar, indicating that they share certain elements in common. An initial temperature optimum of 30 degrees is observed for pyrodoxine transport and, at this temperature, an "overshoot" in intracellular vitamin levels, with subsequent decrease to a constant level, occurs with time. It appears that intracellular vitamin, or a derivative, activates the exit mechanism for the vitamin. Exit rates also depend on the resuspension buffer and are increased in the presence of glucose and decreased by azide. Above 30 degrees net uptake of pyridoxine drops initially, then rapidly increases to a second optimum at 50 degrees; the uptake system is inactivated at about 55 degrees. The optimum at 50 degrees apparently results from activation of inflow as exit is rapid and is accelerated by azide. No overshoot was detected at 50 degrees, so it appears that the exit system is not regulated by intracellular vitamin at this temperature. A phase transition in membrane lipids occurs at 30 degrees and may be responsible for the change in properties of the inflow and exit mechanisms above this temperature. | Transport and metabolism of vitamin B6 in the yeast Saccharomyces carlsbergensis 4228. Active transport of pyridoxine, pyridoxal, and pyridoxamine occurs in resting cells of Saccharomyces carlsbergensis 4228 and can lead to intracellular concentrations of free vitamin much higher than those supplied externally. The initial Km for pyridoxine uptake is 3.6 x 10(-7) M at 30 degrees and pH 4.5, which are optimum for growth. Transport is inhibited by many unphosphorylated vitamin analogs, the most effective being 5'-deoxypyridoxine, 5'-deoxypridoxal, toxopyrimidine, 4'-deoxypyridoxine, and 3-amino-3-deoxypyridoxine. Two distinct uptake systems that differ in structural specificity and ionic requirements are present. One, with optimum pH of 3.5, transports pyridoxal effectively, but not pyridoxamine; the other (optimum pH 6.0) transports pyridoxamine effectively, but not pyridoxal. Both systems transport pyridoxine, while neither transports pyridoxal 5'-phosphate. Other properties of these systems are similar, indicating that they share certain elements in common. An initial temperature optimum of 30 degrees is observed for pyrodoxine transport and, at this temperature, an "overshoot" in intracellular vitamin levels, with subsequent decrease to a constant level, occurs with time. It appears that intracellular vitamin, or a derivative, activates the exit mechanism for the vitamin. Exit rates also depend on the resuspension buffer and are increased in the presence of glucose and decreased by azide. Above 30 degrees net uptake of pyridoxine drops initially, then rapidly increases to a second optimum at 50 degrees; the uptake system is inactivated at about 55 degrees. The optimum at 50 degrees apparently results from activation of inflow as exit is rapid and is accelerated by azide. No overshoot was detected at 50 degrees, so it appears that the exit system is not regulated by intracellular vitamin at this temperature. A phase transition in membrane lipids occurs at 30 degrees and may be responsible for the change in properties of the inflow and exit mechanisms above this temperature. | 2,598 |
pubmed23n0001_1349 | Transport and metabolism of vitamin B6 in Salmonella typhimurium LT2. | Salmonella typhimurium LT2 concentrates radioactivity intracellularly from [3H]pyridoxal or [3H]pyridoxine up to 25 times the external concentration. After 1 min of uptake intracellular radioactivity is found as phosphorylated vitamin B6. The process is sensitive to temperature and is maximally active at pH 8.1, but under the conditions tested it is insensitive to monovalent cations or metabolic inhibitors, and does not require an exogenous energy source. The Km values for uptake of pyridoxine and pyridoxal are 2.0 x 10(-7) M and 1.2 x 10(-7) M, respectively; [3H]pyridoxamine is not transported. Evidence is presented for an uptake mechanism involving facilitated diffusion followed by trapping by pyridoxal kinase. S. typhimurium also appears to lack a periplasmic binding protein for vitamin B6. | Transport and metabolism of vitamin B6 in Salmonella typhimurium LT2. Salmonella typhimurium LT2 concentrates radioactivity intracellularly from [3H]pyridoxal or [3H]pyridoxine up to 25 times the external concentration. After 1 min of uptake intracellular radioactivity is found as phosphorylated vitamin B6. The process is sensitive to temperature and is maximally active at pH 8.1, but under the conditions tested it is insensitive to monovalent cations or metabolic inhibitors, and does not require an exogenous energy source. The Km values for uptake of pyridoxine and pyridoxal are 2.0 x 10(-7) M and 1.2 x 10(-7) M, respectively; [3H]pyridoxamine is not transported. Evidence is presented for an uptake mechanism involving facilitated diffusion followed by trapping by pyridoxal kinase. S. typhimurium also appears to lack a periplasmic binding protein for vitamin B6. | 2,599 |
pubmed23n0001_1350 | Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase. | 1. A detailed study of cytochrome c oxidase activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome c concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholiptaining aid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD1 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2. The maximal velocities as low ionic strength increased with pH and were highest above ph 7.5. 3. The presence and properties of the low apparent Km phase of the kinetics were strongly dependent on the nature and concentration of the anions in the medium. The multivalent anions, phosphate, ADP, and ATP, greatly decreased the proportion of this phase and similarly decreased the amount of high affinity cytochrome c-cytochrome oxidase complex formed. The order of effectiveness was ATP greater than ADP greater than P1 and since phosphate binds to cytochrome c more strongly than the nucleotides, it is concluded that the inhibition resulted from anion interaction with the oxidase. 4mat low concentrations bakers' yeast iso-1, bakers' yeast iso-1, horse, and Euglena cytochromes c at high concentrations all attained the same maximal velocity. The different proportions of low apparent Km phase in the kinetic patterns of these cytochromes c correlated with the amounts of high affinity complex formed with purified cytochrome c oxidase. 5. The apparent Km for cytochrome c activity in the succinate-cytochrome c reductase system of Keilin-Hartree particles was identical with that obtained with the oxidase (5 x 10(-8) M), suggesting the same site serves both reactions. 6. It is concluded that the observed kinetics result from two catalytically active sites on the cytochrome c oxidase protein of different affinities for cytochrome c. The high affinity binding of cytochrome c to the mitochondrial membrane is provided by the oxidase and at this site cytochrome c can be reduced by cytochrome c1. Physiological concentrations of ATP decrease the affinity of this binding to the point that interaction of cytochrome c with numerous mitochondrial pholpholipid sites can competitively remove cytochrome c from the oxidase. It is suggested that this effect of ATP represents a possible mechanism for the control of electron flow to the oxidase. | Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase. 1. A detailed study of cytochrome c oxidase activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome c concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholiptaining aid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD1 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2. The maximal velocities as low ionic strength increased with pH and were highest above ph 7.5. 3. The presence and properties of the low apparent Km phase of the kinetics were strongly dependent on the nature and concentration of the anions in the medium. The multivalent anions, phosphate, ADP, and ATP, greatly decreased the proportion of this phase and similarly decreased the amount of high affinity cytochrome c-cytochrome oxidase complex formed. The order of effectiveness was ATP greater than ADP greater than P1 and since phosphate binds to cytochrome c more strongly than the nucleotides, it is concluded that the inhibition resulted from anion interaction with the oxidase. 4mat low concentrations bakers' yeast iso-1, bakers' yeast iso-1, horse, and Euglena cytochromes c at high concentrations all attained the same maximal velocity. The different proportions of low apparent Km phase in the kinetic patterns of these cytochromes c correlated with the amounts of high affinity complex formed with purified cytochrome c oxidase. 5. The apparent Km for cytochrome c activity in the succinate-cytochrome c reductase system of Keilin-Hartree particles was identical with that obtained with the oxidase (5 x 10(-8) M), suggesting the same site serves both reactions. 6. It is concluded that the observed kinetics result from two catalytically active sites on the cytochrome c oxidase protein of different affinities for cytochrome c. The high affinity binding of cytochrome c to the mitochondrial membrane is provided by the oxidase and at this site cytochrome c can be reduced by cytochrome c1. Physiological concentrations of ATP decrease the affinity of this binding to the point that interaction of cytochrome c with numerous mitochondrial pholpholipid sites can competitively remove cytochrome c from the oxidase. It is suggested that this effect of ATP represents a possible mechanism for the control of electron flow to the oxidase. | 2,600 |
pubmed23n0001_1351 | Autooxidation and hydroxylation reactions of oxygenated cytochrome P-450cam. | Oxy-ferrous substrate-bound cytochrome P-450cam (mrsO2) autooxidizes in the absence of its specific effector protein, putidaredoxin, without hydroxylating the substrate, camphor. The autooxidation is first order with an activation energy of 17 kcal mol-1 at 25 degrees, pH 7.0. Substrate removal and low pH accelerate the reaction. The product, 5-exo-OH camphor, and a nonhydroxylated pseudosubstrate, norcamphor, stabilize the complex in a manner similar to camphor. Increased oxidation rate of mrsO2 and substrate hydroxylation are induced by putidaredoxin, rebredoxin, cytochrome b5, and the apoproteins of the latter two. Dihydrolipoic acid and other dithiols also replace putidaredoxin as effector molecules, but 1000-fold higher concentrations are required. Effector molecules do not increase the autooxidation rate of mrsO2 unless camphor, norcamphor, or another pseudosubstrate is present. Kinetic evidence is presented showing that an active complex between mrsO2 and effector is a required intermediate in mixed function oxidation. | Autooxidation and hydroxylation reactions of oxygenated cytochrome P-450cam. Oxy-ferrous substrate-bound cytochrome P-450cam (mrsO2) autooxidizes in the absence of its specific effector protein, putidaredoxin, without hydroxylating the substrate, camphor. The autooxidation is first order with an activation energy of 17 kcal mol-1 at 25 degrees, pH 7.0. Substrate removal and low pH accelerate the reaction. The product, 5-exo-OH camphor, and a nonhydroxylated pseudosubstrate, norcamphor, stabilize the complex in a manner similar to camphor. Increased oxidation rate of mrsO2 and substrate hydroxylation are induced by putidaredoxin, rebredoxin, cytochrome b5, and the apoproteins of the latter two. Dihydrolipoic acid and other dithiols also replace putidaredoxin as effector molecules, but 1000-fold higher concentrations are required. Effector molecules do not increase the autooxidation rate of mrsO2 unless camphor, norcamphor, or another pseudosubstrate is present. Kinetic evidence is presented showing that an active complex between mrsO2 and effector is a required intermediate in mixed function oxidation. | 2,601 |
pubmed23n0001_1352 | Demonstration of a precursor-product relationship between soluble and cross-linked elastin, and the biosynthesis of the desmosines in vitro. | Direct evidence showing that a soluble form of elastin is the precursor of cross-linked elastin was obtained from pulse-chase experiments using chick embryo aortas and by demonstrating the conversion of soluble elastin into cross-linked elastin in a cell-free system. Acetic acid extracts of embryonic chick aorta pulse-labeled with [14C]lysine contain two radioactive proteins of molecular weights 74,000 and 138,000 which have been identified previously as soluble elastin and the pro-alpha chain of collagen, respectively. In pulse-chase experiments, the radioactivity incorporated in the soluble elastin during the pulse with [14C]lysine disappeared during a 24-hour chase with [12C]lysine and 89% of that which disappeared was accounted for in the desmosines of alkali-insoluble elastin. The disappearance of the radioactivity from the soluble fraction and its appearance in the desmosines of elastin were inhibited by beta-aminopropionitrile, a specific inhibitor of the cross-linking enzyme lysyl oxidase. In addition in vitro experiments, it was shown that the radioactivity in the desmosines of elastin can arise from that present in an acid-soluble precursor protein. This precursor protein is soluble elastin, as demonstrated by the formation of desmosines when a homogeneous preparation of soluble elastin was incubated with purified lysyl oxidase. | Demonstration of a precursor-product relationship between soluble and cross-linked elastin, and the biosynthesis of the desmosines in vitro. Direct evidence showing that a soluble form of elastin is the precursor of cross-linked elastin was obtained from pulse-chase experiments using chick embryo aortas and by demonstrating the conversion of soluble elastin into cross-linked elastin in a cell-free system. Acetic acid extracts of embryonic chick aorta pulse-labeled with [14C]lysine contain two radioactive proteins of molecular weights 74,000 and 138,000 which have been identified previously as soluble elastin and the pro-alpha chain of collagen, respectively. In pulse-chase experiments, the radioactivity incorporated in the soluble elastin during the pulse with [14C]lysine disappeared during a 24-hour chase with [12C]lysine and 89% of that which disappeared was accounted for in the desmosines of alkali-insoluble elastin. The disappearance of the radioactivity from the soluble fraction and its appearance in the desmosines of elastin were inhibited by beta-aminopropionitrile, a specific inhibitor of the cross-linking enzyme lysyl oxidase. In addition in vitro experiments, it was shown that the radioactivity in the desmosines of elastin can arise from that present in an acid-soluble precursor protein. This precursor protein is soluble elastin, as demonstrated by the formation of desmosines when a homogeneous preparation of soluble elastin was incubated with purified lysyl oxidase. | 2,602 |
pubmed23n0001_1353 | Chondroitinase C from Flavobacterium heparinum. | A chondroitinase that acts upon chondroitin sulfate C and hyaluronic acid was isolated from Flavobacterium heparinum. This enzyme was seperated from constitutional chondroitinase AC and an induced chondroitinase B also present in extracts of F. heparinum previously grown in the presence of chondroitin sulfates A, B or C. The enzyme acts upon chondroitin sulfate C producing tetrasaccharide plus an unsaturated 6-sulfated disaccharide (delta Di-6S), and upon hyaluronic acid producing unsaturated nonsulfated disaccharide (delta Di-OS). Chondroitin sulfate A is also degraded producing oligosaccharides and delta Di-6S but not delta Di-4S. The chondroitinase C is also distinguished from the chondroitinases B and AC by several properties, such as effect of ions, temperature for optimal activity, and susceptibility to increasing salt concentrations. The substrate specificity of the chondroitinase C is different from that of any other chondroitinase or hyaluronidase described so far. | Chondroitinase C from Flavobacterium heparinum. A chondroitinase that acts upon chondroitin sulfate C and hyaluronic acid was isolated from Flavobacterium heparinum. This enzyme was seperated from constitutional chondroitinase AC and an induced chondroitinase B also present in extracts of F. heparinum previously grown in the presence of chondroitin sulfates A, B or C. The enzyme acts upon chondroitin sulfate C producing tetrasaccharide plus an unsaturated 6-sulfated disaccharide (delta Di-6S), and upon hyaluronic acid producing unsaturated nonsulfated disaccharide (delta Di-OS). Chondroitin sulfate A is also degraded producing oligosaccharides and delta Di-6S but not delta Di-4S. The chondroitinase C is also distinguished from the chondroitinases B and AC by several properties, such as effect of ions, temperature for optimal activity, and susceptibility to increasing salt concentrations. The substrate specificity of the chondroitinase C is different from that of any other chondroitinase or hyaluronidase described so far. | 2,603 |
pubmed23n0001_1354 | Transport mechanisms in isolated plasma membranes. Nucleoside processing by membrane vesicles from mouse fibroblast cells grown in defined medium. | Plasma membrane vesicles were isolated from a subline of L929 mouse fibroblasts grown on defined medium in the absence of serum. These vesicles were not significantly contaminated by mitochondria or endoplasmic reticulum. The isolation procedure, a modification of that originally developed by McKeel and Jarett (McKeel, D.W., and Jarett, L. (1970) J. Cell Biol. 44, 417-432) employs mechanical homogenization in isotonic medium followed by differential centrifugation. The resultant plasma membrane vesicles take up radioactivity when exposed to uniformly labeled nucleosides. Two subfractions of the plasma membrane were isolated, distinguished by their differing activity of 5'-nucleotidase and (Na+,K+)-stimulated ATPase, two well known plasma membrane enzyme markers. Uptake of nucleoside radioactivity was extensively studied in one subfraction; it was linear with time and membrane concentration over ranges used for the studies. Apparent Km values for uptake of radioactivity from adenosine, inosine, and uridine were 7.1 +/- 26 muM, respectively. Uptake of radioactivity from all three nucleosides exhibits a broad pH optimum from pH 7 to pH 9, but falls off rapidly at lower pH. N-Ethylmaleimide was an effective inhibitor of uptake of radioactivity from all three nucleosides; uptake of radioactivity from uridine is more sensitive than uptake of radioactivity from the purine nucleosides. Adenosine inhibited uptake of radioactivity from inosine more than from uridine. Inosine inhibited the uptake of radioactivity from adenosine, but uridine did not. Caffeine and 6-methylaminopurine riboside (6-N-methyladenosine differentially inhibit uptake of radioactivity from adenosine and inosine, and thus the vesicles apparently possess seperate transport systems for uptake of radioactivity from purine nucleosides and from uridine. | Transport mechanisms in isolated plasma membranes. Nucleoside processing by membrane vesicles from mouse fibroblast cells grown in defined medium. Plasma membrane vesicles were isolated from a subline of L929 mouse fibroblasts grown on defined medium in the absence of serum. These vesicles were not significantly contaminated by mitochondria or endoplasmic reticulum. The isolation procedure, a modification of that originally developed by McKeel and Jarett (McKeel, D.W., and Jarett, L. (1970) J. Cell Biol. 44, 417-432) employs mechanical homogenization in isotonic medium followed by differential centrifugation. The resultant plasma membrane vesicles take up radioactivity when exposed to uniformly labeled nucleosides. Two subfractions of the plasma membrane were isolated, distinguished by their differing activity of 5'-nucleotidase and (Na+,K+)-stimulated ATPase, two well known plasma membrane enzyme markers. Uptake of nucleoside radioactivity was extensively studied in one subfraction; it was linear with time and membrane concentration over ranges used for the studies. Apparent Km values for uptake of radioactivity from adenosine, inosine, and uridine were 7.1 +/- 26 muM, respectively. Uptake of radioactivity from all three nucleosides exhibits a broad pH optimum from pH 7 to pH 9, but falls off rapidly at lower pH. N-Ethylmaleimide was an effective inhibitor of uptake of radioactivity from all three nucleosides; uptake of radioactivity from uridine is more sensitive than uptake of radioactivity from the purine nucleosides. Adenosine inhibited uptake of radioactivity from inosine more than from uridine. Inosine inhibited the uptake of radioactivity from adenosine, but uridine did not. Caffeine and 6-methylaminopurine riboside (6-N-methyladenosine differentially inhibit uptake of radioactivity from adenosine and inosine, and thus the vesicles apparently possess seperate transport systems for uptake of radioactivity from purine nucleosides and from uridine. | 2,604 |
pubmed23n0001_1355 | Metallophosphoryl and Apophosphoryl Alkaline Phosphatases. | The noncovalent phosphate (E-P) and covalent phosphory (E-P) complexes of Zn(II), Cd(II), and apoalkaline phosphatases of Escherichia coli have been studied by stopped flow kinetic methods and 32P-labeling techniques. With 2,4-dinitrophenylphosphate as substrate, preincubation of the Zn(II) enzyme with Pi at pH 8 slows the pre-steady state burst rate, but does not affect the burst magnitude of 1 mol of ROH per enzyme dimer. Preincubation of the enzyme with Pi at pH 5.5 reduces the burst magnitude by one-half, as well as reducing the burst rate. Reduction of the burst magnitude as a function of the pH of the preincubation with Pi follows the same function as that previously established for the formation of E-P. Hence, ROP phosphorylates the enzyme by displacing phosphate from E-P during a pre-steady state reaction, while E-P turns over at the steady state velocity. | Metallophosphoryl and Apophosphoryl Alkaline Phosphatases. The noncovalent phosphate (E-P) and covalent phosphory (E-P) complexes of Zn(II), Cd(II), and apoalkaline phosphatases of Escherichia coli have been studied by stopped flow kinetic methods and 32P-labeling techniques. With 2,4-dinitrophenylphosphate as substrate, preincubation of the Zn(II) enzyme with Pi at pH 8 slows the pre-steady state burst rate, but does not affect the burst magnitude of 1 mol of ROH per enzyme dimer. Preincubation of the enzyme with Pi at pH 5.5 reduces the burst magnitude by one-half, as well as reducing the burst rate. Reduction of the burst magnitude as a function of the pH of the preincubation with Pi follows the same function as that previously established for the formation of E-P. Hence, ROP phosphorylates the enzyme by displacing phosphate from E-P during a pre-steady state reaction, while E-P turns over at the steady state velocity. | 2,605 |
pubmed23n0001_1356 | 31P NMR of phosphate and phosphonate complexes of metalloalkaline phosphatases. | 31P NMR spectra of phosphate and phosphonate complexes of Escherichia coli alkaline phosphatase have been obtained by Fourier transform NMR methods. One equivalent of P1i, bound to Zn(II) alkaline phosphatase, pH 8, gives rise to a single 31P resonance 2 ppm downfield from that for Pi, and assignable to the noncovalent complex, E-P. Inorganic phosphate in excess of 1 eq per enzyme dimer gives rise to a resonance at the position expected for free Pi. At pH 5.1, a second resonance appears 8.5 ppm downfield from that for free Pi, and is assignable to the covalent complex, E-P. The large downfield shift suggests that the enzyme phosphoryl group is highly strained with an O-P-O bond angle of under 100 degrees. | 31P NMR of phosphate and phosphonate complexes of metalloalkaline phosphatases. 31P NMR spectra of phosphate and phosphonate complexes of Escherichia coli alkaline phosphatase have been obtained by Fourier transform NMR methods. One equivalent of P1i, bound to Zn(II) alkaline phosphatase, pH 8, gives rise to a single 31P resonance 2 ppm downfield from that for Pi, and assignable to the noncovalent complex, E-P. Inorganic phosphate in excess of 1 eq per enzyme dimer gives rise to a resonance at the position expected for free Pi. At pH 5.1, a second resonance appears 8.5 ppm downfield from that for free Pi, and is assignable to the covalent complex, E-P. The large downfield shift suggests that the enzyme phosphoryl group is highly strained with an O-P-O bond angle of under 100 degrees. | 2,606 |
pubmed23n0001_1357 | Isolation and characterization of indole-3-acetaldehyde reductases from Cucumis sativus. | In a continuing study of the biosynthetic pathway and regulatory mechanisms governing indole-3-acetic acid (auxin) formation, we report the isolation and initial characterization of three distinct indole-3-acetaldehyde reductases from cucumber seedlings. These enzymes catalyze the reduction of indole-3-acetaldehyde to indole-3-ethanol with the concomitant oxidation of NAD(P)H to NAD(P)+. Two of the reductases are specific for NADPH as second substrate, while the third is specific for NADH. The enzymes show a strong specificity for indoleacetaldehyde, with apparent Km values of 73mum, 130mum, and 400mum being calculated for the two NADPH-specific reductases and the NADH-specific reductase, respectively. Under no conditions of substrate concentration, incubation time, or assay method could the reverse reaction be observed. Chromatography on a calibrated Sephadex gel column led to estimated molecualr weights of 52,000 and 17,000 for the NADPH-specific reductases, while a value of 33,000 was obtained for the NADH-specific reductase. Both NADPH-specific reductases showed a pH optimum of 5.2 with a secondary optimum at 7.0, and both enzymes were activated by increasing ionic strength. The NADH-specific reductase showed a pH optimum of 7.0 with a secondary optimum at 6.1 and was slightly inhibited by increasing ionic strength. | Isolation and characterization of indole-3-acetaldehyde reductases from Cucumis sativus. In a continuing study of the biosynthetic pathway and regulatory mechanisms governing indole-3-acetic acid (auxin) formation, we report the isolation and initial characterization of three distinct indole-3-acetaldehyde reductases from cucumber seedlings. These enzymes catalyze the reduction of indole-3-acetaldehyde to indole-3-ethanol with the concomitant oxidation of NAD(P)H to NAD(P)+. Two of the reductases are specific for NADPH as second substrate, while the third is specific for NADH. The enzymes show a strong specificity for indoleacetaldehyde, with apparent Km values of 73mum, 130mum, and 400mum being calculated for the two NADPH-specific reductases and the NADH-specific reductase, respectively. Under no conditions of substrate concentration, incubation time, or assay method could the reverse reaction be observed. Chromatography on a calibrated Sephadex gel column led to estimated molecualr weights of 52,000 and 17,000 for the NADPH-specific reductases, while a value of 33,000 was obtained for the NADH-specific reductase. Both NADPH-specific reductases showed a pH optimum of 5.2 with a secondary optimum at 7.0, and both enzymes were activated by increasing ionic strength. The NADH-specific reductase showed a pH optimum of 7.0 with a secondary optimum at 6.1 and was slightly inhibited by increasing ionic strength. | 2,607 |
pubmed23n0001_1358 | Calcium transport driven by a proton gradient and inverted membrane vesicles of Escherichia coli. | Calcium transport into inverted vesicles of Escherichia coli was observed to occur without an exogenous energy source when an artificial proton gradient was used. The orientation of the proton gradient was acid inside and alkaline outside. Either phosphate or oxalate was necessary for transport, as was found for respiratory-driven or ATP-driven uptake (Tsuchiya, T., and Rosen, B.P. (1975) J. Biol. Chem. 250, 7687-7692). Phosphate accumulation was found to occur in conjunction with calcium accumulation. Calcium transport driven by an artificial proton gradient was stimulated by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ATPase (EC 3.6.1.3). Valinomycin, which catalyzes electrogenic potassium movement, stimulated calcium accumulation, while nigericin, which catalyzes electroneutral exchange of potassium and protons, inhibited both artificial proton gradient-driven transport and respiratory-driven transport. Other properties of the proton gradient-driven system and the previously reported energy-linked calcium transport system are similar, indicating that calcium is transported by the same carrier whether energy is supplied through an artificial proton gradient or an energized membrane state. These results suggest the existence of a calcium/proton antiport. | Calcium transport driven by a proton gradient and inverted membrane vesicles of Escherichia coli. Calcium transport into inverted vesicles of Escherichia coli was observed to occur without an exogenous energy source when an artificial proton gradient was used. The orientation of the proton gradient was acid inside and alkaline outside. Either phosphate or oxalate was necessary for transport, as was found for respiratory-driven or ATP-driven uptake (Tsuchiya, T., and Rosen, B.P. (1975) J. Biol. Chem. 250, 7687-7692). Phosphate accumulation was found to occur in conjunction with calcium accumulation. Calcium transport driven by an artificial proton gradient was stimulated by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ATPase (EC 3.6.1.3). Valinomycin, which catalyzes electrogenic potassium movement, stimulated calcium accumulation, while nigericin, which catalyzes electroneutral exchange of potassium and protons, inhibited both artificial proton gradient-driven transport and respiratory-driven transport. Other properties of the proton gradient-driven system and the previously reported energy-linked calcium transport system are similar, indicating that calcium is transported by the same carrier whether energy is supplied through an artificial proton gradient or an energized membrane state. These results suggest the existence of a calcium/proton antiport. | 2,608 |
pubmed23n0001_1359 | Stoichiometry of H+ ejection during respiration-dependent accumulation of Ca2+ by rat liver mitochondria. | We have investigated the energy-dependent uptake of Ca2+ by rat liver mitochondria with succinate as respiratory substrate with rotenone added to block NAD-linked electron transport. In the presence of 3-hydroxybutyric or other permeant monocarboxylic acids Ca2+ was taken up to extents approaching those seen in the presence of phosphate. The quantitative relationship between cation and anion uptake was determined from the slope of a plot of 3-hydroxybutyrate uptake against Ca2+ uptake, a method which allowed determination of the stoichiometry without requiring ambiguous corrections for early nonenergized or nonstoichiometric binding events. This procedure showed that 2 molecules of 3-hydroxtbutyrate were accumulated with each Ca2+ ion. Under these conditions close to 2 Ca2+ ions and 4 molecules of 3-hydroxybutyrate were accumulated per pair of electrons per energy-conserving site of the respiratory chain. Since 3-hydroxybutyrate must be protonated to pass the membrane as the undissociated free acid, it is concluded that 4 protons were ejected (and subsequently reabsorbed) per pair of electrons per energy-conserving site, in contrast to the value 2.0 postulated by the chemiosmotic hypothesis. | Stoichiometry of H+ ejection during respiration-dependent accumulation of Ca2+ by rat liver mitochondria. We have investigated the energy-dependent uptake of Ca2+ by rat liver mitochondria with succinate as respiratory substrate with rotenone added to block NAD-linked electron transport. In the presence of 3-hydroxybutyric or other permeant monocarboxylic acids Ca2+ was taken up to extents approaching those seen in the presence of phosphate. The quantitative relationship between cation and anion uptake was determined from the slope of a plot of 3-hydroxybutyrate uptake against Ca2+ uptake, a method which allowed determination of the stoichiometry without requiring ambiguous corrections for early nonenergized or nonstoichiometric binding events. This procedure showed that 2 molecules of 3-hydroxtbutyrate were accumulated with each Ca2+ ion. Under these conditions close to 2 Ca2+ ions and 4 molecules of 3-hydroxybutyrate were accumulated per pair of electrons per energy-conserving site of the respiratory chain. Since 3-hydroxybutyrate must be protonated to pass the membrane as the undissociated free acid, it is concluded that 4 protons were ejected (and subsequently reabsorbed) per pair of electrons per energy-conserving site, in contrast to the value 2.0 postulated by the chemiosmotic hypothesis. | 2,609 |
pubmed23n0001_1360 | Identification of albumin as the serum factor essential for the growth of activated human lymphocytes. | Albumin from human, bovine, or rabbit serum supported the growth of concanavalin A-stimulated human thymus-derived lymphocytes equally well. This activity was completely abolished by pepsin digestion. It was shown for bovine serum albumin that the albumin molecule itself, and neither an impurity nor a factor bound to albumin was essential for the growth of lymphocytes. This conclusion was based on observations that the growth-promoting activity could not be removed from albumin, and that the specific activity of albumin remained unaltered after the following procedures: molecular sieving at pH 7.5 at pH 3.0, and in 8 M urea at pH 6.6; ion exchange chromatography at pH 4.3 and in 8 M urea at pH 7.2; isoelectric focusing; charcoal treatment; acetone precipitation; and reduction with 2-mercaptoethanol in the presence of 8 M urea. Dimeric albumin was found to support growth of lymphocytes as well as monomeric albumin, and mercaptalbumin and non-mercaptalbumin were shown to have equal activity. | Identification of albumin as the serum factor essential for the growth of activated human lymphocytes. Albumin from human, bovine, or rabbit serum supported the growth of concanavalin A-stimulated human thymus-derived lymphocytes equally well. This activity was completely abolished by pepsin digestion. It was shown for bovine serum albumin that the albumin molecule itself, and neither an impurity nor a factor bound to albumin was essential for the growth of lymphocytes. This conclusion was based on observations that the growth-promoting activity could not be removed from albumin, and that the specific activity of albumin remained unaltered after the following procedures: molecular sieving at pH 7.5 at pH 3.0, and in 8 M urea at pH 6.6; ion exchange chromatography at pH 4.3 and in 8 M urea at pH 7.2; isoelectric focusing; charcoal treatment; acetone precipitation; and reduction with 2-mercaptoethanol in the presence of 8 M urea. Dimeric albumin was found to support growth of lymphocytes as well as monomeric albumin, and mercaptalbumin and non-mercaptalbumin were shown to have equal activity. | 2,610 |
pubmed23n0001_1361 | The fluorimetric detection of pesticides on aluminium oxide layers. | Several pesticides have been investigated for their fluorigenic properties on acidic and basic aluminium oxide layers. Fluorescence was obtained in several instances and the relative intensities were observed. Fluorescence spectra were recorded for the best fluorescence obtained before and after heat treatment of the chromatogram. The results are compared with those already reported for silica gel layers. | The fluorimetric detection of pesticides on aluminium oxide layers. Several pesticides have been investigated for their fluorigenic properties on acidic and basic aluminium oxide layers. Fluorescence was obtained in several instances and the relative intensities were observed. Fluorescence spectra were recorded for the best fluorescence obtained before and after heat treatment of the chromatogram. The results are compared with those already reported for silica gel layers. | 2,611 |
pubmed23n0001_1362 | Determination of indomethacin in serum and urine by electron-capture gas-liquid chromatography. | A specific and sensitive method for the quantitative determination of indomethacin in serum and urine is described. The drug is extracted at pH 5.0 with 1,2-dichloroethane and a portion of the organic extract is concentrated and made to react with diazoethane in diethyl ether. The ethyl ester derivative is analyzed by electron-capture gas-liquid chromatography, quantitation being achieved by comparison of peak areas for samples and standards, which are prepared in serum or urine and treated in the same manner as the samples. The limit of sensitivity is 50 ng/ml and the relative standard derivation for repeat determinations on the same sample is about 3%. | Determination of indomethacin in serum and urine by electron-capture gas-liquid chromatography. A specific and sensitive method for the quantitative determination of indomethacin in serum and urine is described. The drug is extracted at pH 5.0 with 1,2-dichloroethane and a portion of the organic extract is concentrated and made to react with diazoethane in diethyl ether. The ethyl ester derivative is analyzed by electron-capture gas-liquid chromatography, quantitation being achieved by comparison of peak areas for samples and standards, which are prepared in serum or urine and treated in the same manner as the samples. The limit of sensitivity is 50 ng/ml and the relative standard derivation for repeat determinations on the same sample is about 3%. | 2,612 |
pubmed23n0001_1363 | Separation of some typical Krebs cycle acids by high-speed isotachophoresis. | In order to perform a required separation, an experimental procedure for the selection of suitable operating conditions is suggested and discussed. It is based on measurements of the dependences of the relative effective mobilities of the components under investigation on the pH of the leading electrolyte. The procedure was applied to a set of typical Krebs cycle acids and the values of the relative effective mobilities measured are given in tables and graphs. A pH of 3.8 was selected as the most suitable. At this value, the acids investigated were successfully separated in less than 4 min using 0.011 M hydrochloric acid + beta-alanine as the leading electrolyte. | Separation of some typical Krebs cycle acids by high-speed isotachophoresis. In order to perform a required separation, an experimental procedure for the selection of suitable operating conditions is suggested and discussed. It is based on measurements of the dependences of the relative effective mobilities of the components under investigation on the pH of the leading electrolyte. The procedure was applied to a set of typical Krebs cycle acids and the values of the relative effective mobilities measured are given in tables and graphs. A pH of 3.8 was selected as the most suitable. At this value, the acids investigated were successfully separated in less than 4 min using 0.011 M hydrochloric acid + beta-alanine as the leading electrolyte. | 2,614 |
pubmed23n0001_1364 | Rapid gas chromatographic determination of underivatized theophylline in whole blood. | A rapid gas chromatographic method for the determination of underivatized theophylline in whole blood is described. Theophylline is extracted from acidified blood and chromatographed directly using cyheptamide as an internal standard. Concentrations of the drug down to 2 mug/ml in blood could be determined with recoveries ranging from 90 to 110%. | Rapid gas chromatographic determination of underivatized theophylline in whole blood. A rapid gas chromatographic method for the determination of underivatized theophylline in whole blood is described. Theophylline is extracted from acidified blood and chromatographed directly using cyheptamide as an internal standard. Concentrations of the drug down to 2 mug/ml in blood could be determined with recoveries ranging from 90 to 110%. | 2,615 |
pubmed23n0001_1365 | Plasma androgen responce to hCG stimulation in prepubertal boys with hypospadias and cryptorchidism. | Serum levels of testosterone, androstenedione and dehydroepiandrosterone were measured before and after 5 days of treatment with hCG (2000 IU/d) in 36 prepubertal boys with cryptorchidism and 11 with hypospadias in order to determine whether a defect in androgen synthesis could be a common cause for these disorders. Baseline and stimulated levels of testosterone, androstenedione and dehydroepiandrosterone were similar in patients with unilateral cryptorchidism, monorchism and hypospadias; baseline and stimulated levels of testosterone were lower in boys with bilateral cryptorchidism. Testosterone levels did not correlate with either the anatomical location of the testis in patients with unilateral cryptorchidism or with the site of the urethra in boys with hypospadias. Seven of 36 patients with cryptorchidism had a positive family history of a similar disorder; testosterone levels were similar in patients with and without a family history. It is concluded: 1) in all patients studied, the gonadotropin dependent phase of testosterone production is present; 2) hCG stimulation cannot detect unilateral Leydig cell dysfunction; and 3) in familial cases of cryptorchidism, some factor other than an abnormality in androgen synthesis may be responsible for the hereditary tendency. | Plasma androgen responce to hCG stimulation in prepubertal boys with hypospadias and cryptorchidism. Serum levels of testosterone, androstenedione and dehydroepiandrosterone were measured before and after 5 days of treatment with hCG (2000 IU/d) in 36 prepubertal boys with cryptorchidism and 11 with hypospadias in order to determine whether a defect in androgen synthesis could be a common cause for these disorders. Baseline and stimulated levels of testosterone, androstenedione and dehydroepiandrosterone were similar in patients with unilateral cryptorchidism, monorchism and hypospadias; baseline and stimulated levels of testosterone were lower in boys with bilateral cryptorchidism. Testosterone levels did not correlate with either the anatomical location of the testis in patients with unilateral cryptorchidism or with the site of the urethra in boys with hypospadias. Seven of 36 patients with cryptorchidism had a positive family history of a similar disorder; testosterone levels were similar in patients with and without a family history. It is concluded: 1) in all patients studied, the gonadotropin dependent phase of testosterone production is present; 2) hCG stimulation cannot detect unilateral Leydig cell dysfunction; and 3) in familial cases of cryptorchidism, some factor other than an abnormality in androgen synthesis may be responsible for the hereditary tendency. | 2,616 |
pubmed23n0001_1366 | A rabbit reticulocyte model for the role of hemin-controlled repressor in hypochromic anemias. | Hemin allows maximal protein synthesis in intact rabbit reticulocytes and their cell-free lysate preparations by retarding the formation of a translational repressor (HCR) found in the postribosomal supernate. In order to evaluate the role of HCR in the pathogenesis of hypochromic anemias, HCR was isolated and partially purified from intact rabbit reticulocytes incubated in vitro with either 0.1 mM alpha,alpha-dipyridyl (an iron-chelating agent) or 0.1 M ethanol. Both of these agents inhibit reticulocyte protein synthesis. Hemin (50 muM) protects against the inhibition by both agents. A ferrous iron-transferrin mixture, however, protects only against alpha,alpha-dipyridyl. Both alpha,alpha-dipyridyl and ethanol inhibit heme synthesis before the time that protein synthesis is affected, while neither lowers either ATP or GSH levels. These results indicate that while both agents inhibit heme synthesis, alpha,alpha-dipyridyl does so by inducing iron deficiency while ethanol works at a non-iron-requiring step. When HCR was isolated from intact cells and assayed in the reticulocyte cell-free systems, plus and minus hemin, premature appearance of HCR was found in cells incubated in vitro with alpha,alpha-dipyridyl or ethanol. When hemin was present in the intact cell incubation, the appearance of HCR was retarded. The HCR from alpha,alpha-dipyridyl ethanol-treated cells was partially purified and eluted at the same location on a Sephadex G-200 column (molecular weight approximately 3 x 10(5)) as that from postribosomal supernates incubated minus hemin. In addition rabbits with phenylhydrazine-induced hemolytic anemia were given intravenous ethanol in vivo at a dose of 0.4 ml/kg. This concentration of alcohol resulted in an inhibition of the rate of heme synthesis and protein synthesis as well as an acceleration of HCR formation in reticulocytes. The HCR from these in vivo treated rabbits was isolated, partially purified, and assayed in an identical fashion as the in vitro experiments. These in vivo experiments further support the physiological and pathophysiological role of HCR in reticulocytes. On the basis of these results a model for a role of HCR in some of the hypochromic anemias is proposed. In iron deficiency or chronic disease (where iron is not available to the erythroblast for heme synthesis) HCR appears prematurely and inhibits protein synthesis. When heme synthesis is inhibited by ethanol but there is sufficient intracellular iron, HCR appears prematurely and inhibits protein synthesis, iron accumulates in the erythroblast, and the end result is sideroblastic anemia. | A rabbit reticulocyte model for the role of hemin-controlled repressor in hypochromic anemias. Hemin allows maximal protein synthesis in intact rabbit reticulocytes and their cell-free lysate preparations by retarding the formation of a translational repressor (HCR) found in the postribosomal supernate. In order to evaluate the role of HCR in the pathogenesis of hypochromic anemias, HCR was isolated and partially purified from intact rabbit reticulocytes incubated in vitro with either 0.1 mM alpha,alpha-dipyridyl (an iron-chelating agent) or 0.1 M ethanol. Both of these agents inhibit reticulocyte protein synthesis. Hemin (50 muM) protects against the inhibition by both agents. A ferrous iron-transferrin mixture, however, protects only against alpha,alpha-dipyridyl. Both alpha,alpha-dipyridyl and ethanol inhibit heme synthesis before the time that protein synthesis is affected, while neither lowers either ATP or GSH levels. These results indicate that while both agents inhibit heme synthesis, alpha,alpha-dipyridyl does so by inducing iron deficiency while ethanol works at a non-iron-requiring step. When HCR was isolated from intact cells and assayed in the reticulocyte cell-free systems, plus and minus hemin, premature appearance of HCR was found in cells incubated in vitro with alpha,alpha-dipyridyl or ethanol. When hemin was present in the intact cell incubation, the appearance of HCR was retarded. The HCR from alpha,alpha-dipyridyl ethanol-treated cells was partially purified and eluted at the same location on a Sephadex G-200 column (molecular weight approximately 3 x 10(5)) as that from postribosomal supernates incubated minus hemin. In addition rabbits with phenylhydrazine-induced hemolytic anemia were given intravenous ethanol in vivo at a dose of 0.4 ml/kg. This concentration of alcohol resulted in an inhibition of the rate of heme synthesis and protein synthesis as well as an acceleration of HCR formation in reticulocytes. The HCR from these in vivo treated rabbits was isolated, partially purified, and assayed in an identical fashion as the in vitro experiments. These in vivo experiments further support the physiological and pathophysiological role of HCR in reticulocytes. On the basis of these results a model for a role of HCR in some of the hypochromic anemias is proposed. In iron deficiency or chronic disease (where iron is not available to the erythroblast for heme synthesis) HCR appears prematurely and inhibits protein synthesis. When heme synthesis is inhibited by ethanol but there is sufficient intracellular iron, HCR appears prematurely and inhibits protein synthesis, iron accumulates in the erythroblast, and the end result is sideroblastic anemia. | 2,617 |
pubmed23n0001_1367 | Arylsulfatase B of human lung. Isolation, characterization, and interaction with slow-reacting substance of anaphylaxis. | Arylsulfatase B was separated from arylsulfatase A in extracts of human lung tissue by anion exchange chromatography and further purified by gel filtration and cation exchange chromatography. Arylsulfatase B of human lung was similar to that enzyme in other tissues and species, exhibiting an apparent mol wt of approximately 60,000, a pH optimum for cleavage of 4-nitrocatechol sulfate (pNCS) of 5.5-6.0, and a sensitivity to inhibition by phosphate ions and especially pyrophosphate in the presence of NaCl. Human lung arylsulfatase B inactivated slow-reacting substance of anaphylaxix (SRS-A) in a linear time-dependent reaction in which the rate was determined by the enzyme-to-substrate ratio. Cleavage of pNCS by human lung arylsulfatase B was competitively suppressed by SRS-A. The finding that human lung tissue contains predominately arylsulfatase B discloses a potential regulatory mechanism for inactivation of SRS-A at or near the site of its generation. | Arylsulfatase B of human lung. Isolation, characterization, and interaction with slow-reacting substance of anaphylaxis. Arylsulfatase B was separated from arylsulfatase A in extracts of human lung tissue by anion exchange chromatography and further purified by gel filtration and cation exchange chromatography. Arylsulfatase B of human lung was similar to that enzyme in other tissues and species, exhibiting an apparent mol wt of approximately 60,000, a pH optimum for cleavage of 4-nitrocatechol sulfate (pNCS) of 5.5-6.0, and a sensitivity to inhibition by phosphate ions and especially pyrophosphate in the presence of NaCl. Human lung arylsulfatase B inactivated slow-reacting substance of anaphylaxix (SRS-A) in a linear time-dependent reaction in which the rate was determined by the enzyme-to-substrate ratio. Cleavage of pNCS by human lung arylsulfatase B was competitively suppressed by SRS-A. The finding that human lung tissue contains predominately arylsulfatase B discloses a potential regulatory mechanism for inactivation of SRS-A at or near the site of its generation. | 2,618 |
pubmed23n0001_1368 | Credibility ratings for desensitization and pseudotherapy among moderately and mildly snake-avoidant college students. | Twenty moderately avoidant and 20 mildly avoidant Ss were chosen as though they were to participate in a snake-avoidance desensitization experiment. After relaxation training, Ss listened to a taped excerpt of either desensitization or pseudotherapy and responded to a 5-item questionnaire that assessed the credibility of the treatment. Mildly avoidant Ss rated desensitization as more credible than pseudotherapy. The implication of this finding is that a great many experimental studies of systematic desensitization have failed to control satisfactorily for the operation of expectancy effects and nonspecific forms of behavioral influence. | Credibility ratings for desensitization and pseudotherapy among moderately and mildly snake-avoidant college students. Twenty moderately avoidant and 20 mildly avoidant Ss were chosen as though they were to participate in a snake-avoidance desensitization experiment. After relaxation training, Ss listened to a taped excerpt of either desensitization or pseudotherapy and responded to a 5-item questionnaire that assessed the credibility of the treatment. Mildly avoidant Ss rated desensitization as more credible than pseudotherapy. The implication of this finding is that a great many experimental studies of systematic desensitization have failed to control satisfactorily for the operation of expectancy effects and nonspecific forms of behavioral influence. | 2,619 |
pubmed23n0001_1369 | A comparison of female and male neurotic depressives. | Female and male neurotic depressives in an outpatient psychiatric clinic were compared on measures of subjective distress, interpersonal concerns, and types of treatment given. There were essentially no differences between the two groups on the patient self-report measures. However, female patients were found to have significantly more therapy sessions that the male neurotic depressives. Also, the female patients were more likely than the male patients to receive psychotropic medication, especially the more potent antidepressant variety. | A comparison of female and male neurotic depressives. Female and male neurotic depressives in an outpatient psychiatric clinic were compared on measures of subjective distress, interpersonal concerns, and types of treatment given. There were essentially no differences between the two groups on the patient self-report measures. However, female patients were found to have significantly more therapy sessions that the male neurotic depressives. Also, the female patients were more likely than the male patients to receive psychotropic medication, especially the more potent antidepressant variety. | 2,620 |
pubmed23n0001_1370 | Counter-current immunoelectrophoresis for the diagnosis of pneumococcal chest infection. | Counter-current immunoelectrohporesis is a rapid sensitive method for detecting pneumococcal capsular antigens in sputum. A result can be obtained within 45 minutes. The optimum conditions for performing the test are given. Counter-current immunoelectrophoresis works with all the 33 pneumococcal antigens tested except type 14. Better precipitin lines are obtained when the gel-support is acid (pH 6-6) than when it is alkaline (pH 8-6). Omniserum is as effective as group-specific sera for the identification of pneumococcal capsular antigens. The findings suggest that pneumococcal infection was present in 44% of 300 unselected suspected chest infections. Culture for pneumococci was positive in only 15% of these cases. The clinical importance of these findings is still being studied but our experience has shown that patients with chest infections should have effective antipneumococcal antibiotics as part of their regimen and that the laboratory diagnosis may be made quickly and accurately by counter-current immunoelectrophoresis. | Counter-current immunoelectrophoresis for the diagnosis of pneumococcal chest infection. Counter-current immunoelectrohporesis is a rapid sensitive method for detecting pneumococcal capsular antigens in sputum. A result can be obtained within 45 minutes. The optimum conditions for performing the test are given. Counter-current immunoelectrophoresis works with all the 33 pneumococcal antigens tested except type 14. Better precipitin lines are obtained when the gel-support is acid (pH 6-6) than when it is alkaline (pH 8-6). Omniserum is as effective as group-specific sera for the identification of pneumococcal capsular antigens. The findings suggest that pneumococcal infection was present in 44% of 300 unselected suspected chest infections. Culture for pneumococci was positive in only 15% of these cases. The clinical importance of these findings is still being studied but our experience has shown that patients with chest infections should have effective antipneumococcal antibiotics as part of their regimen and that the laboratory diagnosis may be made quickly and accurately by counter-current immunoelectrophoresis. | 2,621 |
pubmed23n0001_1371 | Use of anaerobic culture for the improved isolation of Streptococcus pneumoniae. | The following report compares the results of aerobic and anaerobic cultivation for the primary isolation of Streptococcus pneumoniae (the pneumococcus) from the respiratory tract of children. Of 414 specimens of respiratory tract secretions cultured, 65 (15-7%) yielded pneumococci; 31 (47-7%) grew both aerobically and anaerobically, but 34 (52-3%) strains were isolated only from the anaerobic culture. Pneumococci cultured anaerobically with added carbon dioxide characteristically produce large mucoid colonies which are more easily distinguished than the 'normal' colonies commonly seen in the mixed flora isolated from respiratory sites. This advantage justifies the inclusion of anaerobic culture when attempting to isolate Str. pneumoniae from clinical material. | Use of anaerobic culture for the improved isolation of Streptococcus pneumoniae. The following report compares the results of aerobic and anaerobic cultivation for the primary isolation of Streptococcus pneumoniae (the pneumococcus) from the respiratory tract of children. Of 414 specimens of respiratory tract secretions cultured, 65 (15-7%) yielded pneumococci; 31 (47-7%) grew both aerobically and anaerobically, but 34 (52-3%) strains were isolated only from the anaerobic culture. Pneumococci cultured anaerobically with added carbon dioxide characteristically produce large mucoid colonies which are more easily distinguished than the 'normal' colonies commonly seen in the mixed flora isolated from respiratory sites. This advantage justifies the inclusion of anaerobic culture when attempting to isolate Str. pneumoniae from clinical material. | 2,622 |
pubmed23n0001_1372 | Factors affecting the assay of gentamicin by the plate diffusion method. | Standard curves were prepared by plotting log gentamicin concentration against zone diameter using a conventional plate diffusion method. Results were obtained at varying concentrations of sodium chloride and at different pHs. Under optimum conditions the range in zone diameters was markedly increased, thus considerably improving the potential accuracy of the plate assay method. | Factors affecting the assay of gentamicin by the plate diffusion method. Standard curves were prepared by plotting log gentamicin concentration against zone diameter using a conventional plate diffusion method. Results were obtained at varying concentrations of sodium chloride and at different pHs. Under optimum conditions the range in zone diameters was markedly increased, thus considerably improving the potential accuracy of the plate assay method. | 2,623 |
pubmed23n0001_1373 | An optimized semi-automatic rate method for serum glutathione reductase activity and its application to patients with malignant disease. | An improved and optimized method for serum glutathione reductase is described. The reference range for normal subjects is 47-79 IU/1. The method is more sensitive than conventional enzyme tests in the detection of malignant disease. It was not raised more frequently in patients with clinical evidence of metastases than in those clinically free of such metastases, and it did not seem to correlate with prognosis among those patients who failed to survive six months from the time the analysis was first conducted. | An optimized semi-automatic rate method for serum glutathione reductase activity and its application to patients with malignant disease. An improved and optimized method for serum glutathione reductase is described. The reference range for normal subjects is 47-79 IU/1. The method is more sensitive than conventional enzyme tests in the detection of malignant disease. It was not raised more frequently in patients with clinical evidence of metastases than in those clinically free of such metastases, and it did not seem to correlate with prognosis among those patients who failed to survive six months from the time the analysis was first conducted. | 2,624 |
pubmed23n0001_1374 | Chemical modulation of agonistic display in Betta splendens. | Mirror-induced aggressive display of male Siamese fighting fish was reduced by water-borne traces of (a) a pair of male combatants, (b) a nondisplaying fish of either sex, and (c) bits of torn cadual fin. These findings may indicate a mechanism whereby males tolerate the presence of conspecifics to permit nonagressive social interactions. | Chemical modulation of agonistic display in Betta splendens. Mirror-induced aggressive display of male Siamese fighting fish was reduced by water-borne traces of (a) a pair of male combatants, (b) a nondisplaying fish of either sex, and (c) bits of torn cadual fin. These findings may indicate a mechanism whereby males tolerate the presence of conspecifics to permit nonagressive social interactions. | 2,626 |
pubmed23n0001_1375 | Qualitative and quantitative changes in carbohydrates during the manufacture of yogurt. | The average lactose content of yogurt mix was 8.50% and decreased during fermentation to 5.75%. The initial galactose content of the mix was a trace but increased to 1.20% during fermentation. Glucose content remained a trace throughout fermentation. Several brands of commercial yogurt were purchased from local supermarkets and analyzed for carbohydrate content. Lactose ranged from 3.31 to 4.74%, galactose varied from 1.48 to 2.50%, and glucose was only a trace in all samples. Several samples of buttermilk also exhibited the near absence of glucose. | Qualitative and quantitative changes in carbohydrates during the manufacture of yogurt. The average lactose content of yogurt mix was 8.50% and decreased during fermentation to 5.75%. The initial galactose content of the mix was a trace but increased to 1.20% during fermentation. Glucose content remained a trace throughout fermentation. Several brands of commercial yogurt were purchased from local supermarkets and analyzed for carbohydrate content. Lactose ranged from 3.31 to 4.74%, galactose varied from 1.48 to 2.50%, and glucose was only a trace in all samples. Several samples of buttermilk also exhibited the near absence of glucose. | 2,628 |
pubmed23n0001_1376 | Effects of adrenalectomy and glucocorticoid therapy on enzyme activities in mammary and adipose tissues from lactating rats. | Several mammary and adipose enzymes were measured in normal, adrenal-ectomized, adrenalectomized cortisol-treated, and intake-restricted lactating rats. Acetyl-CoA carboxylase, lipoprotein lipase, and triglyceride synthetase complex activities in mammary tissue were unchanged by intake restriction, decreased by adrenalectomy, and increased by glucocorticoid-replacement therapy. Malic dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and lipoprotein lipase activities in adipose were unchanged after adrenalectomy. | Effects of adrenalectomy and glucocorticoid therapy on enzyme activities in mammary and adipose tissues from lactating rats. Several mammary and adipose enzymes were measured in normal, adrenal-ectomized, adrenalectomized cortisol-treated, and intake-restricted lactating rats. Acetyl-CoA carboxylase, lipoprotein lipase, and triglyceride synthetase complex activities in mammary tissue were unchanged by intake restriction, decreased by adrenalectomy, and increased by glucocorticoid-replacement therapy. Malic dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and lipoprotein lipase activities in adipose were unchanged after adrenalectomy. | 2,627 |
pubmed23n0001_1377 | B-complex vitamins in cultured and acidified yogurt. | Studies were to determine the effect of various factors upon B-vitamin content of cultured yogurt and to compare the B-vitamin contents of cultured and direct acidified yogurt. Incubation of yogurt culture at 42 C for 3 h yielded maximum vitamin synthesis concurrent with optimal flavor and texture qualities. A method was standardized for the manufacture of direct acidified yogurt involving the use of Stabilac acidulant and nonfat dry milk, Carboxymethyl cellulose, gelatin, and Starite. Acidified yogurt showed a slightly higher content of certain B-vitamins than the cultured yogurt due to the contribution made by various food additives. Both cultured and acidified yogurt showed good keeping quality and freedom from microbial contaminants during storage at 5 C for 16 days. However, folic acid and vitamin B12 contents decreased 29 and 60% in cultured yogurt and 48 and 54% in acidified yogurt. | B-complex vitamins in cultured and acidified yogurt. Studies were to determine the effect of various factors upon B-vitamin content of cultured yogurt and to compare the B-vitamin contents of cultured and direct acidified yogurt. Incubation of yogurt culture at 42 C for 3 h yielded maximum vitamin synthesis concurrent with optimal flavor and texture qualities. A method was standardized for the manufacture of direct acidified yogurt involving the use of Stabilac acidulant and nonfat dry milk, Carboxymethyl cellulose, gelatin, and Starite. Acidified yogurt showed a slightly higher content of certain B-vitamins than the cultured yogurt due to the contribution made by various food additives. Both cultured and acidified yogurt showed good keeping quality and freedom from microbial contaminants during storage at 5 C for 16 days. However, folic acid and vitamin B12 contents decreased 29 and 60% in cultured yogurt and 48 and 54% in acidified yogurt. | 2,629 |
pubmed23n0001_1378 | Microorganisms and characteristics of laban. | Laban had a titratable acidity of about 1.0%, a pH of 4.25, an ethanol content of 1.25%, and contained 4.2 mug acetaldehyde and 34 mug acetoin/ml. There was no diacetyl. Five microorganisms, classified as Streptococcus thermophilus, Lactobacillus acidophilus, Leuconostoc lactis, Kluyveromyces fragilis, and Saccharomyces cerevisiae, were responsible for the fermentation. Streptococcus thermophilus and L. acidophilus were responsible for acid production with S. thermophilus producing acid more rapidly. Most of the acetaldehyde was produced by K. fragilis, little ethanol was found in absence of S. cerevisiae, and the acetoin was producted by S. thermophilus. | Microorganisms and characteristics of laban. Laban had a titratable acidity of about 1.0%, a pH of 4.25, an ethanol content of 1.25%, and contained 4.2 mug acetaldehyde and 34 mug acetoin/ml. There was no diacetyl. Five microorganisms, classified as Streptococcus thermophilus, Lactobacillus acidophilus, Leuconostoc lactis, Kluyveromyces fragilis, and Saccharomyces cerevisiae, were responsible for the fermentation. Streptococcus thermophilus and L. acidophilus were responsible for acid production with S. thermophilus producing acid more rapidly. Most of the acetaldehyde was produced by K. fragilis, little ethanol was found in absence of S. cerevisiae, and the acetoin was producted by S. thermophilus. | 2,630 |
pubmed23n0001_1379 | A mechanism for tobacco smoke-induced allergy. | Normal CFW mice, when exposed to tobacco smoke, showed a significantly increased susceptibility to the lethal effects of histamine. The LD50 for mice subjected to smoke was 45 mg/kg of histamine, whereas in normal CFW mice the LD50 was 1,100 mg/kg. The histamine susceptibility of smoked mice was markedly diminished by injecting the animals with isoproterenol. Normal CFW mice, as well as sham control mice, exhibited an epinephrine-induced hyperglycemia, whereas the blood glucose values for smoked mice given epinephrine were essentially the same as those for sham mice given only saline. This observation indicates that tobacco smoke may contain a component which causes an autonomic imbalance, hence rendering the mice more susceptible to histamine. This tobacco smoke-induced allergy is probably related to a blockade of adrenergic receptors and not to an immunologic phenomenon. | A mechanism for tobacco smoke-induced allergy. Normal CFW mice, when exposed to tobacco smoke, showed a significantly increased susceptibility to the lethal effects of histamine. The LD50 for mice subjected to smoke was 45 mg/kg of histamine, whereas in normal CFW mice the LD50 was 1,100 mg/kg. The histamine susceptibility of smoked mice was markedly diminished by injecting the animals with isoproterenol. Normal CFW mice, as well as sham control mice, exhibited an epinephrine-induced hyperglycemia, whereas the blood glucose values for smoked mice given epinephrine were essentially the same as those for sham mice given only saline. This observation indicates that tobacco smoke may contain a component which causes an autonomic imbalance, hence rendering the mice more susceptible to histamine. This tobacco smoke-induced allergy is probably related to a blockade of adrenergic receptors and not to an immunologic phenomenon. | 2,631 |
pubmed23n0001_1380 | Regulation of the antibody response to type III pneumococcal polysaccharide. V. Ontogeny of factors influencing the magnitude of the plaque-forming cell response. | Mice of different ages were evaluated with respect to their ability to give a plaque-forming cell (PFC) response to Type III pneumococcal polysaccharide (SSSIII), as well as the degree of amplifier and suppressor thymus-derived(T) cell activity present. Although the magnitude of the PFC response to an optimally immunogenic dose of SSS-III for 2-and 3-week old mice was only 7% and 14%, respectively, of that produced by adult (8-week old) mice, values comparable to those of adult animals were attained by 4 weeks of age; no significant changes in the ability to respond to SSS-III occurred thereafter. Amplifier T cell activity, which was minimal at 2 to 4 weeks of age, matured slowly and did not reach a maximum until 8 to 10 weeks of age. By contrast, suppressor T cell activity appeared to be fully developed at least as early as 2 weeks of age; here, the inhibitory effects produced could by abrogated by depletion of T cells, indicating that the unresponsiveneness induced by such cells does not result in the depletion ot irreversible inactivation of B cells capable of responding to SSS-III. These findings suggest that the inhibitory effects of suppressor T cells are predominant in young mice and that such cells may play an important role in determining the ease with which unresponsiveness is induced in neonates, and in the prevention of autoimmune disease. Also, studies conducted with adult-thymectomized mice showed that both amplifier and suppressor T cells, once seeded to the periphery, are stable and do not depend upon the presence of intact thymus for the expression or renewal of their activity. | Regulation of the antibody response to type III pneumococcal polysaccharide. V. Ontogeny of factors influencing the magnitude of the plaque-forming cell response. Mice of different ages were evaluated with respect to their ability to give a plaque-forming cell (PFC) response to Type III pneumococcal polysaccharide (SSSIII), as well as the degree of amplifier and suppressor thymus-derived(T) cell activity present. Although the magnitude of the PFC response to an optimally immunogenic dose of SSS-III for 2-and 3-week old mice was only 7% and 14%, respectively, of that produced by adult (8-week old) mice, values comparable to those of adult animals were attained by 4 weeks of age; no significant changes in the ability to respond to SSS-III occurred thereafter. Amplifier T cell activity, which was minimal at 2 to 4 weeks of age, matured slowly and did not reach a maximum until 8 to 10 weeks of age. By contrast, suppressor T cell activity appeared to be fully developed at least as early as 2 weeks of age; here, the inhibitory effects produced could by abrogated by depletion of T cells, indicating that the unresponsiveneness induced by such cells does not result in the depletion ot irreversible inactivation of B cells capable of responding to SSS-III. These findings suggest that the inhibitory effects of suppressor T cells are predominant in young mice and that such cells may play an important role in determining the ease with which unresponsiveness is induced in neonates, and in the prevention of autoimmune disease. Also, studies conducted with adult-thymectomized mice showed that both amplifier and suppressor T cells, once seeded to the periphery, are stable and do not depend upon the presence of intact thymus for the expression or renewal of their activity. | 2,633 |
pubmed23n0001_1381 | The role of the capsular polysaccharide in the activation of the alternative pathway by the pneumococcus. | Previous studies have shown that when pneumococci are incubated in normal, nonimmune serum, they activate the alternative pathway and opsonically active C3b is fixed to the surface of the organism. Other studies have demonstrated that C3-dependent opsonization via the alternative pathway plays a significant role in the nonimmune host's defense against the pneumococcus. The present studies concern the role of the capsular polysaccharide in initiating the activation of the alternative pathway by the pneumococcus. Some pneumococcal capsular polysaccharide types, but not all, are able to activate the alternative pathway. Soluble purified capsular polysaccharide types 1, 4 and 25 activate the alternative pathway, whereas types 2, 3, 14, and 19 do not. Since the capsular polysaccharides exist in their native form attached to the pneumococcal surface, selected capsular polysaccharides were also tested for their ability to activate the alternative pathway when attached to a particulate carrier, sheep erythrocytes. Capsular polysaccharide types 2 and 3 failed to activate the alternative pathway when attached to sheep erythrocytes, paralleling the results obtained when these capsular polysaccharides were in solution. In contrast, the type 25 capsular polysaccharide not only activated the alternative pathway when attached to sheep erythrocytes, as it had when in solution, but it also initiated alternative pathway-mediated lysis of the erythrocytes. The capsular polysaccharide is not required for the activation of the alternative pathway by the pneumococcus. Although all types of encapsulated pneumococci are able to activate the alternative pathway, not all the purified capsular polysaccharide types are able to do so. In addition, a nonencapsulated pneumococcus, derived originally from a type 2 organism, activates the alternative pathway as well as a fully encapsulated type 2 pneumococcus. | The role of the capsular polysaccharide in the activation of the alternative pathway by the pneumococcus. Previous studies have shown that when pneumococci are incubated in normal, nonimmune serum, they activate the alternative pathway and opsonically active C3b is fixed to the surface of the organism. Other studies have demonstrated that C3-dependent opsonization via the alternative pathway plays a significant role in the nonimmune host's defense against the pneumococcus. The present studies concern the role of the capsular polysaccharide in initiating the activation of the alternative pathway by the pneumococcus. Some pneumococcal capsular polysaccharide types, but not all, are able to activate the alternative pathway. Soluble purified capsular polysaccharide types 1, 4 and 25 activate the alternative pathway, whereas types 2, 3, 14, and 19 do not. Since the capsular polysaccharides exist in their native form attached to the pneumococcal surface, selected capsular polysaccharides were also tested for their ability to activate the alternative pathway when attached to a particulate carrier, sheep erythrocytes. Capsular polysaccharide types 2 and 3 failed to activate the alternative pathway when attached to sheep erythrocytes, paralleling the results obtained when these capsular polysaccharides were in solution. In contrast, the type 25 capsular polysaccharide not only activated the alternative pathway when attached to sheep erythrocytes, as it had when in solution, but it also initiated alternative pathway-mediated lysis of the erythrocytes. The capsular polysaccharide is not required for the activation of the alternative pathway by the pneumococcus. Although all types of encapsulated pneumococci are able to activate the alternative pathway, not all the purified capsular polysaccharide types are able to do so. In addition, a nonencapsulated pneumococcus, derived originally from a type 2 organism, activates the alternative pathway as well as a fully encapsulated type 2 pneumococcus. | 2,634 |
pubmed23n0001_1382 | The effects of age on the immune response to type III pneumococcal polysaccharide (SIII) and bacterial lipopolysaccharide (LPS) in BALB/c, SJL/J, and C3H mice. | Type III pneumococcal polysaccharide (SIII) and bacterial lipopolysaccharide (LPS) were used to evaluate B cell and T cell regulatory functions in BALB/c, SJL/J, and C3H mice of various ages. It was found that the BALB/c and C3H mice could mount high level plaque-forming cell (PFC) responses to SIII at various ages through 110 weeks whereas the levels of the SJL/J PFC responses had begun to decline by the age of 42 weeks through the age of 80 weeks. BALB/c mice were also capable of producing strong PFC responses to LPS at various ages through 110 weeks whereas the comparable SJL/J PFC responses to LPS had declined by 80 weeks of age. By using anti-lymphocyte serum (ALS) and low-dose paralysis to SIII, it was shown that suppressor T cell activity was apparently greater in young BALB/c mice than in older BALB/c mice. It was also found that paralysis to SIII in BALB/c mice was easier to achieve at an early age. SJL/J mice were found to have the necessary B cell activity to respond to SIII through 80 weeks of age and the PFC responses could be greatly enhanced by ALS. Implications of the roles of regulatory T cells in aging are discussed. | The effects of age on the immune response to type III pneumococcal polysaccharide (SIII) and bacterial lipopolysaccharide (LPS) in BALB/c, SJL/J, and C3H mice. Type III pneumococcal polysaccharide (SIII) and bacterial lipopolysaccharide (LPS) were used to evaluate B cell and T cell regulatory functions in BALB/c, SJL/J, and C3H mice of various ages. It was found that the BALB/c and C3H mice could mount high level plaque-forming cell (PFC) responses to SIII at various ages through 110 weeks whereas the levels of the SJL/J PFC responses had begun to decline by the age of 42 weeks through the age of 80 weeks. BALB/c mice were also capable of producing strong PFC responses to LPS at various ages through 110 weeks whereas the comparable SJL/J PFC responses to LPS had declined by 80 weeks of age. By using anti-lymphocyte serum (ALS) and low-dose paralysis to SIII, it was shown that suppressor T cell activity was apparently greater in young BALB/c mice than in older BALB/c mice. It was also found that paralysis to SIII in BALB/c mice was easier to achieve at an early age. SJL/J mice were found to have the necessary B cell activity to respond to SIII through 80 weeks of age and the PFC responses could be greatly enhanced by ALS. Implications of the roles of regulatory T cells in aging are discussed. | 2,635 |
pubmed23n0001_1383 | Production of IgG antibodies and enhanced response of nude mice to DNP-AE-dextran. | DNP-AE-dextran, prepared by the binding of DNP-epsilon-aminocaproyl residues to animoethylated dextran (predominantly alpha-1,6-linked), induced a T-independent anti-DNP antibody response in mice. However, certain differences were observed between the response to this antigen in normal andnude mice. Thus, the antibody titers of nu/nu mice from day 10 to 38 after immunization were significantly higher than those of nu/+ controls. Furthermore, DNP-AE-dextran induced a weak secondary response in nu/+ but not in nu/nu mice. For both thymusless and normal mice the production of IgG in addition to IgM antibodies to DNP-AE-dextran could be established. The former included antibodies of the IgG1 subclass which were considered to be particularly thymus dependent (1). The higher response of nu/nu mice was reflected mainly in the increased production of IgG antibodies. Under the influence of a graft-vs-host reaction, a 10-fold increase in antibody titers to DNP-AE-dextran was observed, due entirely to an enhanced IgG response. | Production of IgG antibodies and enhanced response of nude mice to DNP-AE-dextran. DNP-AE-dextran, prepared by the binding of DNP-epsilon-aminocaproyl residues to animoethylated dextran (predominantly alpha-1,6-linked), induced a T-independent anti-DNP antibody response in mice. However, certain differences were observed between the response to this antigen in normal andnude mice. Thus, the antibody titers of nu/nu mice from day 10 to 38 after immunization were significantly higher than those of nu/+ controls. Furthermore, DNP-AE-dextran induced a weak secondary response in nu/+ but not in nu/nu mice. For both thymusless and normal mice the production of IgG in addition to IgM antibodies to DNP-AE-dextran could be established. The former included antibodies of the IgG1 subclass which were considered to be particularly thymus dependent (1). The higher response of nu/nu mice was reflected mainly in the increased production of IgG antibodies. Under the influence of a graft-vs-host reaction, a 10-fold increase in antibody titers to DNP-AE-dextran was observed, due entirely to an enhanced IgG response. | 2,636 |
pubmed23n0001_1384 | Lymphoid cell fractionation by aggregated immunoglobulin-agarose columns. | Fractionation by columns of aggregated rat immunoglobulin (Agg Ig)-agarose was investigated as a method of separating different populations of lymphoid cells. With rat spleen cells, Agg Ig columns retained phagocytes, IgM- and IgG-antibody-forming-cells, cells mediating antibody- or PHA-induced lysis of chicken erythrocytes, and specifically immune splenocytes lytic to chicken erythrocytes without exogenous antibody. Agg Ig columns did not selectively remove 'B lymphocytes' (surface-Ig-bearing lymphocytes with or without EAC' receptors), or T lymphocytes capable of PHA-induced proliferation or graft-versus-host reactivity. With mouse spleen cells, Agg Ig columns retained alloimmune cytotoxic T cells. | Lymphoid cell fractionation by aggregated immunoglobulin-agarose columns. Fractionation by columns of aggregated rat immunoglobulin (Agg Ig)-agarose was investigated as a method of separating different populations of lymphoid cells. With rat spleen cells, Agg Ig columns retained phagocytes, IgM- and IgG-antibody-forming-cells, cells mediating antibody- or PHA-induced lysis of chicken erythrocytes, and specifically immune splenocytes lytic to chicken erythrocytes without exogenous antibody. Agg Ig columns did not selectively remove 'B lymphocytes' (surface-Ig-bearing lymphocytes with or without EAC' receptors), or T lymphocytes capable of PHA-induced proliferation or graft-versus-host reactivity. With mouse spleen cells, Agg Ig columns retained alloimmune cytotoxic T cells. | 2,637 |
pubmed23n0001_1385 | Rheological studies of Hb SS blood: influence of hematocrit, hypertonicity, separation of cells, deoxygenation, and mixture with normal cells. | Studies of the rheological properties of Hb SS blood indicate that the marked increase in viscosity with deoxygenation is primarily due to cell-cell interaction of cells which were not permanently deformed. The permanently deformed cells, the bottom fraction of cells separated by centrifugation, show only a fraction of the increase in viscosity compared to top cells, when each was deoxygenated. Top cells showed a greater degree of morphologic change with deoxygenation compared to bottom cells. The viscosity of deoxygenated Hb SS blood was disproportionately reduced by the addition of compatible deoxygenated Hb AA cells. A mixture of 1/4 Hb AA cells and 3/4 Hb SS cells reduced the viscosity of deoxygenated Hb SS blood 50 per cent. Studies of Hb SS cells in hypertonic media indicate that hypertonicity per se does not cause sickling. Normal and Hb SS erythrocytes both show identical changes in rheological properties when suspended in hypertonic serum. However, changes in oxygen saturation due to a decrease in intracellular pH with certain hypertonic media may lead to sickling when Hb SS blood is at intermediate PO2 levels. The addition of x-ray contrast material to blood results in the changes due to hypertonicity but does not cause sickling of Hb SS cells. The contrast material lowers the pH of the media and raises intracellular pH. | Rheological studies of Hb SS blood: influence of hematocrit, hypertonicity, separation of cells, deoxygenation, and mixture with normal cells. Studies of the rheological properties of Hb SS blood indicate that the marked increase in viscosity with deoxygenation is primarily due to cell-cell interaction of cells which were not permanently deformed. The permanently deformed cells, the bottom fraction of cells separated by centrifugation, show only a fraction of the increase in viscosity compared to top cells, when each was deoxygenated. Top cells showed a greater degree of morphologic change with deoxygenation compared to bottom cells. The viscosity of deoxygenated Hb SS blood was disproportionately reduced by the addition of compatible deoxygenated Hb AA cells. A mixture of 1/4 Hb AA cells and 3/4 Hb SS cells reduced the viscosity of deoxygenated Hb SS blood 50 per cent. Studies of Hb SS cells in hypertonic media indicate that hypertonicity per se does not cause sickling. Normal and Hb SS erythrocytes both show identical changes in rheological properties when suspended in hypertonic serum. However, changes in oxygen saturation due to a decrease in intracellular pH with certain hypertonic media may lead to sickling when Hb SS blood is at intermediate PO2 levels. The addition of x-ray contrast material to blood results in the changes due to hypertonicity but does not cause sickling of Hb SS cells. The contrast material lowers the pH of the media and raises intracellular pH. | 2,640 |
pubmed23n0001_1386 | Detection of pneumococcal polysaccharide in the sputum of patients with pneumococcal pneumonia by counterimmunoelectrophoresis. | Each of 41 patients with bacterial pneumonia was placed into 1 of 4 categories based on the relative clinical certainty of the diagnosis of pneumococcal pneumonia. The frequency of pneumococcal polysaccharide in the sputum by counterimmunoelectrophoresis (CIE) was then noted for each diagnostic category of patients. Detection of pneumococcal polysaccharide in sputum correlated with the diagnostic certainty of pneumococcal pneumonia, while results of culture of sputum were less indicative of pneumococcal infection. Saliva of 83 normal individuals failed to give positive tests for pneumococcal polysaccharide despite the presence of alpha-hemolytic streptococci on culture. Furthermore, the mere presence of pneumococci in cultures did not predict a positive test for polysaccharide by CIE nor did the absence of pneumococci mean that polysaccharide would not be detected. This study suggests that detection of pneumococcal polysaccharide appears more rapid, more sensitive, and more specific than sputum cultures in diagnosing pneumococcal infection of the lung. | Detection of pneumococcal polysaccharide in the sputum of patients with pneumococcal pneumonia by counterimmunoelectrophoresis. Each of 41 patients with bacterial pneumonia was placed into 1 of 4 categories based on the relative clinical certainty of the diagnosis of pneumococcal pneumonia. The frequency of pneumococcal polysaccharide in the sputum by counterimmunoelectrophoresis (CIE) was then noted for each diagnostic category of patients. Detection of pneumococcal polysaccharide in sputum correlated with the diagnostic certainty of pneumococcal pneumonia, while results of culture of sputum were less indicative of pneumococcal infection. Saliva of 83 normal individuals failed to give positive tests for pneumococcal polysaccharide despite the presence of alpha-hemolytic streptococci on culture. Furthermore, the mere presence of pneumococci in cultures did not predict a positive test for polysaccharide by CIE nor did the absence of pneumococci mean that polysaccharide would not be detected. This study suggests that detection of pneumococcal polysaccharide appears more rapid, more sensitive, and more specific than sputum cultures in diagnosing pneumococcal infection of the lung. | 2,641 |
pubmed23n0001_1387 | The myofilament lattice: studies on isolated fibers. IV. Lattice equilibria in striated muscle. | Accounts of similarities between the thick filament lattice of striated muscle and smectic liquid-crystalline structures have focused upon an equilibrium between electrostatic (repulsive) and van der Waal's (attractive) forces. In living, intact muscle the fiber volume constitutes an additional important parameter which influences the amount of interaxial separation between the filaments. This is demonstrable by comparison of the lattice behavior of living fibers with that of fibers from which the sarcolemma has either been removed or made leaky by glycerination. These comparisons were made mainly by low-angle X-ray diffraction under conditions of changes in sarcomere length, ionic strength or osmolarity, and pH. Single fibers with the sarcolemma removed and glycerinated muscle have lattices which behave in accord with equilibrium liquid-crystalline systems in which the thick filament spacing is determined by the balance between electrostatic and van der Waal's forces. Conversely, osmotic and shortening studies demonstrate that the living, intact muscle has a lattice which behaves in accord with the so-called non-equilibrium (volume-constrained) liquid-crystalline condition in which the interaxial separation between the thick filaments is solely due to the amount of volume available as determined by the Donnan steady-state across the sarcolemma. | The myofilament lattice: studies on isolated fibers. IV. Lattice equilibria in striated muscle. Accounts of similarities between the thick filament lattice of striated muscle and smectic liquid-crystalline structures have focused upon an equilibrium between electrostatic (repulsive) and van der Waal's (attractive) forces. In living, intact muscle the fiber volume constitutes an additional important parameter which influences the amount of interaxial separation between the filaments. This is demonstrable by comparison of the lattice behavior of living fibers with that of fibers from which the sarcolemma has either been removed or made leaky by glycerination. These comparisons were made mainly by low-angle X-ray diffraction under conditions of changes in sarcomere length, ionic strength or osmolarity, and pH. Single fibers with the sarcolemma removed and glycerinated muscle have lattices which behave in accord with equilibrium liquid-crystalline systems in which the thick filament spacing is determined by the balance between electrostatic and van der Waal's forces. Conversely, osmotic and shortening studies demonstrate that the living, intact muscle has a lattice which behaves in accord with the so-called non-equilibrium (volume-constrained) liquid-crystalline condition in which the interaxial separation between the thick filaments is solely due to the amount of volume available as determined by the Donnan steady-state across the sarcolemma. | 2,642 |
pubmed23n0001_1388 | The identification and subcellular localization of thrombosthenin "M", the myosin-like component of pig platelets. | A protein has been studied which spontaneously precipitates from stored fractions of platelet soluble phase prepared by density gradient centrifugation. It is rich in a Ca2+ ATPase activity which displays an activity/pH profile resembling that of skeletal muscle myosin. Adjustment of freshly prepared soluble phase fractions to 0.6 M with respect to KCl and dilution 1 in 3 results in the precipitations of a protein fraction with essentially the same enzymatic properties as the spontaneously precipitable protein. These two similar proteins represent between 9 and 13% of the soluble phase total protein and each account for almost the whole of divalent cation activated ATPase activity of the soluble phases from which they were derived. The Mg2+ ATPase activity is only about twice purified with respect to the soluble phase enzyme activity, but the Ca2+ ATPase shows a 10-13-fold enrichment. Synthetic actomyosins can be prepared from the two proteins by addition of either platelet or skeletal muscle actin. These show significant increases in Mg2+ ATPase at the most favourable combination ratios. The ratio between the yield of soluble phase protein obtained by dilution precipitation and the lactate dehydrogenase activity of the soluble phase remains constant under a wide range of homogenization and sonication conditions applied to the original whole platelet suspensions. This confirms our earlier view that the soluble phase is a valid intracellular compartment for a considerable proportion of the platelet contractile protein and that in the complex the myosin-like component predominates. | The identification and subcellular localization of thrombosthenin "M", the myosin-like component of pig platelets. A protein has been studied which spontaneously precipitates from stored fractions of platelet soluble phase prepared by density gradient centrifugation. It is rich in a Ca2+ ATPase activity which displays an activity/pH profile resembling that of skeletal muscle myosin. Adjustment of freshly prepared soluble phase fractions to 0.6 M with respect to KCl and dilution 1 in 3 results in the precipitations of a protein fraction with essentially the same enzymatic properties as the spontaneously precipitable protein. These two similar proteins represent between 9 and 13% of the soluble phase total protein and each account for almost the whole of divalent cation activated ATPase activity of the soluble phases from which they were derived. The Mg2+ ATPase activity is only about twice purified with respect to the soluble phase enzyme activity, but the Ca2+ ATPase shows a 10-13-fold enrichment. Synthetic actomyosins can be prepared from the two proteins by addition of either platelet or skeletal muscle actin. These show significant increases in Mg2+ ATPase at the most favourable combination ratios. The ratio between the yield of soluble phase protein obtained by dilution precipitation and the lactate dehydrogenase activity of the soluble phase remains constant under a wide range of homogenization and sonication conditions applied to the original whole platelet suspensions. This confirms our earlier view that the soluble phase is a valid intracellular compartment for a considerable proportion of the platelet contractile protein and that in the complex the myosin-like component predominates. | 2,643 |
pubmed23n0001_1389 | Control of mouth opening and pharynx protrusion during feeding in the sea anemone Calliactis parasitica. | 1. Activity in all three known conducting systems (the nerve net, SS1, and SS2) may accompany feeding in Calliactis. The most marked response is an increase in pulse frequency in the SS2 (the endodermal slow conducting system) during mouth opening and pharynx protrusion. 2. Electrical stimulation of the SS2 at a frequency of one shock every 5 s elicits mouth opening and pharynx protrusion in the absence of food. 3. A rise in SS2 pulse frequency is also evoked by food extracts, some amino acids, and in particular by the tripeptide reduced glutathione, which produces a response at a concentration of 10(-5) M. 4. Although the SS2 is an endodermal system, the receptors involved in the response to food appear to be ectodermal. 5. The epithelium that lines the pharynx conducts SS1 pulses, but there is some evidence for polarization of conduction. | Control of mouth opening and pharynx protrusion during feeding in the sea anemone Calliactis parasitica. 1. Activity in all three known conducting systems (the nerve net, SS1, and SS2) may accompany feeding in Calliactis. The most marked response is an increase in pulse frequency in the SS2 (the endodermal slow conducting system) during mouth opening and pharynx protrusion. 2. Electrical stimulation of the SS2 at a frequency of one shock every 5 s elicits mouth opening and pharynx protrusion in the absence of food. 3. A rise in SS2 pulse frequency is also evoked by food extracts, some amino acids, and in particular by the tripeptide reduced glutathione, which produces a response at a concentration of 10(-5) M. 4. Although the SS2 is an endodermal system, the receptors involved in the response to food appear to be ectodermal. 5. The epithelium that lines the pharynx conducts SS1 pulses, but there is some evidence for polarization of conduction. | 2,645 |
pubmed23n0001_1390 | Blood oxygen transport and metabolism of the confined lugworm Arenicola marina (L.). | Oxygen consumption (MO2), haemoglobin oxygen saturation level (SVO2) and pH (pHv) in prebranchial blood were measured in lugworms experimentally confined in sea water at 15 degrees C. Total blood flow through the gills (Vb) was estimated. For sea water oxygen partial pressure (PwO2) between 120 and 150 Torr MO2, SVO2 and Vb were high and nearly constant. For PwO2 less than 120 Torr, Vb fell quickly, MO2 progressively dropped, and metabolism remained aerobic at the expense of the prebrancial blood oxygen store. For PwO2 less than 50 Torr, Vb and SvO2 values were extremely low, and the low pHv and the modified buffer power of the surrounding sea water showed that anaerobic metabolism was occurring. Changes in respiratory gas exchanges and metabolism during the tidal cycle are deduced from the comparison of these results with data obtained in the field. | Blood oxygen transport and metabolism of the confined lugworm Arenicola marina (L.). Oxygen consumption (MO2), haemoglobin oxygen saturation level (SVO2) and pH (pHv) in prebranchial blood were measured in lugworms experimentally confined in sea water at 15 degrees C. Total blood flow through the gills (Vb) was estimated. For sea water oxygen partial pressure (PwO2) between 120 and 150 Torr MO2, SVO2 and Vb were high and nearly constant. For PwO2 less than 120 Torr, Vb fell quickly, MO2 progressively dropped, and metabolism remained aerobic at the expense of the prebrancial blood oxygen store. For PwO2 less than 50 Torr, Vb and SvO2 values were extremely low, and the low pHv and the modified buffer power of the surrounding sea water showed that anaerobic metabolism was occurring. Changes in respiratory gas exchanges and metabolism during the tidal cycle are deduced from the comparison of these results with data obtained in the field. | 2,646 |
pubmed23n0001_1391 | Multiple sedimenting species of properdin in human serum and interaction of purified properdin with the third component of complement. | Normal human serum subjected to sucrose density gradient analysis exhibited multiple sedimenting species of properdin antigen. Properdin antigen distribution was dependent on serum concentration, ionic strength, temperature, and the presence of C3, and was not dependent on the presence of divalent metal cations or blood coagulation. In mixtures of purified components, properdin sedimented heavier in the presence of C3, C3b, or C3c. Addition of factor B to mixtures containing C3 and properdin was without effect. These data provide insights into earlier discrepancies concerning the sedimentation behavior of partially purified properdin, indicate a propensity of some constituents of the alternative pathway to form protein-protein complexes, and suggest caution in interpretation of immunopathological studies in which properdin deposits are found in the presence of C3. | Multiple sedimenting species of properdin in human serum and interaction of purified properdin with the third component of complement. Normal human serum subjected to sucrose density gradient analysis exhibited multiple sedimenting species of properdin antigen. Properdin antigen distribution was dependent on serum concentration, ionic strength, temperature, and the presence of C3, and was not dependent on the presence of divalent metal cations or blood coagulation. In mixtures of purified components, properdin sedimented heavier in the presence of C3, C3b, or C3c. Addition of factor B to mixtures containing C3 and properdin was without effect. These data provide insights into earlier discrepancies concerning the sedimentation behavior of partially purified properdin, indicate a propensity of some constituents of the alternative pathway to form protein-protein complexes, and suggest caution in interpretation of immunopathological studies in which properdin deposits are found in the presence of C3. | 2,647 |
pubmed23n0001_1392 | [Cathepsin, phosphoprotein-phosphatase and acid phosphatase in the soluble fraction of the cattle brain cortex: purification and properties (author's transl)]. | Cattle brain cortex was homogenised in 0, 29 mol/1 sucrose and centrifuged at 101 000 X g. The supernatant contains the majority of 3 enzymes participating in protein turnover: cathepsin (EC 3.4.4.23), phosphoprotein phosphatase (EC 3.1.3.16) and acid phosphatase (EC 3.1.3.2). They were separated by chromatography on Sephadex G 200 in neutral buffer. The cathepsin was purified up to 380 fold by gel filtration on Sephadex and column electrophoresis. The pH optimum of cathepsin was 5.7. At 37 degrees C no decrease of activity was measurable during 30 min. The Km was found to be 2.75 mg/ml Casein Hammarsten. The molecular weight by gel filtration and exclusion-gel electrophoresis was about 45 000, corresponding to the cathepsin from human liver (Barrett, A.J. (1970) Biochem. J. 117, 601-607). The sedimentation constant 3.0 S20,W is comparable with the values of proteinase of different origin, and the composition is similar with respect to the high proportion of acidic amino acids. The phosphoprotein phosphatase can be further purified by chromatography on hydroxyapatite and by column electrophoresis. The pH optimum of phosphoprotein phosphatase was about pH 5.5. At 45 degrees C no decrease of activity was measurable during 20 min; the Km was 1.43 mg/ml casein isoelectric. The pH optimum of acid phosphatase was about 5.6. At 54 degrees C NO DECREASE OF ACTIVITY WAs measurable during 30 min; the Km was 2 mumol/1 for Sodium phenolphthalein diphosphate. All three enzymes slowly lost their activity during several weeks at - 4 degrees C, apparently by self digestion in the cold. | [Cathepsin, phosphoprotein-phosphatase and acid phosphatase in the soluble fraction of the cattle brain cortex: purification and properties (author's transl)]. Cattle brain cortex was homogenised in 0, 29 mol/1 sucrose and centrifuged at 101 000 X g. The supernatant contains the majority of 3 enzymes participating in protein turnover: cathepsin (EC 3.4.4.23), phosphoprotein phosphatase (EC 3.1.3.16) and acid phosphatase (EC 3.1.3.2). They were separated by chromatography on Sephadex G 200 in neutral buffer. The cathepsin was purified up to 380 fold by gel filtration on Sephadex and column electrophoresis. The pH optimum of cathepsin was 5.7. At 37 degrees C no decrease of activity was measurable during 30 min. The Km was found to be 2.75 mg/ml Casein Hammarsten. The molecular weight by gel filtration and exclusion-gel electrophoresis was about 45 000, corresponding to the cathepsin from human liver (Barrett, A.J. (1970) Biochem. J. 117, 601-607). The sedimentation constant 3.0 S20,W is comparable with the values of proteinase of different origin, and the composition is similar with respect to the high proportion of acidic amino acids. The phosphoprotein phosphatase can be further purified by chromatography on hydroxyapatite and by column electrophoresis. The pH optimum of phosphoprotein phosphatase was about pH 5.5. At 45 degrees C no decrease of activity was measurable during 20 min; the Km was 1.43 mg/ml casein isoelectric. The pH optimum of acid phosphatase was about 5.6. At 54 degrees C NO DECREASE OF ACTIVITY WAs measurable during 30 min; the Km was 2 mumol/1 for Sodium phenolphthalein diphosphate. All three enzymes slowly lost their activity during several weeks at - 4 degrees C, apparently by self digestion in the cold. | 2,648 |
pubmed23n0001_1393 | Polydipsia, hyponatremia, and seizures in psychotic patients. | Case histories are presented for four psychotic patients who ingested large quantities of water and subsequently developed grand mal seizures and serum sodium levels of less than 121 meq/liter. The physiology of psychogenic polydipsia and related disorders is reviewed. The relation of this disorder to temporal lobe seizures and to the use of phenothiazines is considered. | Polydipsia, hyponatremia, and seizures in psychotic patients. Case histories are presented for four psychotic patients who ingested large quantities of water and subsequently developed grand mal seizures and serum sodium levels of less than 121 meq/liter. The physiology of psychogenic polydipsia and related disorders is reviewed. The relation of this disorder to temporal lobe seizures and to the use of phenothiazines is considered. | 2,649 |
pubmed23n0001_1394 | Isolation from bovine brain of a fraction containing capillaries and a fraction containing membrane fragments of the choroid plexus. | Combined differential and density gradient centrifugation was used for the isolation of a capillary-rich fraction from the cerebral cortex and a brush border containing fraction from the bovine choroid plexus. The activities of gamma-glutamyl transpeptidase and several other marker enzymes were monitored during the fractionation procedure. Electron microscopic examination showed a membrane-rich fraction in the choroid plexus high in the gamma-glutamyl transpeptidase and 5'-nucleotidase activities. From the brain cortex, a capillary-rich fraction was obtained which was high in gamma-glutamyl transpeptidase and alkaline phosphatase activities. A histochemical examination showed gamma-glutamyl transpeptidase activity localized in the capillary walls. | Isolation from bovine brain of a fraction containing capillaries and a fraction containing membrane fragments of the choroid plexus. Combined differential and density gradient centrifugation was used for the isolation of a capillary-rich fraction from the cerebral cortex and a brush border containing fraction from the bovine choroid plexus. The activities of gamma-glutamyl transpeptidase and several other marker enzymes were monitored during the fractionation procedure. Electron microscopic examination showed a membrane-rich fraction in the choroid plexus high in the gamma-glutamyl transpeptidase and 5'-nucleotidase activities. From the brain cortex, a capillary-rich fraction was obtained which was high in gamma-glutamyl transpeptidase and alkaline phosphatase activities. A histochemical examination showed gamma-glutamyl transpeptidase activity localized in the capillary walls. | 2,650 |
pubmed23n0001_1395 | Ionic mechanism of 5-hydroxytryptamine induced hyperpolarization and inhibitory junctional potential in body wall muscle cells of Hirudo medicinalis. | Five-hydroxytryptamine (5-HT) causes a hyperpolarization and increased conductance of the leech body wall muscle cell membrane. If 5-HT is applied in the absence of the Cl minus ion, the response appears as a depolarization, whereas if 5-HT is applied in the absence of the K+ ion, the response is a hyperpolarization. In both cases, the conductance of the muscle cell membrane is increased. Stimulation of the peripheral nerve to the body wall muscle produces a complex junctional potential in muscle cells. Exposing the muscle to d-tubocurarine (d-TC) eliminates the excitatory component (EJP) of the complex potential. The inhibitory potential (IJP) that remains has an equilibrium potential at approximately 65 mV. Furthermore, this IJP appears as a depolarization when the nerve is stimulated in the presence of d-TC and low CL minus, whereas this is not the case if the nerve is stimulated in the presence of d-TC and low K+. The drugs BOL-148 and cyproheptadine block the IJP's in the body wall muscle. These data are interpreted as indicating that 5-HT acts on leech body wall muscle cells by increasing the conductance to the Cl minus ion and that the IJP's caused by nerve stimulation are probably the result of 5-HT release at nerve terminals. As a final point, it has been shown that the inhibition by 5-HT of the spontaneous EJP's that occur on the leech body wall muscle results from an inhibition of central neurons and not from any direct effect on the muscle cell or on peripheral synapses. | Ionic mechanism of 5-hydroxytryptamine induced hyperpolarization and inhibitory junctional potential in body wall muscle cells of Hirudo medicinalis. Five-hydroxytryptamine (5-HT) causes a hyperpolarization and increased conductance of the leech body wall muscle cell membrane. If 5-HT is applied in the absence of the Cl minus ion, the response appears as a depolarization, whereas if 5-HT is applied in the absence of the K+ ion, the response is a hyperpolarization. In both cases, the conductance of the muscle cell membrane is increased. Stimulation of the peripheral nerve to the body wall muscle produces a complex junctional potential in muscle cells. Exposing the muscle to d-tubocurarine (d-TC) eliminates the excitatory component (EJP) of the complex potential. The inhibitory potential (IJP) that remains has an equilibrium potential at approximately 65 mV. Furthermore, this IJP appears as a depolarization when the nerve is stimulated in the presence of d-TC and low CL minus, whereas this is not the case if the nerve is stimulated in the presence of d-TC and low K+. The drugs BOL-148 and cyproheptadine block the IJP's in the body wall muscle. These data are interpreted as indicating that 5-HT acts on leech body wall muscle cells by increasing the conductance to the Cl minus ion and that the IJP's caused by nerve stimulation are probably the result of 5-HT release at nerve terminals. As a final point, it has been shown that the inhibition by 5-HT of the spontaneous EJP's that occur on the leech body wall muscle results from an inhibition of central neurons and not from any direct effect on the muscle cell or on peripheral synapses. | 2,651 |
pubmed23n0001_1396 | Cerebrospinal fluid lactate and lactate/pyruvate ratios in hydrocephalus. | Cerebral metabolism in 21 hydrocephalic patients was studied. Preoperative and postoperative specimens of cerebrospinal fluid (CSF) were obtained and the cerebral perfusion pressure (CPP) was calculated in each instance. The specimens of CSF were analyzed for lactate and pyruvate and the lactate/pyruvate (L/P) ratio was calculated for each sample. The L/P ratio, which reflects the redox state of the cell, was used to determine the extent of anaerobic metabolism. An inverse relationship was noted between CPP and lactate as well as the L/P ratio. In general, the level of anaerobic metabolism was decreased after insertion of a shunt. | Cerebrospinal fluid lactate and lactate/pyruvate ratios in hydrocephalus. Cerebral metabolism in 21 hydrocephalic patients was studied. Preoperative and postoperative specimens of cerebrospinal fluid (CSF) were obtained and the cerebral perfusion pressure (CPP) was calculated in each instance. The specimens of CSF were analyzed for lactate and pyruvate and the lactate/pyruvate (L/P) ratio was calculated for each sample. The L/P ratio, which reflects the redox state of the cell, was used to determine the extent of anaerobic metabolism. An inverse relationship was noted between CPP and lactate as well as the L/P ratio. In general, the level of anaerobic metabolism was decreased after insertion of a shunt. | 2,652 |
pubmed23n0001_1397 | Effects of hyperventilation, CO2, and CSF pressure on internal carotid blood flow in the baboon. | The combined effect upon cerebral blood flow (CBF) of an elevation of cerebrospinal fluid pressure (CSFP) and changes in respiratory CO2 was studied in nine baboons under chloralose anesthesia. The animals were mildly hyperventilated and provided with increasing amounts of CO2 in O2-air. Arterial CO2 tensions (PaCO2) increased from 17 to 58 mm Hg. Internal carotid blood flow (ICBF) was measured at normal CSFP and at hydrostatically maintained 50 mm Hg CSFP. It was found that: 1) end-tidal CO2 may be used as a substitute for arterial PaCO2 determinations; 2) this elevation of CSFP has little effect on ICBF during hypercapnia and normocapnia; however, 3) during hypocapnia the ICBF is reduced an additional 20% when CSFP is elevated; that is, ICBF is reduced 50% from normal when end-tidal CO2 is reduced to 2% at this elevated level of CSFP. Caution should be exercised during hyperventilation therapy particularly if the elevated CSFP or intracranial pressure (ICP) is not reduced to approach normal levels; in these conditions, the combination of decreasing PaCO2 and elevated ICP may reduce CBF below critical levels and thus lead to cerebral hypoxia. | Effects of hyperventilation, CO2, and CSF pressure on internal carotid blood flow in the baboon. The combined effect upon cerebral blood flow (CBF) of an elevation of cerebrospinal fluid pressure (CSFP) and changes in respiratory CO2 was studied in nine baboons under chloralose anesthesia. The animals were mildly hyperventilated and provided with increasing amounts of CO2 in O2-air. Arterial CO2 tensions (PaCO2) increased from 17 to 58 mm Hg. Internal carotid blood flow (ICBF) was measured at normal CSFP and at hydrostatically maintained 50 mm Hg CSFP. It was found that: 1) end-tidal CO2 may be used as a substitute for arterial PaCO2 determinations; 2) this elevation of CSFP has little effect on ICBF during hypercapnia and normocapnia; however, 3) during hypocapnia the ICBF is reduced an additional 20% when CSFP is elevated; that is, ICBF is reduced 50% from normal when end-tidal CO2 is reduced to 2% at this elevated level of CSFP. Caution should be exercised during hyperventilation therapy particularly if the elevated CSFP or intracranial pressure (ICP) is not reduced to approach normal levels; in these conditions, the combination of decreasing PaCO2 and elevated ICP may reduce CBF below critical levels and thus lead to cerebral hypoxia. | 2,653 |
pubmed23n0001_1398 | The effect of dietary vanadium on fatty acid and cholesterol synthesis and turnover in the chick. | Day-old male, broiler type chicks were used to study the effect of 100 ppm dietary vanadium on fatty acid and cholesterol synthesis and turnover in vivo. After feeding the experimental diets for 4 weeks body weight and liver weight of chicks fed 100 ppm vanadium were significantly less than those of the control chicks and liver total lipid and cholesterol tended to be slightly higher than the levels of the control chicks. [1-14C] Acetate was administered intravenously and the specific activities of plasma and liver total lipid, cholesterol and fatty acid were determined at 0.25, 0.50, 1.0, 4.0, 8.0 and 15.0 hours after the injection. Plasma total lipid and cholesterol were significantly higher than the levels in the control chicks. The rate of incorporation of [1-14C]acetate into plasma and liver total lipid, cholesterol and fatty acid was higher in chicks fed vanadium than the control group at any of the time being tested after the injection. There was a significant increase in the hepatic citrate cleavage enzyme activity among chicks fed 100 ppm vanadium, whereas, there was no significant change in acetate thiokinase activity. Turnover rate of plasma total lipid and fatty acid in vanadium fed chicks was lower than the control. The turnover rate of plasma cholesterol determined by administering [4-14C]cholesterol and periodically measuring the specific activity of plasma cholesterol was higher in chicks fed vanadium than in those fed the basal diet. | The effect of dietary vanadium on fatty acid and cholesterol synthesis and turnover in the chick. Day-old male, broiler type chicks were used to study the effect of 100 ppm dietary vanadium on fatty acid and cholesterol synthesis and turnover in vivo. After feeding the experimental diets for 4 weeks body weight and liver weight of chicks fed 100 ppm vanadium were significantly less than those of the control chicks and liver total lipid and cholesterol tended to be slightly higher than the levels of the control chicks. [1-14C] Acetate was administered intravenously and the specific activities of plasma and liver total lipid, cholesterol and fatty acid were determined at 0.25, 0.50, 1.0, 4.0, 8.0 and 15.0 hours after the injection. Plasma total lipid and cholesterol were significantly higher than the levels in the control chicks. The rate of incorporation of [1-14C]acetate into plasma and liver total lipid, cholesterol and fatty acid was higher in chicks fed vanadium than the control group at any of the time being tested after the injection. There was a significant increase in the hepatic citrate cleavage enzyme activity among chicks fed 100 ppm vanadium, whereas, there was no significant change in acetate thiokinase activity. Turnover rate of plasma total lipid and fatty acid in vanadium fed chicks was lower than the control. The turnover rate of plasma cholesterol determined by administering [4-14C]cholesterol and periodically measuring the specific activity of plasma cholesterol was higher in chicks fed vanadium than in those fed the basal diet. | 2,654 |
pubmed23n0001_1399 | Changes of pH-values during storage of fetal blood samples. | In recent years increased attention has been given to the degree of acidity of fetal blood during the course of labor or immediately after delivery. As it is not always possible to measure the blood pH immediately after sampling, it is important to know whether or not the pH is likely to change if the blood sample is stored for some time. 30 heparinized whole blood samples were collected from the umbilical artery of 30 newborn infants immediately after delivery and were stored at room temperature. pH -measurements were made every 5 minutes up to 80 minutes. In a second group the same number of samples were stored in a refrigator. pH-measurements were performed after 30 minutes and then every hour for up to 7 hours, and finally after 24 hours. The pHact-values did not change at room temperature (about 24 degrees C) for up to 25 minutes; pHqu40-values decreased initially more rapidly but at 50 minutes the decrease was the same as in pHact-values namely 0.02 units. Afterwards the pHact-values decreased at a mena rate of about 0.03 units per hour and pHqu40-values at a mean rate of about 0.017 units per hour. If the storage temperature was lowered by refrigeration (about 6 degrees C) the pHact-values remained between 0 and -0.02 units for 6 hours and pHqu40-values for 3 hours. From these experiments it can be concluded that fetal blood samples must be kept in a refrigerator in order to inhibit autoxidation, if they have to be stored for longer than 50 minutes. | Changes of pH-values during storage of fetal blood samples. In recent years increased attention has been given to the degree of acidity of fetal blood during the course of labor or immediately after delivery. As it is not always possible to measure the blood pH immediately after sampling, it is important to know whether or not the pH is likely to change if the blood sample is stored for some time. 30 heparinized whole blood samples were collected from the umbilical artery of 30 newborn infants immediately after delivery and were stored at room temperature. pH -measurements were made every 5 minutes up to 80 minutes. In a second group the same number of samples were stored in a refrigator. pH-measurements were performed after 30 minutes and then every hour for up to 7 hours, and finally after 24 hours. The pHact-values did not change at room temperature (about 24 degrees C) for up to 25 minutes; pHqu40-values decreased initially more rapidly but at 50 minutes the decrease was the same as in pHact-values namely 0.02 units. Afterwards the pHact-values decreased at a mena rate of about 0.03 units per hour and pHqu40-values at a mean rate of about 0.017 units per hour. If the storage temperature was lowered by refrigeration (about 6 degrees C) the pHact-values remained between 0 and -0.02 units for 6 hours and pHqu40-values for 3 hours. From these experiments it can be concluded that fetal blood samples must be kept in a refrigerator in order to inhibit autoxidation, if they have to be stored for longer than 50 minutes. | 2,655 |
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