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PMID:21042
|
Activity and interference effects in measurement of ionized calcium with ion-selective electrodes.
|
We assessed the magnitude of the errors in ionized-calcium measurements resulting from changes in electrolyte composition for three different ion-selective electrodes: the Orion Model 93-20, the Radiometer F2112, and the Simon neutral carrier membrane electrode. We attempted to distinguish between errors arising from changes in calcium ion activity and those due to interferences in the electrode response. Variation in sodium ion concentration over the range 100--180 mmol/liter produces changes in apparent ionized-calcium concentration that are largely attributable to activity effects for the Radiometer and Simon electrodes. The Orion electrode is subject to an additional sodium-interference effect. Apparent ionized calcium concentration measurements are independent of pH for the Radiometer electrode but not for the Orion electrode; the Simon electrode exhibits intermediate pH response, which is probably clinically negligible. Magnesium and potassium ions have little effect on ionized calcium concentration measurements, particularly when these ions are incorporated into calibration standards.
|
Activity and interference effects in measurement of ionized calcium with ion-selective electrodes. We assessed the magnitude of the errors in ionized-calcium measurements resulting from changes in electrolyte composition for three different ion-selective electrodes: the Orion Model 93-20, the Radiometer F2112, and the Simon neutral carrier membrane electrode. We attempted to distinguish between errors arising from changes in calcium ion activity and those due to interferences in the electrode response. Variation in sodium ion concentration over the range 100--180 mmol/liter produces changes in apparent ionized-calcium concentration that are largely attributable to activity effects for the Radiometer and Simon electrodes. The Orion electrode is subject to an additional sodium-interference effect. Apparent ionized calcium concentration measurements are independent of pH for the Radiometer electrode but not for the Orion electrode; the Simon electrode exhibits intermediate pH response, which is probably clinically negligible. Magnesium and potassium ions have little effect on ionized calcium concentration measurements, particularly when these ions are incorporated into calibration standards.
|
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] |
PMID:21043
|
Effects of ethanol (0.75 g/kg body weight) on the activities of selected enzymes in sera of healthy young adults: 2. Interindividual variations in response of gamma-glutamyltransferase to repeated ethanol challenges.
|
We measured individual temporal changes in gamma-glutamyltransferase activity in serum after four consecutive ethanol challenges in each of eight apparently healthy volunteers. Each challenge consisted of ingesting 0.75 g of ethanol per kilogram of body weight on two evenings, followed by five days of abstention during which the activity was assessed at regular intervals. A nine-day abstention period preceded the study, and initial baseline activity was determined. The highest values for the group mean activity were +12%, +11%, +22%, and +18% greater than baseline after the first, second, third, and fourth ethanol challenges, respectively. We noted marked intra-individual and interindividual variation in the enzymic response; peak values ranged from +1% in one subject to +57% in another. The possible causes for these biological variations are discussed.
|
Effects of ethanol (0.75 g/kg body weight) on the activities of selected enzymes in sera of healthy young adults: 2. Interindividual variations in response of gamma-glutamyltransferase to repeated ethanol challenges. We measured individual temporal changes in gamma-glutamyltransferase activity in serum after four consecutive ethanol challenges in each of eight apparently healthy volunteers. Each challenge consisted of ingesting 0.75 g of ethanol per kilogram of body weight on two evenings, followed by five days of abstention during which the activity was assessed at regular intervals. A nine-day abstention period preceded the study, and initial baseline activity was determined. The highest values for the group mean activity were +12%, +11%, +22%, and +18% greater than baseline after the first, second, third, and fourth ethanol challenges, respectively. We noted marked intra-individual and interindividual variation in the enzymic response; peak values ranged from +1% in one subject to +57% in another. The possible causes for these biological variations are discussed.
|
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] |
PMID:21044
|
Albumin/calcium association at different pH, as determined by potentiometry.
|
Calcium binding by albumin was determined potentiometrically at physiological ionic strength and temperature as a function of pH. The binding data indicate at least 30 different binding sites with different association constants and different H+ interaction. One site appears to be responsible for the major binding at physiological pH and substance concentration of free calcium, together with three other sites that bind with less affinity.
|
Albumin/calcium association at different pH, as determined by potentiometry. Calcium binding by albumin was determined potentiometrically at physiological ionic strength and temperature as a function of pH. The binding data indicate at least 30 different binding sites with different association constants and different H+ interaction. One site appears to be responsible for the major binding at physiological pH and substance concentration of free calcium, together with three other sites that bind with less affinity.
|
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] |
PMID:21045
|
The use of tears for diagnosis of GM1 gangliosidosis.
|
The properties of beta-galactosidase of tears were investigated and the standard assay system was accomplished. The pH optimum was 4.2. The enzyme had a KM of 8.3 X 10(-4) M. The activity was stimulated by chloride ions and slightly stabilized by bovine serum albumin. The activities of normal individuals were 205 +/- 80 (S.D.) nmol/h/ml. The activity in the tears of the patient with GM1 gangliosidosis decreased to about 20% of normal control and this disease could be diagnosed by the assay of beta-galactosidase in tears.
|
The use of tears for diagnosis of GM1 gangliosidosis. The properties of beta-galactosidase of tears were investigated and the standard assay system was accomplished. The pH optimum was 4.2. The enzyme had a KM of 8.3 X 10(-4) M. The activity was stimulated by chloride ions and slightly stabilized by bovine serum albumin. The activities of normal individuals were 205 +/- 80 (S.D.) nmol/h/ml. The activity in the tears of the patient with GM1 gangliosidosis decreased to about 20% of normal control and this disease could be diagnosed by the assay of beta-galactosidase in tears.
|
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] |
PMID:21046
|
Assay of serum free fatty acids by extraction-photometric procedure.
|
Reaction conditions for extraction-photometric determination of free fatty acids in serum were studied. The results were found to be influenced by (a) sodium chloride concentration in the copper reagent, (b) the kind of standard solution used, (c) centrifugation of the reaction mixture and (d) serum phospholipids. A micromethod based on the formation of free fatty acids-copper soaps with a stable copper reagent and sensitive indicator for copper, 2-(2-thiazolylazo)-4-methoxyphenol, is described.
|
Assay of serum free fatty acids by extraction-photometric procedure. Reaction conditions for extraction-photometric determination of free fatty acids in serum were studied. The results were found to be influenced by (a) sodium chloride concentration in the copper reagent, (b) the kind of standard solution used, (c) centrifugation of the reaction mixture and (d) serum phospholipids. A micromethod based on the formation of free fatty acids-copper soaps with a stable copper reagent and sensitive indicator for copper, 2-(2-thiazolylazo)-4-methoxyphenol, is described.
|
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] |
PMID:21047
|
Sweat testing for cystic fibrosis: errors associated with the in-situ sweat test using chloride ion selective electrodes.
|
The in-situ sweat test is prone to errors from various sources. This paper examines errors due to evaporation, absorption of water into the skin, and pressure of the electrode on the skin. Only evaporation caused serious errors. The accuracy and precision when measuring small chloride/sweat samples on the skin are not significantly worse than when measuring bulk solutions.
|
Sweat testing for cystic fibrosis: errors associated with the in-situ sweat test using chloride ion selective electrodes. The in-situ sweat test is prone to errors from various sources. This paper examines errors due to evaporation, absorption of water into the skin, and pressure of the electrode on the skin. Only evaporation caused serious errors. The accuracy and precision when measuring small chloride/sweat samples on the skin are not significantly worse than when measuring bulk solutions.
|
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] |
PMID:21048
|
Influence of ions on the antigen-antibody complex formation as measured by radioimmunoassay.
|
In this study, using radioimmunoassay techniques, we found that ions at concentrations in the order of 0.1 molar influence the antigen-antibody complex formation. The angiotensin I/anti-angiotensin I reaction was studied in detail. Particularly bivalent cations and anions with a strong chaotropic effect (SCN-, I- and ClO4-) were found to influence strongly the specific immunological reaction. However, NO3- had also a remarkably strong influence. We found that the equilibrium constant, rather than the number of binding sites of the antibody, is influenced by the ions. It should be borne in mind that relatively high concentrations of electrolyte (as compared with the concentrations of antigen and antibody) show this effect. Consequently, this effect is of less practical importance for routine radioimmunoassay than is, for example, the effect of pH. However, this phenomenon shows that the radioimmunoassay technique might be valuable not only for quantization of very low hormone concentrations in biological fluids, but has also important potential applications in physical and protein chemistry. Particularly, the high sensitivity of this technique and the possibility of studying a homogeneous reaction system might give it advantages over other techniques.
|
Influence of ions on the antigen-antibody complex formation as measured by radioimmunoassay. In this study, using radioimmunoassay techniques, we found that ions at concentrations in the order of 0.1 molar influence the antigen-antibody complex formation. The angiotensin I/anti-angiotensin I reaction was studied in detail. Particularly bivalent cations and anions with a strong chaotropic effect (SCN-, I- and ClO4-) were found to influence strongly the specific immunological reaction. However, NO3- had also a remarkably strong influence. We found that the equilibrium constant, rather than the number of binding sites of the antibody, is influenced by the ions. It should be borne in mind that relatively high concentrations of electrolyte (as compared with the concentrations of antigen and antibody) show this effect. Consequently, this effect is of less practical importance for routine radioimmunoassay than is, for example, the effect of pH. However, this phenomenon shows that the radioimmunoassay technique might be valuable not only for quantization of very low hormone concentrations in biological fluids, but has also important potential applications in physical and protein chemistry. Particularly, the high sensitivity of this technique and the possibility of studying a homogeneous reaction system might give it advantages over other techniques.
|
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] |
PMID:21049
|
Comparison of soluble and membrane-bound neutral arylamidases from renal cell carcinoma.
|
Soluble and membrane-bound neutral arylamidases from renal cell carcinoma were partially purified and their properties were compared. Soluble neutral arylamidase was a heat labile and SH-dependent metalloenzyme. Membrane-bound neutral arylamidase was a rather heat stable metalloenzyme and was activated by Co2+ with changing KM and VMAX. KM values for soluble and membrane-bound neutral arylamidases were different. L-Methionine (5mM) did not affect the enzymic activity of soluble neutral arylamidase, but inhibited 82 percent of the enzymic activity of membrane-bound neutral arylamidase. Molecular weights of soluble and membrane-bound neutral arylamidases by Sephadex G-200 gel filtration were 140000 and 240000, respectively. Soluble and membrane-bound neutral arylamidases from renal cell carcinoma appear to be distinct enzymes.
|
Comparison of soluble and membrane-bound neutral arylamidases from renal cell carcinoma. Soluble and membrane-bound neutral arylamidases from renal cell carcinoma were partially purified and their properties were compared. Soluble neutral arylamidase was a heat labile and SH-dependent metalloenzyme. Membrane-bound neutral arylamidase was a rather heat stable metalloenzyme and was activated by Co2+ with changing KM and VMAX. KM values for soluble and membrane-bound neutral arylamidases were different. L-Methionine (5mM) did not affect the enzymic activity of soluble neutral arylamidase, but inhibited 82 percent of the enzymic activity of membrane-bound neutral arylamidase. Molecular weights of soluble and membrane-bound neutral arylamidases by Sephadex G-200 gel filtration were 140000 and 240000, respectively. Soluble and membrane-bound neutral arylamidases from renal cell carcinoma appear to be distinct enzymes.
|
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] |
PMID:21050
|
Comparative activity of sulphatases in human liver on two synthetic substrates.
|
Ion-exchange chromatography and gel chromatography manifested the presence of three different forms of sulphatase activity in human liver using 4-methylumbelliferyl sulphate as substrate. Studies with the substrate 4-nitrocatechol sulphate and inhibition experiments with AgNO3 were also performed on these three different forms of sulphatases. This makes it possible to identify the three forms as arylsulphatase A, B, and C. The conclusion is drawn that the 4-methylumbelliferyl sulphate substrate is inadequate to measure the lysosomal arylsulphatases A and B, but it could be used to measure the microsomal arylsulphatase C.
|
Comparative activity of sulphatases in human liver on two synthetic substrates. Ion-exchange chromatography and gel chromatography manifested the presence of three different forms of sulphatase activity in human liver using 4-methylumbelliferyl sulphate as substrate. Studies with the substrate 4-nitrocatechol sulphate and inhibition experiments with AgNO3 were also performed on these three different forms of sulphatases. This makes it possible to identify the three forms as arylsulphatase A, B, and C. The conclusion is drawn that the 4-methylumbelliferyl sulphate substrate is inadequate to measure the lysosomal arylsulphatases A and B, but it could be used to measure the microsomal arylsulphatase C.
|
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] |
PMID:21051
|
Some kinetic properites of liver ornithine carbamoyl transferase (OCT) in a patient with OCT deficiency.
|
Some kinetic properties of liver OCT from a patient with OCT deficiency were studied. Contrary to controls, in which two pH optima were observed (pH 7.7 and pH 8.5), only the pH optimum of 8.5 could be demonstrated in our patient. From KM studies at pH 7.7 and pH 8.5, the most striking abnormalities in comparison with human controls were (a) a strongly increased KM (ornithine) at pH 7.7, but less pronounced at pH 8.5, (b) a higher VMAX at pH 8.5 compared with the VMAX at pH 7.7 and (c) the absence of substate inhibition at pH 8.5 to ornithine was elevated up to a concentration above approximately 1.5 mM.
|
Some kinetic properites of liver ornithine carbamoyl transferase (OCT) in a patient with OCT deficiency. Some kinetic properties of liver OCT from a patient with OCT deficiency were studied. Contrary to controls, in which two pH optima were observed (pH 7.7 and pH 8.5), only the pH optimum of 8.5 could be demonstrated in our patient. From KM studies at pH 7.7 and pH 8.5, the most striking abnormalities in comparison with human controls were (a) a strongly increased KM (ornithine) at pH 7.7, but less pronounced at pH 8.5, (b) a higher VMAX at pH 8.5 compared with the VMAX at pH 7.7 and (c) the absence of substate inhibition at pH 8.5 to ornithine was elevated up to a concentration above approximately 1.5 mM.
|
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] |
PMID:21056
|
The effect of anti-inflammatory and antirheumatic drugs on inflammation in the rat.
|
The anti-inflammatory effects of flurbiprofen and a number of other antirheumatic agents were studied in the rat using a model of inflammation, which involved the subcutaneous implantation of polyurethane cubes impregnated with an irritant. Results of acute and chronic studies indicated that flurbiprofen was extremely effective in suppressing both fluid and cellular phases of inflammation and, in this model, was comparable in potency to prednisolone.
|
The effect of anti-inflammatory and antirheumatic drugs on inflammation in the rat. The anti-inflammatory effects of flurbiprofen and a number of other antirheumatic agents were studied in the rat using a model of inflammation, which involved the subcutaneous implantation of polyurethane cubes impregnated with an irritant. Results of acute and chronic studies indicated that flurbiprofen was extremely effective in suppressing both fluid and cellular phases of inflammation and, in this model, was comparable in potency to prednisolone.
|
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] |
PMID:21054
|
The effects of urine pH and plasma protein binding on the renal clearance of disopyramide.
|
To ascertain whether the renal clearance of disopyramide (pKa = 8.36) is affected by urine pH, the disposition kinetics of disopyramide were compared during excretion of acidic and alkaline urine following both single dose intravenous (2mg/kg) and oral (5 mg/kg) administration to 4 healthy male volunteers. No significant difference was observed in the plasma concentration-time curve of disopyramide. The mean 72 hour recovery of disopyramide and its N-deisopropyl metabolite (MND) in urine was 55.1 and 20.3% of the dose respectively, with no apparent difference between the two routes of administration or pH of urine. Renal clearance of disopyramide was found to vary with time, which is partly the result of a concentration dependent change in plasma protein binding. The unbound fraction of drug in plasma varied from 0.32 to 0.72 between 0.4 to 4microgram/ml concentration. However, time-dependent change in renal clearance of disopyramide persists even after correction for plasma protein binding.
|
The effects of urine pH and plasma protein binding on the renal clearance of disopyramide. To ascertain whether the renal clearance of disopyramide (pKa = 8.36) is affected by urine pH, the disposition kinetics of disopyramide were compared during excretion of acidic and alkaline urine following both single dose intravenous (2mg/kg) and oral (5 mg/kg) administration to 4 healthy male volunteers. No significant difference was observed in the plasma concentration-time curve of disopyramide. The mean 72 hour recovery of disopyramide and its N-deisopropyl metabolite (MND) in urine was 55.1 and 20.3% of the dose respectively, with no apparent difference between the two routes of administration or pH of urine. Renal clearance of disopyramide was found to vary with time, which is partly the result of a concentration dependent change in plasma protein binding. The unbound fraction of drug in plasma varied from 0.32 to 0.72 between 0.4 to 4microgram/ml concentration. However, time-dependent change in renal clearance of disopyramide persists even after correction for plasma protein binding.
|
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] |
PMID:21059
|
Use of EMG biofeedback in behavioral treatment of an obsessive-phobic-depressive syndrome.
|
A 25 year old woman who suffered from depression (associated with obsessive, phobic thoughts of animal abuse or neglect) was treated with systematic desensitization in which EMG biofeedback was used to achieve relaxation. EMG responses were also used to aid both the therapist and the patient in the pairing of hierarchical imagery and muscular relaxation during desensitization, EMG responses were particularly useful in detecting rising muscular tension, indicating when visualization of a depression-provoking situation should be terminated. Ratings concerning frequency and intensity of depressive thoughts (made by the patient before and after therapy), as well as 6-month follow-up information, attest to the success of the therapeutic program.
|
Use of EMG biofeedback in behavioral treatment of an obsessive-phobic-depressive syndrome. A 25 year old woman who suffered from depression (associated with obsessive, phobic thoughts of animal abuse or neglect) was treated with systematic desensitization in which EMG biofeedback was used to achieve relaxation. EMG responses were also used to aid both the therapist and the patient in the pairing of hierarchical imagery and muscular relaxation during desensitization, EMG responses were particularly useful in detecting rising muscular tension, indicating when visualization of a depression-provoking situation should be terminated. Ratings concerning frequency and intensity of depressive thoughts (made by the patient before and after therapy), as well as 6-month follow-up information, attest to the success of the therapeutic program.
|
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] |
PMID:21065
|
The permissive role of glucocorticoids in the development of ethanol dependence and tolerance.
|
In mice chronically treated with ethanol (in a liquid diet containing 6% ethanol ad libitum for 2 weeks), brain tryptophan hydroxylase (TPH) activity was increased (by 30-45% in whole brain), while brain tyrosine hydroxylase activity remained unchanged. Such chronic ethanol treatment also induced susceptibility to audiogenic seizures during withdrawal (60% incidence). When ethanol treatment was given to adrenalectomized (Adx) mice, the increase of brain TPH activity and the development of withdrawal audiogenic seizures were both prevented. In Adx mice receiving daily injections of corticosterone (0.5 mg/mouse), the ethanol-induced increase of brain TPH activity and the occurrence of withdrawal audiogenic seizures were both restored. Similarly, the ethanol-induced increase of liver alcohol dehydrogenase activity (by 60%) was prevented in Adx mice and restored by corticosterone replacement. It was noted that in all three cases replacement with such large doses of the corticoid did not enhance the ethanol effects, but merely restored the effects to the levels observed in intact mice. Apparently, glucocorticoids are required in a permissive role in order for the ethanol effects to occur.
|
The permissive role of glucocorticoids in the development of ethanol dependence and tolerance. In mice chronically treated with ethanol (in a liquid diet containing 6% ethanol ad libitum for 2 weeks), brain tryptophan hydroxylase (TPH) activity was increased (by 30-45% in whole brain), while brain tyrosine hydroxylase activity remained unchanged. Such chronic ethanol treatment also induced susceptibility to audiogenic seizures during withdrawal (60% incidence). When ethanol treatment was given to adrenalectomized (Adx) mice, the increase of brain TPH activity and the development of withdrawal audiogenic seizures were both prevented. In Adx mice receiving daily injections of corticosterone (0.5 mg/mouse), the ethanol-induced increase of brain TPH activity and the occurrence of withdrawal audiogenic seizures were both restored. Similarly, the ethanol-induced increase of liver alcohol dehydrogenase activity (by 60%) was prevented in Adx mice and restored by corticosterone replacement. It was noted that in all three cases replacement with such large doses of the corticoid did not enhance the ethanol effects, but merely restored the effects to the levels observed in intact mice. Apparently, glucocorticoids are required in a permissive role in order for the ethanol effects to occur.
|
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] |
PMID:21068
|
Alclofenac: a review of its pharmacological properties and therapeutic efficacy in rheumatoid arthritis and allied rheumatic disorders.
|
Alclofenac is a non-steroidal anti-inflammatory agent advocated for use in rheumatoid arthritis, degenerative joint disease and ankylosing spondylitis. Published data to date, suggest that alclofenac 3g daily is comparable in efficacy with aspirin 4.8g daily, phenylbutazone 300 to 600mg daily and indomethacin 150mg daily. In Welsh patients, gastro-intestinal side-effects have generally been less frequent and milder than with the standard comparison drugs, but in other populations differences in the overall incidence of these side-effects have been less marked. Results of a long-term trial, as evidenced by alterations in certain biochemical indications of disease activity, suggest that alclofenac may possibly reduce the severity of the disease itself, but further studies will be needed to confirm this. However, at present alclofenac should be considered along with the other drugs of its type in the initial treatment of the arthritic patient. Skin rash is the most frequent side-effect, which in a small proportion of affected patients may be associated with systemic effects. A cutaneous reaction appears to be more likely in patients with a history of previous allergy to penicillin and other drugs.
|
Alclofenac: a review of its pharmacological properties and therapeutic efficacy in rheumatoid arthritis and allied rheumatic disorders. Alclofenac is a non-steroidal anti-inflammatory agent advocated for use in rheumatoid arthritis, degenerative joint disease and ankylosing spondylitis. Published data to date, suggest that alclofenac 3g daily is comparable in efficacy with aspirin 4.8g daily, phenylbutazone 300 to 600mg daily and indomethacin 150mg daily. In Welsh patients, gastro-intestinal side-effects have generally been less frequent and milder than with the standard comparison drugs, but in other populations differences in the overall incidence of these side-effects have been less marked. Results of a long-term trial, as evidenced by alterations in certain biochemical indications of disease activity, suggest that alclofenac may possibly reduce the severity of the disease itself, but further studies will be needed to confirm this. However, at present alclofenac should be considered along with the other drugs of its type in the initial treatment of the arthritic patient. Skin rash is the most frequent side-effect, which in a small proportion of affected patients may be associated with systemic effects. A cutaneous reaction appears to be more likely in patients with a history of previous allergy to penicillin and other drugs.
|
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] |
PMID:21070
|
Treatment of chronic schizophrenia.
|
The comprehensive treatment of schizophrenia requires the full resources of a clinical team that is able to offer treatment for the acute psychotic state in a hospital environment, and appropriate rehabilitation following resolution of the florid symptoms. Following a second or subsequent relapse, maintenance therapy with long-acting injections of depot antipsychotics will be required for an unknown period. Although drugs form an essential part of all treatments, it is essential to examine the environment for precipitating factors and to involve the patient's family in the rehabilitation. Recent studies have reported that some patients still have a relatively poor prognosis; although this proportion may be in the minority, the strain on the whole family of an even moderately handicapped patient can be enormous, and it is important to examine the needs of the whole family in evaluating care within the community. The effect of antipsychotic drugs is much wider that the mere control of acute symptoms and can influence the patterns of social behaviour and rehabilitation, in addition to offering protection against stress. The proper use of depot injections requires that they be kept under constant review. The current widespread practice of prescribing anti-cholinergic drugs on a prophylactic basis, or even as the inital treatment of extrapyramidal side-effects, needs revision.
|
Treatment of chronic schizophrenia. The comprehensive treatment of schizophrenia requires the full resources of a clinical team that is able to offer treatment for the acute psychotic state in a hospital environment, and appropriate rehabilitation following resolution of the florid symptoms. Following a second or subsequent relapse, maintenance therapy with long-acting injections of depot antipsychotics will be required for an unknown period. Although drugs form an essential part of all treatments, it is essential to examine the environment for precipitating factors and to involve the patient's family in the rehabilitation. Recent studies have reported that some patients still have a relatively poor prognosis; although this proportion may be in the minority, the strain on the whole family of an even moderately handicapped patient can be enormous, and it is important to examine the needs of the whole family in evaluating care within the community. The effect of antipsychotic drugs is much wider that the mere control of acute symptoms and can influence the patterns of social behaviour and rehabilitation, in addition to offering protection against stress. The proper use of depot injections requires that they be kept under constant review. The current widespread practice of prescribing anti-cholinergic drugs on a prophylactic basis, or even as the inital treatment of extrapyramidal side-effects, needs revision.
|
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] |
PMID:21073
|
Physical studies on the conformation of ribosomal protein S4 from Escherichia coli.
|
Proton magnetic resonance, circular dichroism and infrared spectroscopy were used to investigate the secondary and tertiary structure of the 16-S RNA binding protein S4 from Escherichia coli ribosomes. The proton magnetic resonance spectra of protein S4 in ribosomal reconstitution and low-salt buffers were identical and showed little dipolar broadening of the peaks, suggesting that the protein had an open extended structure. A ring-current-shifted apolar methyl resonance in the high-field region of the spectrum, together with a perturbation of the tyrosine ring proton resonance in the low-field region, indicated the existence of a specific tertiary fold in the polypeptide chain. This structure disappeared on lowering the pH below 5 or on heating above 30 degrees C, both processes being reversible. Circular dichroism measurements on protein S4 showed an alpha-helix content of 32% in reconstitution buffer compared with 26% in low-salt buffer. Heating the protein solution in reconstitution buffer above 35 degrees C reversibly disrupted this extra helix. Infrared studies on both solid films and solutions of protein S4 indicated the presence of little or no beta-structure. These results correlate well with the known RNA binding properties of protein S4.
|
Physical studies on the conformation of ribosomal protein S4 from Escherichia coli. Proton magnetic resonance, circular dichroism and infrared spectroscopy were used to investigate the secondary and tertiary structure of the 16-S RNA binding protein S4 from Escherichia coli ribosomes. The proton magnetic resonance spectra of protein S4 in ribosomal reconstitution and low-salt buffers were identical and showed little dipolar broadening of the peaks, suggesting that the protein had an open extended structure. A ring-current-shifted apolar methyl resonance in the high-field region of the spectrum, together with a perturbation of the tyrosine ring proton resonance in the low-field region, indicated the existence of a specific tertiary fold in the polypeptide chain. This structure disappeared on lowering the pH below 5 or on heating above 30 degrees C, both processes being reversible. Circular dichroism measurements on protein S4 showed an alpha-helix content of 32% in reconstitution buffer compared with 26% in low-salt buffer. Heating the protein solution in reconstitution buffer above 35 degrees C reversibly disrupted this extra helix. Infrared studies on both solid films and solutions of protein S4 indicated the presence of little or no beta-structure. These results correlate well with the known RNA binding properties of protein S4.
|
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] |
PMID:21074
|
Influence of 1,2,3-benzene-tricarboxylate on pyruvate metabolism in rat-liver mitochondria.
|
1,2,3-Benzene-tricarboxylate, a known inhibitor of the mitochondrial tricarboxylate carrier, was found to inhibit pyruvate carboxylation as well as the transport of citrate out of the matrix in rat liver mitochondria incubated with pyruvate. The inhibition of pyruvate carboxylation was observed with both intact mitochondria and with the solubilized pyruvate carboxylase. The inhibition of the pyruvate carboxylase by 1,2,3-benzene-tricarboxylase was not mediated via one of the parameters known to regulate the activity of the enzyme and therefore a direct inhibition of the enzyme by the tricarboxylate was assumed. Since the pyruvate carboxylase is exclusively localized in the mitochondrial matrix space it was concluded that 1,2,3-benzene-tricarboxylate penetrates into this compartment.
|
Influence of 1,2,3-benzene-tricarboxylate on pyruvate metabolism in rat-liver mitochondria. 1,2,3-Benzene-tricarboxylate, a known inhibitor of the mitochondrial tricarboxylate carrier, was found to inhibit pyruvate carboxylation as well as the transport of citrate out of the matrix in rat liver mitochondria incubated with pyruvate. The inhibition of pyruvate carboxylation was observed with both intact mitochondria and with the solubilized pyruvate carboxylase. The inhibition of the pyruvate carboxylase by 1,2,3-benzene-tricarboxylase was not mediated via one of the parameters known to regulate the activity of the enzyme and therefore a direct inhibition of the enzyme by the tricarboxylate was assumed. Since the pyruvate carboxylase is exclusively localized in the mitochondrial matrix space it was concluded that 1,2,3-benzene-tricarboxylate penetrates into this compartment.
|
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] |
PMID:21076
|
Prostatic binding protein. A steriod-binding protein secreted by rat prostate.
|
Rat prostatic cytosol contains a high concentration of a prostatic binding protein with peculiar steroid-binding properties. Indeed, in spite of a relatively low affinity, charcoal adsorption can be used for its measurement. Furthermore, the binding is not specific for particular steroids and increases very strongly after delipidation. In delipidated cytosol the concentration of the binding site is 3.1 micronmol/g protein and the apparent affinity for pregenolone 1.7 X 10(6) M-1. The high concentration of prostatic binding protein in prostatic fluid shows that this substance is secreted by the prostate. Prostatic binding protein has the following physicochemical characteristics: it is precipitated by ammonium sulfate between 50 and 70% saturation; the elution position from a Sephadex G-100 column corresponds to a molecular weight of 51000; it sediments in sucrose density gradients at 3.7 S and is eluted from DEAE-cellulose columns at about 0.25 M KCl. On polyacrylamide gel electrophoresis the binding activity coincides with the major cytosolic protein band. This band has the same mobility as serum albumin in 7% gels, but a higher mobility in more concentrated gels.
|
Prostatic binding protein. A steriod-binding protein secreted by rat prostate. Rat prostatic cytosol contains a high concentration of a prostatic binding protein with peculiar steroid-binding properties. Indeed, in spite of a relatively low affinity, charcoal adsorption can be used for its measurement. Furthermore, the binding is not specific for particular steroids and increases very strongly after delipidation. In delipidated cytosol the concentration of the binding site is 3.1 micronmol/g protein and the apparent affinity for pregenolone 1.7 X 10(6) M-1. The high concentration of prostatic binding protein in prostatic fluid shows that this substance is secreted by the prostate. Prostatic binding protein has the following physicochemical characteristics: it is precipitated by ammonium sulfate between 50 and 70% saturation; the elution position from a Sephadex G-100 column corresponds to a molecular weight of 51000; it sediments in sucrose density gradients at 3.7 S and is eluted from DEAE-cellulose columns at about 0.25 M KCl. On polyacrylamide gel electrophoresis the binding activity coincides with the major cytosolic protein band. This band has the same mobility as serum albumin in 7% gels, but a higher mobility in more concentrated gels.
|
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] |
PMID:21079
|
Yeast pyruvate kinase: a mutant from catalytically insensitive to fructose 1,6-bisphosphate.
|
The paper describes some of the characteristic properties of an altered form of pyruvate kinase from a mutant of Saccharomyces cerevisiae. The partially purified enzyme does not require fructose 1,6-bisphosphate for activity but is stabilised in its presence both at low and at high temperatures. The enzyme displays in the absence of fructose 1,6-bisphosphate hyperbolic kinetics with phosphoenolpyruvate (Km, 0.11 mM), ADP (Km, 0.12 mM) and K+ (Km, 11 mM). Sedimentation velocity experiments indicate that the mutated enzyme and the wild type enzyme have S20,w values of 8.9 and 8.6 S respectively. The mutant with the pyruvate insensitive to fructose 1.6-bisphosphate is capable of growing on synthetic media with alcohol or malate as the sole carbon source. The steady-state intracellular levels of phosphoenolpyruvate in the mutant suggest mechanisms that prevent depletion of this metabolite despite an active pyruvate kinase. Spontaneous reversion of this mutant yields clones with normal enzyme activated by fructose 1,6-bisphosphate.
|
Yeast pyruvate kinase: a mutant from catalytically insensitive to fructose 1,6-bisphosphate. The paper describes some of the characteristic properties of an altered form of pyruvate kinase from a mutant of Saccharomyces cerevisiae. The partially purified enzyme does not require fructose 1,6-bisphosphate for activity but is stabilised in its presence both at low and at high temperatures. The enzyme displays in the absence of fructose 1,6-bisphosphate hyperbolic kinetics with phosphoenolpyruvate (Km, 0.11 mM), ADP (Km, 0.12 mM) and K+ (Km, 11 mM). Sedimentation velocity experiments indicate that the mutated enzyme and the wild type enzyme have S20,w values of 8.9 and 8.6 S respectively. The mutant with the pyruvate insensitive to fructose 1.6-bisphosphate is capable of growing on synthetic media with alcohol or malate as the sole carbon source. The steady-state intracellular levels of phosphoenolpyruvate in the mutant suggest mechanisms that prevent depletion of this metabolite despite an active pyruvate kinase. Spontaneous reversion of this mutant yields clones with normal enzyme activated by fructose 1,6-bisphosphate.
|
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] |
PMID:21083
|
A new factor from enteric bacteria of rats amplifying induction of liver enzyme by glucocorticoid. 1. Purification, properties and biological action.
|
1. A factor, which amplifies the inductions of several liver enzymes by glucocorticoid, was partially purified from Proteus mirabilis from rat intestine. The factor (amplifier) was completely inactivated by alpha-glucosidase, but not by other glycoside hydrolases, proteases, nucleases or phosphatases tested; it was also hydrolysed by HCl with liberation of reducing sugars. Thus the oligosaccharide in this factor seems to be essential for the amplification. 2. In adrenalectomized rats the amplifier increased the inductions of several liver enzymes, such as tyrosine aminotransferase and leucine aminotransferase, by glucocorticoid. But it did not amplify the induction of tyrosine aminotransferase by glucagon or insulin or the activities of enzymes that are not induced by glucocorticoid. The amplifier by itself did not have any glucocorticoid-like action in adrenalectomized rat. These results show that the amplifier specifically increases the inductions of liver enzymes by glucocorticoid. 3. Since similar amplification was also observed in isolated perfused liver and cultured hepatoma cells in vitro, the amplifier seems to act directly on the target organ or cells.
|
A new factor from enteric bacteria of rats amplifying induction of liver enzyme by glucocorticoid. 1. Purification, properties and biological action. 1. A factor, which amplifies the inductions of several liver enzymes by glucocorticoid, was partially purified from Proteus mirabilis from rat intestine. The factor (amplifier) was completely inactivated by alpha-glucosidase, but not by other glycoside hydrolases, proteases, nucleases or phosphatases tested; it was also hydrolysed by HCl with liberation of reducing sugars. Thus the oligosaccharide in this factor seems to be essential for the amplification. 2. In adrenalectomized rats the amplifier increased the inductions of several liver enzymes, such as tyrosine aminotransferase and leucine aminotransferase, by glucocorticoid. But it did not amplify the induction of tyrosine aminotransferase by glucagon or insulin or the activities of enzymes that are not induced by glucocorticoid. The amplifier by itself did not have any glucocorticoid-like action in adrenalectomized rat. These results show that the amplifier specifically increases the inductions of liver enzymes by glucocorticoid. 3. Since similar amplification was also observed in isolated perfused liver and cultured hepatoma cells in vitro, the amplifier seems to act directly on the target organ or cells.
|
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] |
PMID:21084
|
A new factor from enteric bacteria of rats amplifying induction of liver enzyme by glucocorticoid. 2. Mechanism of action.
|
1. An amplifier of the action of glucocorticoid was purified from Proteus mirabilis as described previously. It was found that it amplified the induction of liver tyrosine aminotransferase by dexamethasone markedly with doses of dexamethasone that caused minimal enzyme induction, but had little effect with doses that caused maximal induction. Thus the amplification may represent a saving of glucocorticoid. The amplification of enzyme activity was brought about by increase in amount of enzyme. 2. The amplification was observed when the amplifier was administered before or with dexamethasone, but not when it was given 2 h after dexamethasone. These results and the finding that actinomycin D inhibited the amplification indicate that the amplifier does not act on the translational level of enzyme induction. 3. It was found that the amplifier increased both incorporation of [3H]dexamethasone into the cytosol and binding of [3H]dexamethasone of cytosol protein and that it decreased decay of the [3H]dexamethasone-protein complex.
|
A new factor from enteric bacteria of rats amplifying induction of liver enzyme by glucocorticoid. 2. Mechanism of action. 1. An amplifier of the action of glucocorticoid was purified from Proteus mirabilis as described previously. It was found that it amplified the induction of liver tyrosine aminotransferase by dexamethasone markedly with doses of dexamethasone that caused minimal enzyme induction, but had little effect with doses that caused maximal induction. Thus the amplification may represent a saving of glucocorticoid. The amplification of enzyme activity was brought about by increase in amount of enzyme. 2. The amplification was observed when the amplifier was administered before or with dexamethasone, but not when it was given 2 h after dexamethasone. These results and the finding that actinomycin D inhibited the amplification indicate that the amplifier does not act on the translational level of enzyme induction. 3. It was found that the amplifier increased both incorporation of [3H]dexamethasone into the cytosol and binding of [3H]dexamethasone of cytosol protein and that it decreased decay of the [3H]dexamethasone-protein complex.
|
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] |
PMID:21085
|
gamma-Glutamyl transpeptidase in rat ascites tumor cell LY-5. Lack of functional correlation of its catalytic activity with the amino acid transport.
|
gamma-Glutamyl transpeptidase activity was detected in rat ascites tumor cells (LY-5) suspended in Hanks' balanced saline solution using L-gamma-glutamyl-p-nitroanilide as a substrate. Whole-cell suspension of the tumor cells exhibited full activity of the enzyme without detectable cell disruption under the conditions examined. Various amino acids, transported through specific membrane carriers, did not affect the accessibility of substrate for the enzyme. An inhibitor of sodium-dependent transport systems of amino acids caused no significant change in the rate of enzyme catalysis. Like glutathione or S-methylglutathione, S-acetyldextran (mol. wt 215000) derivative of glutathione, which is believed to be unable to penetrate into intact cells, caused marked inhibition of the rate of p-nitroaniline release from the synthetic substrate by the tumor cells. These data indicated that the active site of the enzyme faced to the outer surface of the cells. gamma-Glutamyl transpeptidase of the tumor cells was successfully affinity-labeled by 6-diazo-5-oxo-L-norleucine, a glutamine analog, without causing detectable change in the viability of the cells under the conditions examined. The rate of transport of alanine, leucine, glycine and glutamine into cells was not affected by the inactivation of this enzyme with the affinity label. Thus, the activity of gamma-glutamyl transpeptidase located on the outer surface of tumor cell membrane does not seem to be requisite for the transport process of amino acids.
|
gamma-Glutamyl transpeptidase in rat ascites tumor cell LY-5. Lack of functional correlation of its catalytic activity with the amino acid transport. gamma-Glutamyl transpeptidase activity was detected in rat ascites tumor cells (LY-5) suspended in Hanks' balanced saline solution using L-gamma-glutamyl-p-nitroanilide as a substrate. Whole-cell suspension of the tumor cells exhibited full activity of the enzyme without detectable cell disruption under the conditions examined. Various amino acids, transported through specific membrane carriers, did not affect the accessibility of substrate for the enzyme. An inhibitor of sodium-dependent transport systems of amino acids caused no significant change in the rate of enzyme catalysis. Like glutathione or S-methylglutathione, S-acetyldextran (mol. wt 215000) derivative of glutathione, which is believed to be unable to penetrate into intact cells, caused marked inhibition of the rate of p-nitroaniline release from the synthetic substrate by the tumor cells. These data indicated that the active site of the enzyme faced to the outer surface of the cells. gamma-Glutamyl transpeptidase of the tumor cells was successfully affinity-labeled by 6-diazo-5-oxo-L-norleucine, a glutamine analog, without causing detectable change in the viability of the cells under the conditions examined. The rate of transport of alanine, leucine, glycine and glutamine into cells was not affected by the inactivation of this enzyme with the affinity label. Thus, the activity of gamma-glutamyl transpeptidase located on the outer surface of tumor cell membrane does not seem to be requisite for the transport process of amino acids.
|
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] |
PMID:21086
|
Hydrogen-ion binding by tobacco-mosaic-virus protein polymers.
|
Hydrogen ion titration curves of tobacco mosaic virus protein have been measured in various conditions of protein concentration, temperature, ionic strength, and rate of pH change. The polymers present at each stage are deduced from turbidity and sedimentation data, plus published information. A simple semi-quantitative analysis of the curves is given, and the pK values of the two abnormal carboxylates in single helix are estimated as 6.4 and about 7.0. Disks, and some faster-forming unknown polymers in the same size range, have been abnormal carboxylate with pK 6.9. These results are most easily interpreted in terms of electrostatic interactions between carboxylates, probably at the axial ends of the protein subunits.
|
Hydrogen-ion binding by tobacco-mosaic-virus protein polymers. Hydrogen ion titration curves of tobacco mosaic virus protein have been measured in various conditions of protein concentration, temperature, ionic strength, and rate of pH change. The polymers present at each stage are deduced from turbidity and sedimentation data, plus published information. A simple semi-quantitative analysis of the curves is given, and the pK values of the two abnormal carboxylates in single helix are estimated as 6.4 and about 7.0. Disks, and some faster-forming unknown polymers in the same size range, have been abnormal carboxylate with pK 6.9. These results are most easily interpreted in terms of electrostatic interactions between carboxylates, probably at the axial ends of the protein subunits.
|
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] |
PMID:21089
|
Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 1. Calorimetric and potentiometric titration of the native apoA-I protein and of the apoA-I protein-dimyristoyl lecithin complex.
|
The ionization behaviour of native apoA-I protein is compare to that of its complex with synthetic dimyristoyl lecithin in studies using calorimetric, potentiometric and spectrophotometric titration. In the presence of phospholipids, 10 out of 21 lysines together with 22 acidic residues are masked in the complex. All tyrosines remain accessible to titration below pH 13. The apparent ionization enthalpy of the 11 lysine residues is not affected by the presence of phospholipids. These data are consistent with discrete binding sites located in the apoprotein helical segments as suggested by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. A tentative localisation of lysine, arginine, aspartic acid and glutamic acid residues directly involved in phospholipid binding is suggested, assuming that such helical regions are involved in apoprotein-phospholipid association.
|
Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 1. Calorimetric and potentiometric titration of the native apoA-I protein and of the apoA-I protein-dimyristoyl lecithin complex. The ionization behaviour of native apoA-I protein is compare to that of its complex with synthetic dimyristoyl lecithin in studies using calorimetric, potentiometric and spectrophotometric titration. In the presence of phospholipids, 10 out of 21 lysines together with 22 acidic residues are masked in the complex. All tyrosines remain accessible to titration below pH 13. The apparent ionization enthalpy of the 11 lysine residues is not affected by the presence of phospholipids. These data are consistent with discrete binding sites located in the apoprotein helical segments as suggested by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. A tentative localisation of lysine, arginine, aspartic acid and glutamic acid residues directly involved in phospholipid binding is suggested, assuming that such helical regions are involved in apoprotein-phospholipid association.
|
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] |
PMID:21090
|
Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 2. Potentiometric titration of the native apo-A-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin.
|
A comparison of the ionization behaviour of the human apoA-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin is based on potentiometric titration of the basic and acidic residues and spectrophotometric titration of the phenolic groups. Experimental data suggest that a number of lysine, arginine, aspartic acid and glutamic acid residues are masked in the complexes. For each of these amino acids and in all three proteins the number of masked residues is consistent with the content of those regions predicted to be involved in lipid binding by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. These data taken together with the results of calorimetric and titration experiments with the apoA-I protein reported in the accompanying article [Rosseneu et al. (1977) Eur. J. Biochem. 79, 251-257] strongly support the general nature of the proposed model and further suggest that ionic interactions have some role in the formation of the dimyristoyl lecithin/apolipoprotein complexes.
|
Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 2. Potentiometric titration of the native apo-A-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin. A comparison of the ionization behaviour of the human apoA-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin is based on potentiometric titration of the basic and acidic residues and spectrophotometric titration of the phenolic groups. Experimental data suggest that a number of lysine, arginine, aspartic acid and glutamic acid residues are masked in the complexes. For each of these amino acids and in all three proteins the number of masked residues is consistent with the content of those regions predicted to be involved in lipid binding by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. These data taken together with the results of calorimetric and titration experiments with the apoA-I protein reported in the accompanying article [Rosseneu et al. (1977) Eur. J. Biochem. 79, 251-257] strongly support the general nature of the proposed model and further suggest that ionic interactions have some role in the formation of the dimyristoyl lecithin/apolipoprotein complexes.
|
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] |
PMID:21092
|
Substrate specificity via ternary complex formation with glutamate dehydrogenase.
|
Very littly discrimination is observed in the binary binding of dicarboxylic acid substrate analogues to glutamate dehydrogenase as monitored by proton nuclear magnetic resonance. Variation in length, charge, bulkiness and conformational rigidity resulted in only a factor of five variation in KD and apparent relaxation time, T2. Upon titration of the binary enzyme-ligand complex with coenzyme to form the ternary enzyme-ligand-coenzyme complex strong discrimination is observed. Coenzyme binds tightly only when the correct substrate is present, otherwise it binds 10 to 150 times more weakly.
|
Substrate specificity via ternary complex formation with glutamate dehydrogenase. Very littly discrimination is observed in the binary binding of dicarboxylic acid substrate analogues to glutamate dehydrogenase as monitored by proton nuclear magnetic resonance. Variation in length, charge, bulkiness and conformational rigidity resulted in only a factor of five variation in KD and apparent relaxation time, T2. Upon titration of the binary enzyme-ligand complex with coenzyme to form the ternary enzyme-ligand-coenzyme complex strong discrimination is observed. Coenzyme binds tightly only when the correct substrate is present, otherwise it binds 10 to 150 times more weakly.
|
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] |
PMID:21093
|
Reversible graft versus host reaction as cause of erythrophagic splenomegaly in a child?
|
The case history of a 9 months old infant with hepatosplenomegaly, pancytopnaenia and disturbances of clotting and cellular immune reactivity is reported. The spleen was removed and showed striking erythrophagocytosis by proliferating histiocytes, typical of "familial erythrophagocytic reticulosis" (Farquhar). A graft-versus-host reaction is discussed as a possible underlying cause. The favourable clinical course and full recovery point to an interrelation with primary hypersplenism.
|
Reversible graft versus host reaction as cause of erythrophagic splenomegaly in a child? The case history of a 9 months old infant with hepatosplenomegaly, pancytopnaenia and disturbances of clotting and cellular immune reactivity is reported. The spleen was removed and showed striking erythrophagocytosis by proliferating histiocytes, typical of "familial erythrophagocytic reticulosis" (Farquhar). A graft-versus-host reaction is discussed as a possible underlying cause. The favourable clinical course and full recovery point to an interrelation with primary hypersplenism.
|
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] |
PMID:21094
|
Interstitial cystitis. Observations on diagnosis and on treatment with anti-inflammatory drugs, particularly benzydamine.
|
Interstitial cystitis is often missed on "routine" cystoscopy. If the disease is suspected, the bladder must be observed twice during complete filling. An anti-inflammatory drug, Benzydamine, has given mixed results but in view of its low toxicity it is worth trying in any patient with severe symptoms.
|
Interstitial cystitis. Observations on diagnosis and on treatment with anti-inflammatory drugs, particularly benzydamine. Interstitial cystitis is often missed on "routine" cystoscopy. If the disease is suspected, the bladder must be observed twice during complete filling. An anti-inflammatory drug, Benzydamine, has given mixed results but in view of its low toxicity it is worth trying in any patient with severe symptoms.
|
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] |
PMID:21095
|
Prune belly syndrome: treatment of terminal renal failure by hemodialysis and renal transplantation.
|
The second case of successful renal transplantation in a patient with "prune belly" syndrome is reported. In spite of early aggressive surgical approach in the management of this disease terminal renal failure frequently ensues. Hemodialysis and renal transplantation have offered new possibilities of prolonging life in these patients. The success of renal transplantation depends on the anatomic and functional state of the lower urinary tract. Pretransplant urologic examination is extremely important for the evaluation of urinary tract abnormalities.
|
Prune belly syndrome: treatment of terminal renal failure by hemodialysis and renal transplantation. The second case of successful renal transplantation in a patient with "prune belly" syndrome is reported. In spite of early aggressive surgical approach in the management of this disease terminal renal failure frequently ensues. Hemodialysis and renal transplantation have offered new possibilities of prolonging life in these patients. The success of renal transplantation depends on the anatomic and functional state of the lower urinary tract. Pretransplant urologic examination is extremely important for the evaluation of urinary tract abnormalities.
|
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] |
PMID:21097
|
Acid mucopolysaccharides in fibroblast cultures. 4. 35S-sulfate incorporation in dependence on pH-value cell density and lactate.
|
Cultures of embryonic rat fibroblasts were incubated with 35S-sulfate at pH 6.6 and 7.4 (Eagle medium, Hepes buffer) for 48 hours. The MPS were isolated and fractionated. Determination of hexuronic acid was done according to BITTER and MUIR, measuring of sulfate incorporation by liquid scintillation counting. 1. HS, Ch-6-S and DS showed different synthesis rates. They rised at increasing cell density and were lower at pH 6.6 than at 7.4. 2. At pH 6.6 the sulfate incorporation rates (de novo-synthesis) of HS, Ch-6-S and DS are lowered. The percent increase of the MPS fractions at pH 6.6 and their specific activities are explained by individual differences in the unlabelled MPS pools as well as by differences in the rates of synthesis and catabolism. The cell density also has an influence on that. Increase of the MPS at pH 6.6 is by mainly due to an inhibition of catabolism. 3. Contrary to CH-6-S and DS heparan sulfate is catabolized more slowly at high cell densities. 4. The volume of the unlabelled MPS pool is modified by lactate at pH 6.6, e.g. it effectuates enhancement of the DS pool at low cell densities. De novo-synthesis and specific activity are scarcely influenced. CH-6-S and DS catabolism are considerably inhibited, their total amount is increased. The relative synthesis rate of DS is enhanced by lactate. 5. The possible importance of the results for inflammatory processes and wound healing is mentioned.
|
Acid mucopolysaccharides in fibroblast cultures. 4. 35S-sulfate incorporation in dependence on pH-value cell density and lactate. Cultures of embryonic rat fibroblasts were incubated with 35S-sulfate at pH 6.6 and 7.4 (Eagle medium, Hepes buffer) for 48 hours. The MPS were isolated and fractionated. Determination of hexuronic acid was done according to BITTER and MUIR, measuring of sulfate incorporation by liquid scintillation counting. 1. HS, Ch-6-S and DS showed different synthesis rates. They rised at increasing cell density and were lower at pH 6.6 than at 7.4. 2. At pH 6.6 the sulfate incorporation rates (de novo-synthesis) of HS, Ch-6-S and DS are lowered. The percent increase of the MPS fractions at pH 6.6 and their specific activities are explained by individual differences in the unlabelled MPS pools as well as by differences in the rates of synthesis and catabolism. The cell density also has an influence on that. Increase of the MPS at pH 6.6 is by mainly due to an inhibition of catabolism. 3. Contrary to CH-6-S and DS heparan sulfate is catabolized more slowly at high cell densities. 4. The volume of the unlabelled MPS pool is modified by lactate at pH 6.6, e.g. it effectuates enhancement of the DS pool at low cell densities. De novo-synthesis and specific activity are scarcely influenced. CH-6-S and DS catabolism are considerably inhibited, their total amount is increased. The relative synthesis rate of DS is enhanced by lactate. 5. The possible importance of the results for inflammatory processes and wound healing is mentioned.
|
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] |
PMID:21106
|
[Analysis of the effect of bicarbonate on taste receptor sensitivity to organic acids].
|
Addition of NaHCO3 or NaOH to the solutions of lactic or pyruvic acids decreased threshold concentrations of H+ ions and increased intensity of afferent impulsation in the cat chroda tympani nerve. In spite of the fact that the concentrations of both lactate and pyruvate anions with the presence of NaOH and NaHCO3 are the same at equal pH values the observed changes of the receptors responses were more intensive if NaHCO3 had been added. Therefore the influence of NaHCO3 on the perception of organic acids depends not only on its buffer properties but, probably, on direct action of CO2 appearing as the result of interaction of NaHCO3 and acids, as well. This assumption was confirmed by the data on high sensitivity of the cat tongue receptors for the carbon dioxide.
|
[Analysis of the effect of bicarbonate on taste receptor sensitivity to organic acids]. Addition of NaHCO3 or NaOH to the solutions of lactic or pyruvic acids decreased threshold concentrations of H+ ions and increased intensity of afferent impulsation in the cat chroda tympani nerve. In spite of the fact that the concentrations of both lactate and pyruvate anions with the presence of NaOH and NaHCO3 are the same at equal pH values the observed changes of the receptors responses were more intensive if NaHCO3 had been added. Therefore the influence of NaHCO3 on the perception of organic acids depends not only on its buffer properties but, probably, on direct action of CO2 appearing as the result of interaction of NaHCO3 and acids, as well. This assumption was confirmed by the data on high sensitivity of the cat tongue receptors for the carbon dioxide.
|
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] |
PMID:21115
|
Degradation and liquefaction effect of streptokinase-Streptodornase and stabilized trypsin on necroses, crusts of fibrinoid, purulent exudate and clotted blood from leg ulcers.
|
Necrotic materials most frequently found in leg ulcers, including crusts with fibrinogen, pus and blood clots, were exposed to solutions of streptokinase-streptodornase and stabilized crystalline trypsin respectively. The investigations were performed at the temperatures of 26 degrees and 33 degrees C, representing the extreme values of the temperature range found in leg ulcers (arteriosclerotic ulcers, stasis ulcers) and at the pH corresponding to that of the enzyme preparations in wet dressings. Streptokinase-streptodornase demonstrated in vitro a more potent proteolytic activity than crystalline trypsin on necroses, crusts of fibrinoid and purulent exudate, which were more rapidly and thoroughly broken up. Both enzyme preparations were, however, equally effective on three-day-old blood clots.
|
Degradation and liquefaction effect of streptokinase-Streptodornase and stabilized trypsin on necroses, crusts of fibrinoid, purulent exudate and clotted blood from leg ulcers. Necrotic materials most frequently found in leg ulcers, including crusts with fibrinogen, pus and blood clots, were exposed to solutions of streptokinase-streptodornase and stabilized crystalline trypsin respectively. The investigations were performed at the temperatures of 26 degrees and 33 degrees C, representing the extreme values of the temperature range found in leg ulcers (arteriosclerotic ulcers, stasis ulcers) and at the pH corresponding to that of the enzyme preparations in wet dressings. Streptokinase-streptodornase demonstrated in vitro a more potent proteolytic activity than crystalline trypsin on necroses, crusts of fibrinoid and purulent exudate, which were more rapidly and thoroughly broken up. Both enzyme preparations were, however, equally effective on three-day-old blood clots.
|
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] |
PMID:21116
|
Anxiety states in family practice: an evaluation of the need for antidepressant as well as anxiolytic therapy.
|
Patients attending their family practitioner with emotional disturbance manifesting predominantly as anxiety were treated once daily for 4 weeks with either a pure anxiolytic, potassium clorazepate, or a formulation of a specific antidepressant together with an anxiolytic, fluphenazine/nortriptyline, in accordance with a double-blind, completely randomized design. After the first week the patients receiving fluphenazine/nortriptyline were showing a better response in terms of total symptomatology as well as anxiety, tension and depression taken separately, and after 4 weeks treatment this trend reached statistically significant levels on both the physicans' ratings and the patients' self-ratings for overall symptomatology (p less than 0-05) as well as anxiety and tension on the physicians' scale (p less than 0-01). Side-effects were infrequent, with the exception of drowsiness which was complained of by 42% of the patients receiving clorazepate. Although simple and convenient to take, a once daily benzodiazepine formulation of fixed dose is likely to be too inflexible to achieve optimal therapeutic effect in many patients. These results are in accord with accumulating evidence for the importance of a depressive aetiology underlying the majority of so-called anxiety states in family practice. Anxiolytic, in the absence of specific antidepressant, therapy is unlikely to be adequate for these patients, and may lead to long-term palliative use of benzodiazepines incurring a risk of dependence.
|
Anxiety states in family practice: an evaluation of the need for antidepressant as well as anxiolytic therapy. Patients attending their family practitioner with emotional disturbance manifesting predominantly as anxiety were treated once daily for 4 weeks with either a pure anxiolytic, potassium clorazepate, or a formulation of a specific antidepressant together with an anxiolytic, fluphenazine/nortriptyline, in accordance with a double-blind, completely randomized design. After the first week the patients receiving fluphenazine/nortriptyline were showing a better response in terms of total symptomatology as well as anxiety, tension and depression taken separately, and after 4 weeks treatment this trend reached statistically significant levels on both the physicans' ratings and the patients' self-ratings for overall symptomatology (p less than 0-05) as well as anxiety and tension on the physicians' scale (p less than 0-01). Side-effects were infrequent, with the exception of drowsiness which was complained of by 42% of the patients receiving clorazepate. Although simple and convenient to take, a once daily benzodiazepine formulation of fixed dose is likely to be too inflexible to achieve optimal therapeutic effect in many patients. These results are in accord with accumulating evidence for the importance of a depressive aetiology underlying the majority of so-called anxiety states in family practice. Anxiolytic, in the absence of specific antidepressant, therapy is unlikely to be adequate for these patients, and may lead to long-term palliative use of benzodiazepines incurring a risk of dependence.
|
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] |
PMID:21117
|
Therapy of alveococcosis in man.
|
Diagnostic, therapeutic and prognostic aspects are interpreted on the basis of two cases of Echinococcus alveolaris. Both patients had undergone partial hepatectomy and were subsequently treated chemotherapeutically. In one case dehydroemetine and mebendazole (Vermox, Janssen), and in the other case metrifonate (Bilarcil, Bayer A.G.) were used. The diagnosis of alveococcosis of the liver was demonstrated in one case histologically on the occasion of appendectomy, and in the other case by the indirect immunofluorescence test and passive haemagglutination. In case of suspected alveococcosis these serological are imperative, as they are the most reliable methods of demonstrating this disease. The latest trend in theraphy aims at a combination of surgery and chemotherapy. Mebendazole seems to be promising as an anthelmintic agent for the treatment of alveococcosis. Remission of the disease was obtained in either case.
|
Therapy of alveococcosis in man. Diagnostic, therapeutic and prognostic aspects are interpreted on the basis of two cases of Echinococcus alveolaris. Both patients had undergone partial hepatectomy and were subsequently treated chemotherapeutically. In one case dehydroemetine and mebendazole (Vermox, Janssen), and in the other case metrifonate (Bilarcil, Bayer A.G.) were used. The diagnosis of alveococcosis of the liver was demonstrated in one case histologically on the occasion of appendectomy, and in the other case by the indirect immunofluorescence test and passive haemagglutination. In case of suspected alveococcosis these serological are imperative, as they are the most reliable methods of demonstrating this disease. The latest trend in theraphy aims at a combination of surgery and chemotherapy. Mebendazole seems to be promising as an anthelmintic agent for the treatment of alveococcosis. Remission of the disease was obtained in either case.
|
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] |
PMID:21120
|
[The orthostatic hypotension in patients treated with antihypertensive drugs (author's transl)].
|
Occasional or recurrent episodes of orthostatic hypotension were observed in 58 out of 584 hypertensive patients treated with alpha-methyldopa, beta-blockers and clonidine alone or associated with diuretics and/or hydralazine and/or reserpine. They occurred more frequently in the elderly. In none dicardial ischaemia. In only 3 cases it caused drowsiness and in 2 transient T wave inversion. These results suggest that postural hypotension does not contraindicate the continuation of antihypertensive treatment.
|
[The orthostatic hypotension in patients treated with antihypertensive drugs (author's transl)]. Occasional or recurrent episodes of orthostatic hypotension were observed in 58 out of 584 hypertensive patients treated with alpha-methyldopa, beta-blockers and clonidine alone or associated with diuretics and/or hydralazine and/or reserpine. They occurred more frequently in the elderly. In none dicardial ischaemia. In only 3 cases it caused drowsiness and in 2 transient T wave inversion. These results suggest that postural hypotension does not contraindicate the continuation of antihypertensive treatment.
|
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] |
PMID:21122
|
[Management and monitoring of multiple pregnancies (author's transl)].
|
The fetal prognosis in multiple pregnancies can be improved by a multifaceted antenatal program which includes early diagnosis prior to 28 weeks gestation, follow-up of multiple pregnancies in the high risk antenatal clinic, early discontinuation of work, treatment of pre-eclampsia, bed rest in hospital between 28 and 33 weeks and sometimes cerclage, prophylactic and therapeutic administration of labour inhibiting drugs, speedy delivery of the second twin and immediate pediatric care. Bed rest and administration of labour inhibiting drugs are the most important points of this program. With this combination, the utero-placental perfusion can be improved. The gestation can be prolonged and the incidence of small weight neonates and the incidence of the perinatal mortality can be reduced. Since even a large antenatal clinic only cares for a small number of multiple pregnancies, a multicentre study to determine the optimal management of multiple pregnancies is urgently required. Multiple pregnancies had too little attention in modern perinatal medicine and deserve all our attention for an improvement of their outcome.
|
[Management and monitoring of multiple pregnancies (author's transl)]. The fetal prognosis in multiple pregnancies can be improved by a multifaceted antenatal program which includes early diagnosis prior to 28 weeks gestation, follow-up of multiple pregnancies in the high risk antenatal clinic, early discontinuation of work, treatment of pre-eclampsia, bed rest in hospital between 28 and 33 weeks and sometimes cerclage, prophylactic and therapeutic administration of labour inhibiting drugs, speedy delivery of the second twin and immediate pediatric care. Bed rest and administration of labour inhibiting drugs are the most important points of this program. With this combination, the utero-placental perfusion can be improved. The gestation can be prolonged and the incidence of small weight neonates and the incidence of the perinatal mortality can be reduced. Since even a large antenatal clinic only cares for a small number of multiple pregnancies, a multicentre study to determine the optimal management of multiple pregnancies is urgently required. Multiple pregnancies had too little attention in modern perinatal medicine and deserve all our attention for an improvement of their outcome.
|
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] |
PMID:21123
|
[Clinical polymorphism and ceruloplasmin variants in hepatolenticular degeneration].
|
The relationship between differences in the clinical polymorphism of hepatolenticular degeneration (Wilson's disease) and characteristics of CP (ceruloplasmin) structural changes were investigated. The comparative study of Wilson's disease patients revealed two forms of clinical development of this disease which differ from each other by the expression of the visceral symptoms preceding the establishment of the typical neurological picture. The peptide map analysis of tryptic hydrolysates of the CP from individual patients has demonstrated the altered peptide patterns in five cases. Clinical and genetic heterogeneity of Wilson's disease is discussed.
|
[Clinical polymorphism and ceruloplasmin variants in hepatolenticular degeneration]. The relationship between differences in the clinical polymorphism of hepatolenticular degeneration (Wilson's disease) and characteristics of CP (ceruloplasmin) structural changes were investigated. The comparative study of Wilson's disease patients revealed two forms of clinical development of this disease which differ from each other by the expression of the visceral symptoms preceding the establishment of the typical neurological picture. The peptide map analysis of tryptic hydrolysates of the CP from individual patients has demonstrated the altered peptide patterns in five cases. Clinical and genetic heterogeneity of Wilson's disease is discussed.
|
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0.0735916942358017,
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] |
PMID:21124
|
Ditazole and platelets III. Effect of ditazole on tumor-cell induced thrombocytopenia and on bleeding time in mice.
|
Ditazole (4,5-diphenyl-2-bis-(2-hydroxyethyl)-aminoxazol) inhibits in vivo platelet aggregation induced in mice by intravenous injection of cells derived from an experimental tumor, the Lewis lung carcinoma. Such a protective effect of ditazole could not be observed when the number of circulating platelets dropped slowly following intramuscular implantation and spontaneous dissemination of the same cancer cells. These results support previous observations suggesting a different mechanism for the thrombocytopenia observed after intravenous and intramuscular injection of cancer cells. Tail transection bleeding time of normal mice is significantly prolonged by ditazole, a finding at variance with that reported in rats.
|
Ditazole and platelets III. Effect of ditazole on tumor-cell induced thrombocytopenia and on bleeding time in mice. Ditazole (4,5-diphenyl-2-bis-(2-hydroxyethyl)-aminoxazol) inhibits in vivo platelet aggregation induced in mice by intravenous injection of cells derived from an experimental tumor, the Lewis lung carcinoma. Such a protective effect of ditazole could not be observed when the number of circulating platelets dropped slowly following intramuscular implantation and spontaneous dissemination of the same cancer cells. These results support previous observations suggesting a different mechanism for the thrombocytopenia observed after intravenous and intramuscular injection of cancer cells. Tail transection bleeding time of normal mice is significantly prolonged by ditazole, a finding at variance with that reported in rats.
|
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] |
PMID:21125
|
[Effects of a new minor tranquilizer, 10-chloro-3-methyl-1 1b-(2-chlorophenyl)-2,3,5,6,7,11b-hexahydrobenzo [6,7]-1,4-diazepino [5,4-b]-oxazol-6-one (CS-386), on the after-discharge and behavior induced by electrical stimulation of the amygdala in freely-moving cats (author's transl)].
|
Effects of a new minor tranquilizer, CS-386, on the after-discharge(AD) and behavior induced by amygdaloid electrical stimulation in freely-moving cats were compared with those of cloxazolam, oxazolam, diazepam, chlordiazepoxide, phenobarbital and chlorphromazine. Effects on change of the AD threshold and duration and on facial twitching, salivation and tonic-clonic convulsion were investigated. CS-386, cloxazolam and oxazolam inhibited amygdaloid AD. CS-386 had the most potent inhibitory effect. These drugs depressed all behavior described above. Diazepam had no effects on the AD threshold, but decreased the AD duration and inhibited the behavior. Chlordiazepoxide had no apparent effects on amygdaloid AD and on facial twitching. Salivation was inhibited with high doses of administration. Phenobarbital shortened the AD duration and at a high dose elevated the AD threshold. This drug also inhibited salivation, but inhibitory effects on other behavior required doses as high as 90 mg/kg. These results suggest that CS-386, cloxazolam and oxazolam are compounds belonging to a classification different from that of chlorpromazine. CS-386 in particular, is a more potent drug chlordiazepoxide, diazepam and phenobarbital and acts on the amygdala itself.
|
[Effects of a new minor tranquilizer, 10-chloro-3-methyl-1 1b-(2-chlorophenyl)-2,3,5,6,7,11b-hexahydrobenzo [6,7]-1,4-diazepino [5,4-b]-oxazol-6-one (CS-386), on the after-discharge and behavior induced by electrical stimulation of the amygdala in freely-moving cats (author's transl)]. Effects of a new minor tranquilizer, CS-386, on the after-discharge(AD) and behavior induced by amygdaloid electrical stimulation in freely-moving cats were compared with those of cloxazolam, oxazolam, diazepam, chlordiazepoxide, phenobarbital and chlorphromazine. Effects on change of the AD threshold and duration and on facial twitching, salivation and tonic-clonic convulsion were investigated. CS-386, cloxazolam and oxazolam inhibited amygdaloid AD. CS-386 had the most potent inhibitory effect. These drugs depressed all behavior described above. Diazepam had no effects on the AD threshold, but decreased the AD duration and inhibited the behavior. Chlordiazepoxide had no apparent effects on amygdaloid AD and on facial twitching. Salivation was inhibited with high doses of administration. Phenobarbital shortened the AD duration and at a high dose elevated the AD threshold. This drug also inhibited salivation, but inhibitory effects on other behavior required doses as high as 90 mg/kg. These results suggest that CS-386, cloxazolam and oxazolam are compounds belonging to a classification different from that of chlorpromazine. CS-386 in particular, is a more potent drug chlordiazepoxide, diazepam and phenobarbital and acts on the amygdala itself.
|
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] |
PMID:21126
|
[Studies on monoamine oxidase. (Report 37) Effects of oxygen concentration on rat liver and brain monoamine oxidase (author's transl)].
|
MAO activity in rat brain mitochondria with tyramine as substrate at 100% oxygen concentration was three times as much as that at 20%. When serotonin served as substrate, difference in activities between the two oxygen concentrations was not significant. Similar results were obtained when rat liver MAO was used as the enzyme source. At 100% oxygen concentration, pargyline showed the most potent inhibition of MAO activity in liver mitochondria with tyramine as substrate, but inhibitions caused by pheniprazine and harmaline were not remarkable. At 100% oxygen concentration, harmaline showed the most potent inhibition of MAO activity in the liver when serotonin served as substrate, while inhibitions of the MAO activity by pargyline and pheniprazine were weak. At 20% oxygen concentration, harmaline showed the most potent inhibition of MAO activity in the brain when serotonin was used as substrate. These inhibitions were studied using Lineweaver-Burk plots. Pargyline revealed a noncompetitive inhibition to MAO activity in liver and brain with tyramine and serotonin as substrate, harmaline a competitive inhibition to MAO activity in liver and brain with tyramine as substrate, while noncompetitive inhibition to MAO activity in liver and brain was evident when serotonin was used as the substrate.
|
[Studies on monoamine oxidase. (Report 37) Effects of oxygen concentration on rat liver and brain monoamine oxidase (author's transl)]. MAO activity in rat brain mitochondria with tyramine as substrate at 100% oxygen concentration was three times as much as that at 20%. When serotonin served as substrate, difference in activities between the two oxygen concentrations was not significant. Similar results were obtained when rat liver MAO was used as the enzyme source. At 100% oxygen concentration, pargyline showed the most potent inhibition of MAO activity in liver mitochondria with tyramine as substrate, but inhibitions caused by pheniprazine and harmaline were not remarkable. At 100% oxygen concentration, harmaline showed the most potent inhibition of MAO activity in the liver when serotonin served as substrate, while inhibitions of the MAO activity by pargyline and pheniprazine were weak. At 20% oxygen concentration, harmaline showed the most potent inhibition of MAO activity in the brain when serotonin was used as substrate. These inhibitions were studied using Lineweaver-Burk plots. Pargyline revealed a noncompetitive inhibition to MAO activity in liver and brain with tyramine and serotonin as substrate, harmaline a competitive inhibition to MAO activity in liver and brain with tyramine as substrate, while noncompetitive inhibition to MAO activity in liver and brain was evident when serotonin was used as the substrate.
|
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] |
PMID:21127
|
[Effects of minor tranquilizers and neuroleptics on open-field behavior in rats (author's transl)].
|
Minor tranquilizers (diazepam, nitrazepam, oxazepam, bromazepam, medazepam, fludiazepam, meprobamate) at low doses increased ambulation score to 145 approximately 288% of control rats. Nitrazepam, diazepam and bromazepam which are potent, clinically prescribed minor tranquilizers increased the ambulation at lower doses than was seen with the other drugs. Fludiazepam and nitrazepam showed a maximum increase in ambulation at the same dose. Fludiazepam, nitrazepam and diazepam proved to have potent inhibitory effects on defecation. Trifluperidol, haloperidol and ID-4708 (a new butyrophenone derivative) and chlorpromazine when given at low doses reduced ambulation, while at higher doses defecation was inhibited. These four drugs reduced ambulation and elicited a recover in rates of defecation in methamphetamine treated rats. Clozapine, thioridazine and floropipamide inhibited defecation at nearly the same doses which reduced ambulation in rats not given the methamphetamine tratment. These three durugs reduced ambulation, but did not produce a recovery in the defecation rates in methamphetamine-treated rats. These results indicate that neuroleptics such as clozapine which rarely induce extrapyramidal side-effects when clinically prescribed, inhibit defecation at nearly the same doses which reduce ambulation. In methamphetamine-treated rats, haloperidol was 31 times more potent than chlorpromazine in inhibiting activity noted with ambulation. This ratio in open-field test was close to the ratio of potency of these drugs as antipsychotic clinically prescribed agents.
|
[Effects of minor tranquilizers and neuroleptics on open-field behavior in rats (author's transl)]. Minor tranquilizers (diazepam, nitrazepam, oxazepam, bromazepam, medazepam, fludiazepam, meprobamate) at low doses increased ambulation score to 145 approximately 288% of control rats. Nitrazepam, diazepam and bromazepam which are potent, clinically prescribed minor tranquilizers increased the ambulation at lower doses than was seen with the other drugs. Fludiazepam and nitrazepam showed a maximum increase in ambulation at the same dose. Fludiazepam, nitrazepam and diazepam proved to have potent inhibitory effects on defecation. Trifluperidol, haloperidol and ID-4708 (a new butyrophenone derivative) and chlorpromazine when given at low doses reduced ambulation, while at higher doses defecation was inhibited. These four drugs reduced ambulation and elicited a recover in rates of defecation in methamphetamine treated rats. Clozapine, thioridazine and floropipamide inhibited defecation at nearly the same doses which reduced ambulation in rats not given the methamphetamine tratment. These three durugs reduced ambulation, but did not produce a recovery in the defecation rates in methamphetamine-treated rats. These results indicate that neuroleptics such as clozapine which rarely induce extrapyramidal side-effects when clinically prescribed, inhibit defecation at nearly the same doses which reduce ambulation. In methamphetamine-treated rats, haloperidol was 31 times more potent than chlorpromazine in inhibiting activity noted with ambulation. This ratio in open-field test was close to the ratio of potency of these drugs as antipsychotic clinically prescribed agents.
|
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] |
PMID:21129
|
[Calcium containing antacids and acid rebound. Testing with intragastric titration and extragastric measurement of pH].
|
Intragastric titration with the pH being measured extragastrically is a method both precise and highly qualified for clinical trials of antacids and quantitative analysis of gastric secretion. In 12 patients with duodenal ulcer, acid secretion was measured by intragastric titration both before and after adminstration of a Ca-Mg-containing antacid. Acid secretion was found to be significantly lower after administration of the Ca-Mg-containing antacid, a so-called acid rebound could not be demonstrated.
|
[Calcium containing antacids and acid rebound. Testing with intragastric titration and extragastric measurement of pH]. Intragastric titration with the pH being measured extragastrically is a method both precise and highly qualified for clinical trials of antacids and quantitative analysis of gastric secretion. In 12 patients with duodenal ulcer, acid secretion was measured by intragastric titration both before and after adminstration of a Ca-Mg-containing antacid. Acid secretion was found to be significantly lower after administration of the Ca-Mg-containing antacid, a so-called acid rebound could not be demonstrated.
|
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] |
PMID:21130
|
[The clinical picture of lactate acidosis. 5. Lactatemia without acidosis. Conclusions].
|
Several inherited metabolic diseases are accompanied by a greater or lesser increase in blood lactate concentration under certain metabolic conditions. These diseases are glycogenosis type I (glocuse-6-phosphate deficiency), fructose-1,6-diphosphatase deficiency, glucose-induced hyperlactate emia, idiopathic lactate acidosis. The conditions are discussed when hyperlactate emia develops. Very large increases in blood lactate concentration are found during muscular activity, lactate concentrations can be as much as 20 mmol/l under these conditions. Regarding these values, the increase in blood lactate concentration during intravenous carbohydrate infusion is minimum, even in the case of fructose infusions (1-4 mmol/l). Therapeutical measures for treatment of increased lactate concentration are discussed. A causal therapy is optimum; however, the precondition is a definite diagnosis. Besides bicarbonate infusions (or infusions of other alkalizing substances) dialysis seems to be a favourable therapy in certain cases. In future, prognosis of lactate emia should be better if the diagnostic measures and differential diagnosis are improved.
|
[The clinical picture of lactate acidosis. 5. Lactatemia without acidosis. Conclusions]. Several inherited metabolic diseases are accompanied by a greater or lesser increase in blood lactate concentration under certain metabolic conditions. These diseases are glycogenosis type I (glocuse-6-phosphate deficiency), fructose-1,6-diphosphatase deficiency, glucose-induced hyperlactate emia, idiopathic lactate acidosis. The conditions are discussed when hyperlactate emia develops. Very large increases in blood lactate concentration are found during muscular activity, lactate concentrations can be as much as 20 mmol/l under these conditions. Regarding these values, the increase in blood lactate concentration during intravenous carbohydrate infusion is minimum, even in the case of fructose infusions (1-4 mmol/l). Therapeutical measures for treatment of increased lactate concentration are discussed. A causal therapy is optimum; however, the precondition is a definite diagnosis. Besides bicarbonate infusions (or infusions of other alkalizing substances) dialysis seems to be a favourable therapy in certain cases. In future, prognosis of lactate emia should be better if the diagnostic measures and differential diagnosis are improved.
|
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] |
PMID:21131
|
[Neurological-psychiatrical and muscular manifestations of vasculitis nodosa (author's transl)].
|
A general review on vasculitis nodosa of report in literature since the work of Strammler 1958 is given. The neurological and psychiatric manifestations of the disease are completely dealt with, whereas dermatological, ophthalmological and otological aspects are brieftly mentioned. Varieties of diffuse and localized cerebral, as well as peripheral syndromes are described. The neuropathological findings are also discussed extensively.
|
[Neurological-psychiatrical and muscular manifestations of vasculitis nodosa (author's transl)]. A general review on vasculitis nodosa of report in literature since the work of Strammler 1958 is given. The neurological and psychiatric manifestations of the disease are completely dealt with, whereas dermatological, ophthalmological and otological aspects are brieftly mentioned. Varieties of diffuse and localized cerebral, as well as peripheral syndromes are described. The neuropathological findings are also discussed extensively.
|
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] |
PMID:21140
|
Primary care and physician extenders in affluent countries.
|
The worldwide growth of specialization in medicine has led to a perceived shortage of primary care. A major response in the United States has been the training of physician extenders (both physician assistants and nurse practitioners). Other industrialized countries have rejected this approach, in favor of strengthening general medical practice through continuing education, provision of ancillary personnel, use of health centers, and by other methods. Developing countries use doctor-substitutes as a reasonable adjustment to their lack of economic resources. All countries use ancillary personnel for selected procedures, such as midwifery, which involve only limited judgment and decision making. The American strategy on use of doctor-substitutes for primary care, however, follows from unwillingness to train greater numbers of primary care physicians and to require them to serve in places of need. This results in an inequitable concentration of doctor-substitutes on service to the poor in both urban and rural areas.
|
Primary care and physician extenders in affluent countries. The worldwide growth of specialization in medicine has led to a perceived shortage of primary care. A major response in the United States has been the training of physician extenders (both physician assistants and nurse practitioners). Other industrialized countries have rejected this approach, in favor of strengthening general medical practice through continuing education, provision of ancillary personnel, use of health centers, and by other methods. Developing countries use doctor-substitutes as a reasonable adjustment to their lack of economic resources. All countries use ancillary personnel for selected procedures, such as midwifery, which involve only limited judgment and decision making. The American strategy on use of doctor-substitutes for primary care, however, follows from unwillingness to train greater numbers of primary care physicians and to require them to serve in places of need. This results in an inequitable concentration of doctor-substitutes on service to the poor in both urban and rural areas.
|
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] |
PMID:21142
|
Is the alkaline cleavage of disulfide bonds in peptides an alpha-beta elimination reaction or a hydrolysis?
|
The cleavage of oxidized glutathione by alkali has been studied as representative of the cleavage of protein disulfides. This process is quite different when studied in 10-4N or 2-10-1N NaOH. At low alkali concentration no spectral changes are noted; at higher hydroxyl concentration the appearance of persulfide groups (followed at 335 nm), the formation of thiocyanate (arising from cold cyanolysis of persulfide groups) and the absorbance at 240 nm follow the same kinetics. The amount of half-cystine, recovered as cysteic acid after a 3-h reaction, is significantly lower than calculated. These results confirm that oxidized glutathione undergoes beta-elimination at high pH values, and that persulfide groups absorb not only at 335 nm (as already known) but also at 240 nm where, under our conditions, the contribution of other absorbing species is not very high.
|
Is the alkaline cleavage of disulfide bonds in peptides an alpha-beta elimination reaction or a hydrolysis? The cleavage of oxidized glutathione by alkali has been studied as representative of the cleavage of protein disulfides. This process is quite different when studied in 10-4N or 2-10-1N NaOH. At low alkali concentration no spectral changes are noted; at higher hydroxyl concentration the appearance of persulfide groups (followed at 335 nm), the formation of thiocyanate (arising from cold cyanolysis of persulfide groups) and the absorbance at 240 nm follow the same kinetics. The amount of half-cystine, recovered as cysteic acid after a 3-h reaction, is significantly lower than calculated. These results confirm that oxidized glutathione undergoes beta-elimination at high pH values, and that persulfide groups absorb not only at 335 nm (as already known) but also at 240 nm where, under our conditions, the contribution of other absorbing species is not very high.
|
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-0.011051242239773273,
-0.0723179504275322,
0.01027609221637249,
0.007719225715845823,
-0.005777457728981972,
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0.04192201420664787,
-0.043305277824401855,
0.07523230463266373,
0.041088737547397614
] |
PMID:21143
|
Modification of lipoxygenase by hydrogen peroxide and photooxidation.
|
The kinetic study of fluorescence stopped-flow method suggested that the interaction between lipoxygenase and H2O2 is consistent with a simple irreversible one-step mechanism. The activation energy of the reaction was 7.2 kcal/mol. Participation of an ionizable group with pK about 8.8, possibly a histidine residue, was suggested from the pH-dependence of the rate constant. No further fluorescence quenching of lipoxygenase was observed when the product was added to the lipoxygenase solution before mixing the lipoxygenase and H2O2 solutions. The fluorescence quenching of lipoxygenase by H2O2 was in parallel with the inactivation of the enzyme. Hydroperoxylinoleic acid strongly protects the inactivation of lipoxygenase caused by H2O2. These results are consistent with an interpretation that OH- and/or O- - are produced when the iron of the enzyme is oxidized by H2O2, which in turn will attack some amino acid essential for the enzyme activity. The pH-dependence of the inactivation rate constant of photooxidation of lipoxygenase sensitized by methylene blue indicated that an ionizable group with pK about 8.8 is concerned with the enzymatic activity. In contrast to the inactivation of lipoxygenase by H2O2, the product protected the inactivation of the enzyme by photooxidation only at high concentration.
|
Modification of lipoxygenase by hydrogen peroxide and photooxidation. The kinetic study of fluorescence stopped-flow method suggested that the interaction between lipoxygenase and H2O2 is consistent with a simple irreversible one-step mechanism. The activation energy of the reaction was 7.2 kcal/mol. Participation of an ionizable group with pK about 8.8, possibly a histidine residue, was suggested from the pH-dependence of the rate constant. No further fluorescence quenching of lipoxygenase was observed when the product was added to the lipoxygenase solution before mixing the lipoxygenase and H2O2 solutions. The fluorescence quenching of lipoxygenase by H2O2 was in parallel with the inactivation of the enzyme. Hydroperoxylinoleic acid strongly protects the inactivation of lipoxygenase caused by H2O2. These results are consistent with an interpretation that OH- and/or O- - are produced when the iron of the enzyme is oxidized by H2O2, which in turn will attack some amino acid essential for the enzyme activity. The pH-dependence of the inactivation rate constant of photooxidation of lipoxygenase sensitized by methylene blue indicated that an ionizable group with pK about 8.8 is concerned with the enzymatic activity. In contrast to the inactivation of lipoxygenase by H2O2, the product protected the inactivation of the enzyme by photooxidation only at high concentration.
|
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] |
PMID:21144
|
The effect of additives on the free radical formation in aqueous solutions of ascorbic acid.
|
The relative rate constants for the decay of ascorbate free radical in aqueous solutions in the presence of heavy metal ions, hydrogen peroxide and sulphite were measured the ESR-spectroscopy. The oxidation of ascorbic acid showed a strong pH dependence, reaching a maximum rate at pH 9.6. It was shown that the ascorbic acid radical decay generally follows overall second order kinetics, being however first order in the presence of hydrogen peroxide. The protective effect of sulphur dioxide is proposed to have nutritional implications.
|
The effect of additives on the free radical formation in aqueous solutions of ascorbic acid. The relative rate constants for the decay of ascorbate free radical in aqueous solutions in the presence of heavy metal ions, hydrogen peroxide and sulphite were measured the ESR-spectroscopy. The oxidation of ascorbic acid showed a strong pH dependence, reaching a maximum rate at pH 9.6. It was shown that the ascorbic acid radical decay generally follows overall second order kinetics, being however first order in the presence of hydrogen peroxide. The protective effect of sulphur dioxide is proposed to have nutritional implications.
|
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] |
PMID:21145
|
Comparison of the effects of timolol and other adrenergic agents on intraocular pressure in the rabbit.
|
The effect of timolol, propranolol, epinephrine, and isoproterenol on intraocular pressure (IOP) (measured by tonometry) were compared after topical administration in conscious rabbits. Epinephrine and isoproterenol decreased IOP in normotensive rabbits, whereas propranolol had no effect. Timolol produced only a slight and inconsistent lowering of IOP in normotensive rabbits. All four agents reduced IOP elevated by an oral water load; the adrenergic agonists were substantially more active than the two beta-adrenergic blocking agents. In alpha-chymotrypsin-induced ocular hypertension, epinephrine, isoproterenol, and timolol were essentially equally effective, whereas propranolol exhibited only weak activity. In this latter model, timolol did not lose its effectiveness after multiple instillations (three/day) over an 8-day period. The concentration of timolol in the acqueous humor after topical application of effective hypotensive doses was relatively high as compared to that found in plasma. In addition, topical doses of timolol required to lower IOP were considerably greater than those needed to reduce or block the ocular hypotensive activity of isoproterenol. The mode of action and therapeutic implications of beta-adrenergic blocking agents in glaucoma are discussed.
|
Comparison of the effects of timolol and other adrenergic agents on intraocular pressure in the rabbit. The effect of timolol, propranolol, epinephrine, and isoproterenol on intraocular pressure (IOP) (measured by tonometry) were compared after topical administration in conscious rabbits. Epinephrine and isoproterenol decreased IOP in normotensive rabbits, whereas propranolol had no effect. Timolol produced only a slight and inconsistent lowering of IOP in normotensive rabbits. All four agents reduced IOP elevated by an oral water load; the adrenergic agonists were substantially more active than the two beta-adrenergic blocking agents. In alpha-chymotrypsin-induced ocular hypertension, epinephrine, isoproterenol, and timolol were essentially equally effective, whereas propranolol exhibited only weak activity. In this latter model, timolol did not lose its effectiveness after multiple instillations (three/day) over an 8-day period. The concentration of timolol in the acqueous humor after topical application of effective hypotensive doses was relatively high as compared to that found in plasma. In addition, topical doses of timolol required to lower IOP were considerably greater than those needed to reduce or block the ocular hypotensive activity of isoproterenol. The mode of action and therapeutic implications of beta-adrenergic blocking agents in glaucoma are discussed.
|
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-0.013012431561946869,
0.00949276052415371,
0.02079518511891365,
0.0029528974555432796,
-0.0735049843788147,
-0.05395768582820892,
-0.05311473459005356,
0.05083511397242546,
0.06876511126756668
] |
PMID:21150
|
Studies on avian erythrocyte metabolism. VII. Effect of inositol pentaphosphate and other organic phosphates on oxygen affinity of the embryonic and adult-type hemoglobins of the turkey embryo.
|
The effects of 2, 3-diphosphoglyceric acid (2, 3-DPG), adenosine triphosphate (ATP), inositol tetraphosphate (ITP), inositol pentaphosphate (IPP), and inositol hexaphosphate (IHP) on oxygen affinity of whole stripped hemoglobin (WSH), hemoglobin H (Hb-H; hatching hemoglobin), hemoglobin A (Hb-A), and hemoglobin D (Hb-D) isolated from erythrocytes (RBC) of the 25-day turkey embryo have been studied. The order of the decrease in oxygen affinity induced by these organic phosphates, at molar ratios of phosphate compound to hemoglobin (tetramer) between 2 and 4, is 2, 3-DPG less than ATP less than ITP less than IPP less than IHP. 2, 3-DPG shows a slightly greater effect on reducing oxygen affinity of Hb-H than on either adult-type hemoglobin. The effect of IPP upon lowering the oxygen affinity of either WSH, Hb-H, Hb-A, or Hb-D is approximately 20 percent less than IHP. The effects of the various organic phosphates upon the Hill constant, n, of these purified hemoglobins is variable but appears to reach a maximum when the molar ratio of organic phosphate to hemoglobin (tetramer) is 2 or greater. None of the physiologically occurring organic phosphates has a significant preferential interaction with any specific hemoglobin. These experiments strengthen and support our earlier conclusion, that the changes in whole blood oxygen affinity which occur during avian development result from the changes in composition of the intraerythrocytic organic phosphates.
|
Studies on avian erythrocyte metabolism. VII. Effect of inositol pentaphosphate and other organic phosphates on oxygen affinity of the embryonic and adult-type hemoglobins of the turkey embryo. The effects of 2, 3-diphosphoglyceric acid (2, 3-DPG), adenosine triphosphate (ATP), inositol tetraphosphate (ITP), inositol pentaphosphate (IPP), and inositol hexaphosphate (IHP) on oxygen affinity of whole stripped hemoglobin (WSH), hemoglobin H (Hb-H; hatching hemoglobin), hemoglobin A (Hb-A), and hemoglobin D (Hb-D) isolated from erythrocytes (RBC) of the 25-day turkey embryo have been studied. The order of the decrease in oxygen affinity induced by these organic phosphates, at molar ratios of phosphate compound to hemoglobin (tetramer) between 2 and 4, is 2, 3-DPG less than ATP less than ITP less than IPP less than IHP. 2, 3-DPG shows a slightly greater effect on reducing oxygen affinity of Hb-H than on either adult-type hemoglobin. The effect of IPP upon lowering the oxygen affinity of either WSH, Hb-H, Hb-A, or Hb-D is approximately 20 percent less than IHP. The effects of the various organic phosphates upon the Hill constant, n, of these purified hemoglobins is variable but appears to reach a maximum when the molar ratio of organic phosphate to hemoglobin (tetramer) is 2 or greater. None of the physiologically occurring organic phosphates has a significant preferential interaction with any specific hemoglobin. These experiments strengthen and support our earlier conclusion, that the changes in whole blood oxygen affinity which occur during avian development result from the changes in composition of the intraerythrocytic organic phosphates.
|
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] |
PMID:21147
|
Studies on the physico-chemical properties of alhagain.
|
The protease isolated jawasee shrub was found to hydrolyze egg albumin, casein, haemoglobin and gelatin optimally near neutral pH. Fibrin, bovin serum albumin, skin albumin and skin mucoids were hydrolyzed at slightly alkaline pH, while skin globulins were hydrolyzed at slightly acidic pH. The enzyme had no effect of fibrous collagen. The optimum conditions for the hydrolysis of 50 mg of egg albumin were found to be 50 mg of alhagain at pH 6.0 and 45 degrees C for 30 minutes. A Km value of 4.4 X 10(-3) M was obtained from the Lineweaver-Burk plot for the hydrolysis of egg albumin. The enzyme was found to be comparatively thermostable and was most stable at pH 4.7. Ultraviolet irradiation exhibited no appreciable effect on the enzyme activity. The ultraviolet absorption spectrum of alhagain in bi-distilled water resembles those of bromelain and trypsin. The sugar-containing enzyme was found to have a molecular weight of 20,650. The enzymeconsists of 189 amino acid residues per molecule, neutral and acidic amino acids being present in high concentrations. The partial specific volume of alhagain was calculated to be 0.743 ml/g from its amino acid composition. Phenylalnine and arginine formed the amino terminal amino acids of alhagain, while aspartic acid and serine were identified as its carboxy terminal amino acids. Results are discussed with relation to other plant proteases.
|
Studies on the physico-chemical properties of alhagain. The protease isolated jawasee shrub was found to hydrolyze egg albumin, casein, haemoglobin and gelatin optimally near neutral pH. Fibrin, bovin serum albumin, skin albumin and skin mucoids were hydrolyzed at slightly alkaline pH, while skin globulins were hydrolyzed at slightly acidic pH. The enzyme had no effect of fibrous collagen. The optimum conditions for the hydrolysis of 50 mg of egg albumin were found to be 50 mg of alhagain at pH 6.0 and 45 degrees C for 30 minutes. A Km value of 4.4 X 10(-3) M was obtained from the Lineweaver-Burk plot for the hydrolysis of egg albumin. The enzyme was found to be comparatively thermostable and was most stable at pH 4.7. Ultraviolet irradiation exhibited no appreciable effect on the enzyme activity. The ultraviolet absorption spectrum of alhagain in bi-distilled water resembles those of bromelain and trypsin. The sugar-containing enzyme was found to have a molecular weight of 20,650. The enzymeconsists of 189 amino acid residues per molecule, neutral and acidic amino acids being present in high concentrations. The partial specific volume of alhagain was calculated to be 0.743 ml/g from its amino acid composition. Phenylalnine and arginine formed the amino terminal amino acids of alhagain, while aspartic acid and serine were identified as its carboxy terminal amino acids. Results are discussed with relation to other plant proteases.
|
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] |
PMID:21148
|
Stearoyl-CoA desaturase in mitochondrial membrane fractions.
|
Several arguments suggested the possibility that the stearoyl-CoA desaturase might be located in the outer mitochondrial membrane in addition to its well presence in the microsomes. In the present investigation, preparations of rat liver microsomes and submitochondrial fractions (outer and inner membranes) were comparatively studied with respect to their stearoyl-CoA desaturase activities. Each fraction has been characterized by determinations of enzymic and chemical markers. This study revealed that while the activity of the stearoyl-CoA desaturase in microsomes was comparable to that found in other laboratories, a very low level of activity was detected in the mitochondrial outer membrane. The possible implications of the lack of stearoyl-CoA desaturase in mitochondria are discussed with respect to the lipid metabolism of these organelles.
|
Stearoyl-CoA desaturase in mitochondrial membrane fractions. Several arguments suggested the possibility that the stearoyl-CoA desaturase might be located in the outer mitochondrial membrane in addition to its well presence in the microsomes. In the present investigation, preparations of rat liver microsomes and submitochondrial fractions (outer and inner membranes) were comparatively studied with respect to their stearoyl-CoA desaturase activities. Each fraction has been characterized by determinations of enzymic and chemical markers. This study revealed that while the activity of the stearoyl-CoA desaturase in microsomes was comparable to that found in other laboratories, a very low level of activity was detected in the mitochondrial outer membrane. The possible implications of the lack of stearoyl-CoA desaturase in mitochondria are discussed with respect to the lipid metabolism of these organelles.
|
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] |
PMID:21152
|
Comparison of the activities of some dehydrogenases in the juxtaglomerular complex of kidneys of Wistar rats and desert rats (Meriones unguiculati).
|
The authors compared the enzyme histochemical activities of some dehydrogenases in the macula densa, the Goormaghtigh cells and the epithelioid (or juxtaglomerular cells in the kidneys of desert rodents (Mongolian Gerbils) with those of the Wistar rats. The macula cells (Table 1), which in the Wistar rats are clearly distinct from the non specific epithelial cells of the distal convolution, show, in the desert rats, noticeable fluctuations. Their enzyme histochemical reactions are often weaker than those of the distal convolution cells, with the exception of the NAD-tetrazolium-reductase activity. The Goormaghtigh cells (Table 2) in the kidneys of the Meriones have a much larger enzymatic spectrum than in the Wistar rats. Here also, we find functional variations in the examined desert species. In the epithelioid cells (Table 3) we observed a somewhat weaker enzymatic activity in the Meriones. These cells contain no secretion granules, this making their diagnosis difficult.
|
Comparison of the activities of some dehydrogenases in the juxtaglomerular complex of kidneys of Wistar rats and desert rats (Meriones unguiculati). The authors compared the enzyme histochemical activities of some dehydrogenases in the macula densa, the Goormaghtigh cells and the epithelioid (or juxtaglomerular cells in the kidneys of desert rodents (Mongolian Gerbils) with those of the Wistar rats. The macula cells (Table 1), which in the Wistar rats are clearly distinct from the non specific epithelial cells of the distal convolution, show, in the desert rats, noticeable fluctuations. Their enzyme histochemical reactions are often weaker than those of the distal convolution cells, with the exception of the NAD-tetrazolium-reductase activity. The Goormaghtigh cells (Table 2) in the kidneys of the Meriones have a much larger enzymatic spectrum than in the Wistar rats. Here also, we find functional variations in the examined desert species. In the epithelioid cells (Table 3) we observed a somewhat weaker enzymatic activity in the Meriones. These cells contain no secretion granules, this making their diagnosis difficult.
|
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] |
PMID:21154
|
Influence of temperature on hemoglobin-ligand interaction in whole blood.
|
Temperature-dependent change in hemoglobin-oxygen affinity was measured as a function of hemoglobin-oxygen saturation. In addition, the CO2 Bohr factor and fixed acid Bohr factor were measured as a function of saturation of temperatures of 23, 30, 37, and 44 degrees C. Measurements were made on normal blood and blood with reduced 2,3-diphosphoglycerate (DPG). The influence of temperature is greatest at low saturation and is enhanced slightly by DPG depletion. The CO2 Bohr factor is increased at high temperatures; this is primarily due to increased carbamino formation with rising temperature, especially at lower oxygen saturation. The effect of DPG on oxygen affinity is reduced at a high temperature and elevated at low temperature. These diverse effects of temperature on hemoglobin-ligand interaction require consideration in assessing oxygen delivery when temperature is increased or decreased.
|
Influence of temperature on hemoglobin-ligand interaction in whole blood. Temperature-dependent change in hemoglobin-oxygen affinity was measured as a function of hemoglobin-oxygen saturation. In addition, the CO2 Bohr factor and fixed acid Bohr factor were measured as a function of saturation of temperatures of 23, 30, 37, and 44 degrees C. Measurements were made on normal blood and blood with reduced 2,3-diphosphoglycerate (DPG). The influence of temperature is greatest at low saturation and is enhanced slightly by DPG depletion. The CO2 Bohr factor is increased at high temperatures; this is primarily due to increased carbamino formation with rising temperature, especially at lower oxygen saturation. The effect of DPG on oxygen affinity is reduced at a high temperature and elevated at low temperature. These diverse effects of temperature on hemoglobin-ligand interaction require consideration in assessing oxygen delivery when temperature is increased or decreased.
|
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] |
PMID:21157
|
Regulatory mutations in the Klebsiella aerogenes structural gene for glutamine synthetase.
|
Glutamine synthetase could be repressed several hundredfold rather than 6- to 10-fold as previously reported. Ammonia was not the primary repression signal for glutamine synthetase. Repression appeared to be mediated by a high level of glutamine and probably by a high ratio of glutamine to alpha-ketoglutarate. Mutations in glnA (the structural gene for glutamine synthetase) were seen to fall into three phenotypic groups: glutamine auxotrophs that produced no detectable glnA product; glutamine auxotrophs that produced a glnA product lacking enzymatic activity (and hence repressibility by ammonia) but were repressible under appropriate conditions; and glutamine synthetase regulatory mutants, whose glnA product was enzymatically active and not repressible under any conditions.
|
Regulatory mutations in the Klebsiella aerogenes structural gene for glutamine synthetase. Glutamine synthetase could be repressed several hundredfold rather than 6- to 10-fold as previously reported. Ammonia was not the primary repression signal for glutamine synthetase. Repression appeared to be mediated by a high level of glutamine and probably by a high ratio of glutamine to alpha-ketoglutarate. Mutations in glnA (the structural gene for glutamine synthetase) were seen to fall into three phenotypic groups: glutamine auxotrophs that produced no detectable glnA product; glutamine auxotrophs that produced a glnA product lacking enzymatic activity (and hence repressibility by ammonia) but were repressible under appropriate conditions; and glutamine synthetase regulatory mutants, whose glnA product was enzymatically active and not repressible under any conditions.
|
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] |
PMID:21158
|
Autogenous regulation of the synthesis of glutamine synthetase in Klebsiella aerogenes.
|
We isolated an F' episome of Escherichia coli carrying the glnA+ gene from K. aerogenes and an F' episome of E. coli carrying the glnA4 allele from K. aerogenes responsible for the constitutive synthesis of glutamine synthetase. Complementation tests with these episomes showed that the glnA4 mutation (leading to the constitutive synthesis of active glutamine synthetase) was in the gene identified by mutations glnA20, glnA51, and glnA5 as the structural gene for glutamine synthetase. By using these merodiploid strains we were able to show that the glnA51 mutation lead to the synthesis of a glutamine synthetase that lacked enzymatic activity but fully retained its regulatory properties. Finally, we discuss a model that explains the several phenotypes associated with mutations such as glnA4 located within the structural gene for glutamine synthetase leading to constitutive synthesis of active glutamine synthetase.
|
Autogenous regulation of the synthesis of glutamine synthetase in Klebsiella aerogenes. We isolated an F' episome of Escherichia coli carrying the glnA+ gene from K. aerogenes and an F' episome of E. coli carrying the glnA4 allele from K. aerogenes responsible for the constitutive synthesis of glutamine synthetase. Complementation tests with these episomes showed that the glnA4 mutation (leading to the constitutive synthesis of active glutamine synthetase) was in the gene identified by mutations glnA20, glnA51, and glnA5 as the structural gene for glutamine synthetase. By using these merodiploid strains we were able to show that the glnA51 mutation lead to the synthesis of a glutamine synthetase that lacked enzymatic activity but fully retained its regulatory properties. Finally, we discuss a model that explains the several phenotypes associated with mutations such as glnA4 located within the structural gene for glutamine synthetase leading to constitutive synthesis of active glutamine synthetase.
|
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] |
PMID:21159
|
Carbon monoxide oxidation by methanogenic bacteria.
|
Different species of methanogenic bacteria growing on CO(2) and H(2) were shown to remove CO added to the gas phase. Rates up to 0.2 mumol of CO depleted/min per 10 ml of culture containing approximately 7 mg of cells (wet weight) were observed. Methanobacterium thermoautotrophicum was selected for further study based on its ability to grow rapidly on a completely mineral medium. This species used CO as the sole energy source by disproportionating CO to CO(2) and CH(4) according to the following equation: 4CO + 2H(2)O --> 1CH(4) + 3CO(2). However, growth was slight, and the growth rate on CO was only 1% of that observed on H(2)/CO(2). Growth only occurred with CO concentrations in the gas phase of lower than 50%. Growth on CO agrees with the finding that cell-free extracts of M. thermoautotrophicum contained both an active factor 420 (F(420))-dependent hydrogenase (7.7 mumol/min per mg of protein at 35 degrees C) and a CO-dehydrogenating enzyme (0.2 mumol/min per mg of protein at 35 degrees C) that catalyzed the reduction of F(420) with CO. The properties of the CO-dehydrogenating enzyme are described. In addition to F(420), viologen dyes were effective electron acceptors for the enzyme. The apparent K(m) for CO was higher than 1 mM. The reaction rate increased with increasing pH and displayed an inflection point at pH 6.7. The temperature dependence of the reaction rate followed the Arrhenius equation with an activation energy (DeltaHdouble dagger) of 14.1 kcal/mol (59.0 kJ/mol). The CO dehydrogenase activity was reversibly inactivated by low concentrations of cyanide (2 muM) and was very sensitive to inactivation by oxygen. Carbon monoxide dehydrogenase of M. thermoautotrophicum exhibited several characteristic properties found for the enzyme of Clostridium pasteurianum but differed mainly in that the clostridial enzyme did not utilize F(420) as the electron acceptor.
|
Carbon monoxide oxidation by methanogenic bacteria. Different species of methanogenic bacteria growing on CO(2) and H(2) were shown to remove CO added to the gas phase. Rates up to 0.2 mumol of CO depleted/min per 10 ml of culture containing approximately 7 mg of cells (wet weight) were observed. Methanobacterium thermoautotrophicum was selected for further study based on its ability to grow rapidly on a completely mineral medium. This species used CO as the sole energy source by disproportionating CO to CO(2) and CH(4) according to the following equation: 4CO + 2H(2)O --> 1CH(4) + 3CO(2). However, growth was slight, and the growth rate on CO was only 1% of that observed on H(2)/CO(2). Growth only occurred with CO concentrations in the gas phase of lower than 50%. Growth on CO agrees with the finding that cell-free extracts of M. thermoautotrophicum contained both an active factor 420 (F(420))-dependent hydrogenase (7.7 mumol/min per mg of protein at 35 degrees C) and a CO-dehydrogenating enzyme (0.2 mumol/min per mg of protein at 35 degrees C) that catalyzed the reduction of F(420) with CO. The properties of the CO-dehydrogenating enzyme are described. In addition to F(420), viologen dyes were effective electron acceptors for the enzyme. The apparent K(m) for CO was higher than 1 mM. The reaction rate increased with increasing pH and displayed an inflection point at pH 6.7. The temperature dependence of the reaction rate followed the Arrhenius equation with an activation energy (DeltaHdouble dagger) of 14.1 kcal/mol (59.0 kJ/mol). The CO dehydrogenase activity was reversibly inactivated by low concentrations of cyanide (2 muM) and was very sensitive to inactivation by oxygen. Carbon monoxide dehydrogenase of M. thermoautotrophicum exhibited several characteristic properties found for the enzyme of Clostridium pasteurianum but differed mainly in that the clostridial enzyme did not utilize F(420) as the electron acceptor.
|
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] |
PMID:21160
|
Efficient sporulation of yeast in media buffered near pH6.
|
Diploid cells of Saccharomyces cerevisiae underwent meiosis and sporulation when placed in 1% potassium acetate sporulation medium. In unbuffered sporulation medium the pH rose very rapidly, reaching pH 8.4 after 2 h of sporulation. Under these conditions, the uptake of radioactive adenine and lysine was extremely limited, and ascus formation was insensitive to inhibitors such as 5-fluorouracil and canavanine. By using several different buffers, we showed that an increase in the pH of sporulation media was not necessary for sporulation to occur. Spore viability and the kinetics of ascus and prototroph formation were normal for cells sporulated in several types of media buffered as low as pH 5.5. Incubation of sporulating cells below pH 6.5 did cause separation of small but viable buds from their mother cells. With sporulating cells buffered below pH 6.5, the incorporation of radioactive adenine and lysine was greatly enhanced and cells became sensitive to inhibition by 5-fluorouracil and canavanine.
|
Efficient sporulation of yeast in media buffered near pH6. Diploid cells of Saccharomyces cerevisiae underwent meiosis and sporulation when placed in 1% potassium acetate sporulation medium. In unbuffered sporulation medium the pH rose very rapidly, reaching pH 8.4 after 2 h of sporulation. Under these conditions, the uptake of radioactive adenine and lysine was extremely limited, and ascus formation was insensitive to inhibitors such as 5-fluorouracil and canavanine. By using several different buffers, we showed that an increase in the pH of sporulation media was not necessary for sporulation to occur. Spore viability and the kinetics of ascus and prototroph formation were normal for cells sporulated in several types of media buffered as low as pH 5.5. Incubation of sporulating cells below pH 6.5 did cause separation of small but viable buds from their mother cells. With sporulating cells buffered below pH 6.5, the incorporation of radioactive adenine and lysine was greatly enhanced and cells became sensitive to inhibition by 5-fluorouracil and canavanine.
|
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0.01478533260524273,
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-0.05917773395776749,
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0.0317782461643219,
-0.041675012558698654,
0.035492487251758575,
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] |
PMID:21161
|
Deoxyribonucleic acid synthesis in permeabilized spheroplasts of Saccharomyces cerevisiae.
|
Osmotically shocked spheroplasts from Saccharomyces cerevisiae incorporated deoxynucleoside triphosphates specifically into double-stranded nuclear and mitochondrial deoxyribonucleic acid (DNA). Results with this in vitro system for cells with and without mitochondrial DNA were compared. Strains lacking mitochondrial DNA were used to study nuclear DNA replication. With a temperature-sensitive mutant defective in DNA replication in vivo, DNA synthesis in vitro was temperature sensitive as well. The product of synthesis with all strains after very short labeling times consisted principally of short fragments that sedimented at approximately 4S in alkali; with longer pulse times or a chase with unlabeled nucleotides, they grew to a more heterogenous size, with an average of 6 to 8S and a maximum of 15S. There was little, if any, integration of these DNA fragments into the high-molecular-weight nuclear DNA. Analysis by CsCl density gradient centrifugation after incorporation of bromodeoxyuridine triphosphate showed that most of the product consisted of chains containing both preexisting and newly synthesized material, but there was also a small fraction (ca. 20%) in which the strands were fully synthesized in vitro. (32)P-label transfer ("nearest-neighbor") experiments demonstrated that at least a part of the material synthesized in vitro contained ribonucleic acid-DNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking up thymidine 5'-monophosphate, sedimented in alkali at 4S, as in the case of the in vitro experiments.
|
Deoxyribonucleic acid synthesis in permeabilized spheroplasts of Saccharomyces cerevisiae. Osmotically shocked spheroplasts from Saccharomyces cerevisiae incorporated deoxynucleoside triphosphates specifically into double-stranded nuclear and mitochondrial deoxyribonucleic acid (DNA). Results with this in vitro system for cells with and without mitochondrial DNA were compared. Strains lacking mitochondrial DNA were used to study nuclear DNA replication. With a temperature-sensitive mutant defective in DNA replication in vivo, DNA synthesis in vitro was temperature sensitive as well. The product of synthesis with all strains after very short labeling times consisted principally of short fragments that sedimented at approximately 4S in alkali; with longer pulse times or a chase with unlabeled nucleotides, they grew to a more heterogenous size, with an average of 6 to 8S and a maximum of 15S. There was little, if any, integration of these DNA fragments into the high-molecular-weight nuclear DNA. Analysis by CsCl density gradient centrifugation after incorporation of bromodeoxyuridine triphosphate showed that most of the product consisted of chains containing both preexisting and newly synthesized material, but there was also a small fraction (ca. 20%) in which the strands were fully synthesized in vitro. (32)P-label transfer ("nearest-neighbor") experiments demonstrated that at least a part of the material synthesized in vitro contained ribonucleic acid-DNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking up thymidine 5'-monophosphate, sedimented in alkali at 4S, as in the case of the in vitro experiments.
|
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] |
PMID:21162
|
Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis.
|
Late during sporulation, Bacillus subtilis produces glucose dehydrogenase (GlcDH; EC 1.1.1.47), which can react with D-glucose or 2-deoxy-D-glucose and can use nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor. This enzyme is found mainly in the forespore compartment and is present in spores; it is probably made exclusively in the forespore. The properties of GlcDH were determined both in crude cell extracts and after purification. The enzyme is stable at pH 6.5 but labile at pH 8 or higher; the pH optimum of enzyme activity is 8. After inactivation at pH 8, the activity can be recovered in crude extracts, but not in solutions of the purified enzyme, by incubation with 3 M KCl and 5 mM NAD or NADP. As determined by gel filtration, enzymatically active GlcDH has a molecular weight of about 115,000 (if the enzyme is assumed to be globular). GlcDH is distinct from a catabolite-repressible inositol dehydrogenase (EC 1.1.1.18), which can also react with D-glucose, requires specifically NAD as a cofactor, and has an electrophoretic mobility different from that of GlcDH.
|
Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis. Late during sporulation, Bacillus subtilis produces glucose dehydrogenase (GlcDH; EC 1.1.1.47), which can react with D-glucose or 2-deoxy-D-glucose and can use nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor. This enzyme is found mainly in the forespore compartment and is present in spores; it is probably made exclusively in the forespore. The properties of GlcDH were determined both in crude cell extracts and after purification. The enzyme is stable at pH 6.5 but labile at pH 8 or higher; the pH optimum of enzyme activity is 8. After inactivation at pH 8, the activity can be recovered in crude extracts, but not in solutions of the purified enzyme, by incubation with 3 M KCl and 5 mM NAD or NADP. As determined by gel filtration, enzymatically active GlcDH has a molecular weight of about 115,000 (if the enzyme is assumed to be globular). GlcDH is distinct from a catabolite-repressible inositol dehydrogenase (EC 1.1.1.18), which can also react with D-glucose, requires specifically NAD as a cofactor, and has an electrophoretic mobility different from that of GlcDH.
|
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] |
PMID:21163
|
Purification and characterization of the membrane-bound ferrochelatase from Spirillum itersonii.
|
The membrane-bound enzyme ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) was purified from isolated membrane fragments of Spirillum itersonii approximately 490-fold. Purification was achieved by solubilization with chaotropic salts followed by ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, and gel filtration on Sephadex G-200. The purified enzyme has an apparent minimum molecular weight of approximately 50,000, as determined by gel filtration in the presence of 0.1% Brij 35 and 1 mM dithiothreitol but forms high-molecular-weight aggregates in the absence of detergent. Purified ferrochelatase is strongly stimulated in the presence of copper. The apparent Km for Fe2+ is 20 micrometer in the absence of copper and 9.5 micrometer in the presence of 20 micrometer CuCl2. The apparent Km for protoporphyrin is 50 micrometer, and it is unaltered by copper. Ferrochelatase has a single pH optimum of 7.50, and it is inhibited 50% by 20 micrometer heme. Certain divalent cations and sulfhydryl reagents also inhibit the enzyme.
|
Purification and characterization of the membrane-bound ferrochelatase from Spirillum itersonii. The membrane-bound enzyme ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) was purified from isolated membrane fragments of Spirillum itersonii approximately 490-fold. Purification was achieved by solubilization with chaotropic salts followed by ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, and gel filtration on Sephadex G-200. The purified enzyme has an apparent minimum molecular weight of approximately 50,000, as determined by gel filtration in the presence of 0.1% Brij 35 and 1 mM dithiothreitol but forms high-molecular-weight aggregates in the absence of detergent. Purified ferrochelatase is strongly stimulated in the presence of copper. The apparent Km for Fe2+ is 20 micrometer in the absence of copper and 9.5 micrometer in the presence of 20 micrometer CuCl2. The apparent Km for protoporphyrin is 50 micrometer, and it is unaltered by copper. Ferrochelatase has a single pH optimum of 7.50, and it is inhibited 50% by 20 micrometer heme. Certain divalent cations and sulfhydryl reagents also inhibit the enzyme.
|
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] |
PMID:21164
|
Regulation of superoxide dismutase synthesis in Escherichia coli: glucose effect.
|
Growth of Escherichia coli, based upon the fermentation of glucose, is associated with a low intracellular level of superoxide dismutase. Exhaustion of glucose, or depression of the pH due to accumulation of organic acids, causes these organisms to then obtain energy from the oxidative degradation of other substances present in a rich medium. This shift in metabolism is associated with a marked increase in the rate of synthesis of superoxide dismutase. Depression of the synthesis of superoxide dismutase by glucose is not due to catabolite repression since it is not eliminated by cyclic adenosine 3',5'-monophosphate and since alpha-methyl glucoside does not mimic the effect of glucose. Moreover, glucose itself no longer depresses superoxide dismutase synthesis when the pH has fallen low enough to cause a shift to a non-fermentative metabolism. It appears likely that superoxide dismutase is controlled directly or indirectly by the intracellular level of O2- and that glucose depressed the level of this enzyme because glucose metabolism is not associated with as rapid a production of O2- as is the metabolsim of many other substances. In accord with this view is the observation that paraquat, which can increase the rate of production of O2- by redox cycling, caused a rapid and marked increase in superoxide dismutase.
|
Regulation of superoxide dismutase synthesis in Escherichia coli: glucose effect. Growth of Escherichia coli, based upon the fermentation of glucose, is associated with a low intracellular level of superoxide dismutase. Exhaustion of glucose, or depression of the pH due to accumulation of organic acids, causes these organisms to then obtain energy from the oxidative degradation of other substances present in a rich medium. This shift in metabolism is associated with a marked increase in the rate of synthesis of superoxide dismutase. Depression of the synthesis of superoxide dismutase by glucose is not due to catabolite repression since it is not eliminated by cyclic adenosine 3',5'-monophosphate and since alpha-methyl glucoside does not mimic the effect of glucose. Moreover, glucose itself no longer depresses superoxide dismutase synthesis when the pH has fallen low enough to cause a shift to a non-fermentative metabolism. It appears likely that superoxide dismutase is controlled directly or indirectly by the intracellular level of O2- and that glucose depressed the level of this enzyme because glucose metabolism is not associated with as rapid a production of O2- as is the metabolsim of many other substances. In accord with this view is the observation that paraquat, which can increase the rate of production of O2- by redox cycling, caused a rapid and marked increase in superoxide dismutase.
|
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] |
PMID:21165
|
Obligatory coupling between proton entry and the synthesis of adenosine 5'-triphosphate in Streptococcus lactis.
|
Proton influx was measured after imposition of an electrochemical potential difference for protons (delta muH+) across the cell membrane of the anaerobe, Streptococcus lactis. As delta muH+ was increased, there was an approximately parallel increase in proton entry, until delta muH+ attained 175 to 200 mV. At this point, a new pathway became available for proton entry, allowing an abrupt increase in both the rate and extent of H+ influx. This gated response depended upon the value of delta muH+ itself, and not upon the value of either the membrane potential or the pH gradient. For delta muH+ above 175 to 200 mV, elevated proton entry occurred only in cells having a functional membrane-bound Ca2+-stimulated, Mg2+stimulated adenosine 5'-triphosphatase (EC 3.6.1.3). When present, elevated proton entry coincided with the appearance of net synthesis of adenosine 5'-triphosphate catalyzed by this adenosine 5'-triphosphatase. These observations demonstrate that membrane-bound adenosine 5'-triphosphatase catalyzes an obligatory coupling between the inward movement of protons and synthesis of adenosine 5'-triphosphate.
|
Obligatory coupling between proton entry and the synthesis of adenosine 5'-triphosphate in Streptococcus lactis. Proton influx was measured after imposition of an electrochemical potential difference for protons (delta muH+) across the cell membrane of the anaerobe, Streptococcus lactis. As delta muH+ was increased, there was an approximately parallel increase in proton entry, until delta muH+ attained 175 to 200 mV. At this point, a new pathway became available for proton entry, allowing an abrupt increase in both the rate and extent of H+ influx. This gated response depended upon the value of delta muH+ itself, and not upon the value of either the membrane potential or the pH gradient. For delta muH+ above 175 to 200 mV, elevated proton entry occurred only in cells having a functional membrane-bound Ca2+-stimulated, Mg2+stimulated adenosine 5'-triphosphatase (EC 3.6.1.3). When present, elevated proton entry coincided with the appearance of net synthesis of adenosine 5'-triphosphate catalyzed by this adenosine 5'-triphosphatase. These observations demonstrate that membrane-bound adenosine 5'-triphosphatase catalyzes an obligatory coupling between the inward movement of protons and synthesis of adenosine 5'-triphosphate.
|
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] |
PMID:21166
|
Transformation in pneumococcus: existence and properties of a complex involving donor deoxyribonucleate single strands in eclipse.
|
Donor deoxyribonucleic acid (DNA) single strands exist in a complex during the eclipse phase in pneumococcal transformation. This eclipse complex exhibited specific physical properties distinct from those of both pure DNA single strands and native DNA. These included a lower affinity for diethylaminoethyl-cellulose and hydroxylapatite than that of single-strand DNA, faster sedimentation than the DNA chains that it contains, and a buoyant density in Cs2SO4 lower than that of native DNA. The complex was dissociated by treatments with sodium dodecyl sulfate, NaOH, guanidine-hydrochloride, chloroform, and proteinase K but was insensitive to ribonuclease.
|
Transformation in pneumococcus: existence and properties of a complex involving donor deoxyribonucleate single strands in eclipse. Donor deoxyribonucleic acid (DNA) single strands exist in a complex during the eclipse phase in pneumococcal transformation. This eclipse complex exhibited specific physical properties distinct from those of both pure DNA single strands and native DNA. These included a lower affinity for diethylaminoethyl-cellulose and hydroxylapatite than that of single-strand DNA, faster sedimentation than the DNA chains that it contains, and a buoyant density in Cs2SO4 lower than that of native DNA. The complex was dissociated by treatments with sodium dodecyl sulfate, NaOH, guanidine-hydrochloride, chloroform, and proteinase K but was insensitive to ribonuclease.
|
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] |
PMID:21167
|
Nitrogen and ammonia assimilation in the cyanobacteria: purification of glutamine synthetase from Anabaena sp. strain CA.
|
Glutamine synthetase was purified from the cyanobacterium Anabaena sp. strain CA, a newly isolated marine organism. This organism grows rapidly under nitrogen-fixing conditions and therefore is ideally suited for studies concerning cyanobacterial nitrogen metabolism. Studies were conducted to optimize the production of glutamine synthetase by Anabaena CA. The highest specific activities were obtained from cells grown in the presence of atmospheric N(2) or KNO(3) (13 mM); when NH(4)Cl was used as the nitrogen source, the specific activity was reduced by approximately 40%. Furthermore, through the use of a whole-cell gamma-glutamylhydroxamate transferase assay, it was found that the maximum number of enzyme units is obtained in the late logarithmic stage of growth. Glutamine synthetase purification requires only three steps and results in a preparation that is electrophoretically homogeneous. The transferase specific activity (units per milligram of protein) of the purified enzyme is 78, whereas the biosynthetic specific activity is 2.2. The molecular weight of the native protein was found to be approximately 590,000, and the subunit molecular weight was determined to be about 50,000. Thus, this cyanobacterial enzyme closely resembles the enzyme obtained from other procaryotic sources, at least with regard to size. The purification of glutamine synthetase from Anabaena CA should stimulate a more detailed study of this enzyme and its role in cyanobacterial nitrogen metabolism.
|
Nitrogen and ammonia assimilation in the cyanobacteria: purification of glutamine synthetase from Anabaena sp. strain CA. Glutamine synthetase was purified from the cyanobacterium Anabaena sp. strain CA, a newly isolated marine organism. This organism grows rapidly under nitrogen-fixing conditions and therefore is ideally suited for studies concerning cyanobacterial nitrogen metabolism. Studies were conducted to optimize the production of glutamine synthetase by Anabaena CA. The highest specific activities were obtained from cells grown in the presence of atmospheric N(2) or KNO(3) (13 mM); when NH(4)Cl was used as the nitrogen source, the specific activity was reduced by approximately 40%. Furthermore, through the use of a whole-cell gamma-glutamylhydroxamate transferase assay, it was found that the maximum number of enzyme units is obtained in the late logarithmic stage of growth. Glutamine synthetase purification requires only three steps and results in a preparation that is electrophoretically homogeneous. The transferase specific activity (units per milligram of protein) of the purified enzyme is 78, whereas the biosynthetic specific activity is 2.2. The molecular weight of the native protein was found to be approximately 590,000, and the subunit molecular weight was determined to be about 50,000. Thus, this cyanobacterial enzyme closely resembles the enzyme obtained from other procaryotic sources, at least with regard to size. The purification of glutamine synthetase from Anabaena CA should stimulate a more detailed study of this enzyme and its role in cyanobacterial nitrogen metabolism.
|
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] |
PMID:21168
|
Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci.
|
Inhibition of peptidoglycan synthesis causes an immediate and massive secretion of both newly synthesized and "old" lipids from several species of bacteria, including streptococci, Staphylococcus epidermidis, and Bacillus subtilis. Lipid secretion occurs in the absence of detectable bacterial lysis. This novel phenomenon was examined in more detail in three strains of streptococci: S. sanguis (group H), S. pyogenes (group A), And S. pneumoniae. The secretion of lipids is specifically induced by inhibitors of peptidoglycan synthesis; it is not caused by inhibitors of protein, ribonucleic acid, or deoxyribonucleic acid synthesis. The occurrence appears to be reversible since penicillin-induced secretion comes to a halt upon the timely addition of penicillinase, correlating with resumption of culture growth. All cellular lipids are secreted in essentially the same proportions as those found in the drug treated bacteria. It is suggested that continued peptidoglycan synthesis may be essential for the integration and retention of lipid material in the plasma membrane.
|
Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. Inhibition of peptidoglycan synthesis causes an immediate and massive secretion of both newly synthesized and "old" lipids from several species of bacteria, including streptococci, Staphylococcus epidermidis, and Bacillus subtilis. Lipid secretion occurs in the absence of detectable bacterial lysis. This novel phenomenon was examined in more detail in three strains of streptococci: S. sanguis (group H), S. pyogenes (group A), And S. pneumoniae. The secretion of lipids is specifically induced by inhibitors of peptidoglycan synthesis; it is not caused by inhibitors of protein, ribonucleic acid, or deoxyribonucleic acid synthesis. The occurrence appears to be reversible since penicillin-induced secretion comes to a halt upon the timely addition of penicillinase, correlating with resumption of culture growth. All cellular lipids are secreted in essentially the same proportions as those found in the drug treated bacteria. It is suggested that continued peptidoglycan synthesis may be essential for the integration and retention of lipid material in the plasma membrane.
|
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] |
PMID:21170
|
Isolation and characterization of NADP+-specific isocitrate dehydrogenase from the pupa of Bombyx mori.
|
NADP+-specific isocitrate dehydrogenase was found in several tissues of the pupa of the silkworm, Bombyx mori. This enzyme was highly purified from the whole bodies of pupae. This is the first isolation of the enzyme from insect materials. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis. The reaction catalyzed by the purified enzyme was readily reversible. The pH optimum for the forward reaction (reduction of NADP+) was 7.8, and that for the reverse reaction (oxidation of NADPH) was 6.6. The enzyme had a molecular weight of 86,000 and was found to be composed of two identical subunits, which have a molecular weight of 44,000. The activity of the enzyme in the forward reaction was slightly inhibited by citrate, oxaloacetate, alpha-ketoglutarate, and others. Citrate stabilized the activity over a wide pH region.
|
Isolation and characterization of NADP+-specific isocitrate dehydrogenase from the pupa of Bombyx mori. NADP+-specific isocitrate dehydrogenase was found in several tissues of the pupa of the silkworm, Bombyx mori. This enzyme was highly purified from the whole bodies of pupae. This is the first isolation of the enzyme from insect materials. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis. The reaction catalyzed by the purified enzyme was readily reversible. The pH optimum for the forward reaction (reduction of NADP+) was 7.8, and that for the reverse reaction (oxidation of NADPH) was 6.6. The enzyme had a molecular weight of 86,000 and was found to be composed of two identical subunits, which have a molecular weight of 44,000. The activity of the enzyme in the forward reaction was slightly inhibited by citrate, oxaloacetate, alpha-ketoglutarate, and others. Citrate stabilized the activity over a wide pH region.
|
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] |
PMID:21171
|
Stimulatory effect of n-alkanes on CTP: Cholinephosphate cytidyltransferase activity in rat liver microsomal membranes in vitro.
|
To assess the effect of alteration of membrane structure on the enzymic activities related to phospholipid synthesis in microsomal membrane, the effects of several organic solvents have been studied in an in vitro system, in which the cytoplasmic extract prepared from rat liver incorporated [14C]choline or [14C]CDP-choline into phosphatidycholine (lecithin). The optimum conditions for the incorporation were determined. Among several organic solvents examined, n-alkanes such as n-hexane, n-octane, and n-tetradecane stimulated the incorporation. It was shown that n-alkanes stimulated one of three enzymic steps of lecithin biosynthesis from choline; that is, the formulation of CDP-choline catalyzed by CTP: cholinephosphate cytidyltransferase [EC 2.7.7.15], an enzyme on the microsomal membrane. It was further shown that the same enzyme was also stimulated by preincubation of microsomes in the absence of substrate. It is suggested that alteration of the lipid environment of the microsomal membrane induced by n-alkanes caused activation of this enzymic step.
|
Stimulatory effect of n-alkanes on CTP: Cholinephosphate cytidyltransferase activity in rat liver microsomal membranes in vitro. To assess the effect of alteration of membrane structure on the enzymic activities related to phospholipid synthesis in microsomal membrane, the effects of several organic solvents have been studied in an in vitro system, in which the cytoplasmic extract prepared from rat liver incorporated [14C]choline or [14C]CDP-choline into phosphatidycholine (lecithin). The optimum conditions for the incorporation were determined. Among several organic solvents examined, n-alkanes such as n-hexane, n-octane, and n-tetradecane stimulated the incorporation. It was shown that n-alkanes stimulated one of three enzymic steps of lecithin biosynthesis from choline; that is, the formulation of CDP-choline catalyzed by CTP: cholinephosphate cytidyltransferase [EC 2.7.7.15], an enzyme on the microsomal membrane. It was further shown that the same enzyme was also stimulated by preincubation of microsomes in the absence of substrate. It is suggested that alteration of the lipid environment of the microsomal membrane induced by n-alkanes caused activation of this enzymic step.
|
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] |
PMID:21172
|
H+ translocation in lysozyme-treated cells of the blue-green alga Plectonema boryanum. Difference between isotonically and hypotonically treated preparations and effects of divalent cations.
|
Lysozyme-treated cells of a blue-green alga, Plectonema boryanum, had an internal pH of 7.3+/-0.2 under isotonic and hypotonic conditions. This value was similar to that of untreated cells. The CCCP-induced biphasic H+ change seen in the isotonic cells was not observed in the hypotonically treated cells. The biphasic time course remained in the hypotonic preparation if CaCl2 or MgCl2 was added prior to the osmotic shock. It is suggested that the cells have two compartments of H+ concentration. The outer region may be more acidic than the inner region. A light-induced H+ efflux was observed under isotonic conditions and an influx of H+ under hypotonic conditions. The H+ influx was not observed when lysozyme-treated cells were incubated with CaCl2 or MgCl2 prior to the hypotonic treatment. Two types of effects of divalent cations, one on the rigidity of the outer membrane and another on the permeability characteristics of the inner photosynthetic membrane, are indicated. Rearrangement of the photosynthetic membranes and an apparent inversion of the H+ pump by hypotonic shock are also suggested.
|
H+ translocation in lysozyme-treated cells of the blue-green alga Plectonema boryanum. Difference between isotonically and hypotonically treated preparations and effects of divalent cations. Lysozyme-treated cells of a blue-green alga, Plectonema boryanum, had an internal pH of 7.3+/-0.2 under isotonic and hypotonic conditions. This value was similar to that of untreated cells. The CCCP-induced biphasic H+ change seen in the isotonic cells was not observed in the hypotonically treated cells. The biphasic time course remained in the hypotonic preparation if CaCl2 or MgCl2 was added prior to the osmotic shock. It is suggested that the cells have two compartments of H+ concentration. The outer region may be more acidic than the inner region. A light-induced H+ efflux was observed under isotonic conditions and an influx of H+ under hypotonic conditions. The H+ influx was not observed when lysozyme-treated cells were incubated with CaCl2 or MgCl2 prior to the hypotonic treatment. Two types of effects of divalent cations, one on the rigidity of the outer membrane and another on the permeability characteristics of the inner photosynthetic membrane, are indicated. Rearrangement of the photosynthetic membranes and an apparent inversion of the H+ pump by hypotonic shock are also suggested.
|
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] |
PMID:21173
|
Deoxyribonuclease A, a novel deoxyribonuclease highly active toward Polydeoxyadenylic acid and polythymidylic acid from Achatina fulica.
|
Two novel deoxyribonucleases, termed DNases A and A', have been purified from the hepatopancreas of Achatina fulica (agate snail). DNases A and A' were obtained in 3.6 % and 7.7% yields by acetate buffer extraction and successive chromatography on hydroxyapatite, phosphocellulose and poly(A)-Sepharose. The two DNases are highly active toward poly(dA) and at a salt concentration of 0.15 m and pH 5.0 exhibit 10-fold higher hydrolyzing activities toward poly(dA) than toward calf thymus denature DNA. The enzymes also act considerably on poly(dT) at pH 4.0, but do not appreciably digest other synthetic homopolymers such as poly(dC), poly(dG) and poly(A). The limit products obtained by exhaustive digestion of poly(dA) with DNases A and A' are 98% and 99% acid-soluble and consist of oligonucleotides with an average chain length of about 5 nucleotides containing barely detectable dimers and trimers, respectively. These fragments have terminal 5'-hydroxyl and 3'-phosphate groups. The mode of action appears to be endonucleolytic. Both enzymes have the same pH optimum of 5.0 for poly(dA-hydrolyzing activity. Ionic strength is critical for the maximum activity.
|
Deoxyribonuclease A, a novel deoxyribonuclease highly active toward Polydeoxyadenylic acid and polythymidylic acid from Achatina fulica. Two novel deoxyribonucleases, termed DNases A and A', have been purified from the hepatopancreas of Achatina fulica (agate snail). DNases A and A' were obtained in 3.6 % and 7.7% yields by acetate buffer extraction and successive chromatography on hydroxyapatite, phosphocellulose and poly(A)-Sepharose. The two DNases are highly active toward poly(dA) and at a salt concentration of 0.15 m and pH 5.0 exhibit 10-fold higher hydrolyzing activities toward poly(dA) than toward calf thymus denature DNA. The enzymes also act considerably on poly(dT) at pH 4.0, but do not appreciably digest other synthetic homopolymers such as poly(dC), poly(dG) and poly(A). The limit products obtained by exhaustive digestion of poly(dA) with DNases A and A' are 98% and 99% acid-soluble and consist of oligonucleotides with an average chain length of about 5 nucleotides containing barely detectable dimers and trimers, respectively. These fragments have terminal 5'-hydroxyl and 3'-phosphate groups. The mode of action appears to be endonucleolytic. Both enzymes have the same pH optimum of 5.0 for poly(dA-hydrolyzing activity. Ionic strength is critical for the maximum activity.
|
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] |
PMID:21175
|
Acetyl-CoA-dependent elongation of fatty acids in Mycobacterium smegmatis.
|
An enzyme system of Mycobacterium smegmatis catalyzing the elongation of medium-chain fatty acids with acetyl-CoA was obtained free from de novo fatty acid synthetase by ammonium sulfate fractionation. The system was resolved by gel filtration and DEAE-cellulose chromatography into three fractions, all of which were required for reconstitution of the elongation activity. The three fractions were highly purified enoyl-CoA hydratase, highly purified 3-hydroxyacyl-CoA dehydrogenase, and a fraction containing both enoyl-CoA reductase and thiolase. The reconstituted system was avidin-insenstive, required NADH as a sole hydrogen donor, and was sensitive to pCMB, but not to N-ethylmaleimide or monoiodoacetate. Decanoyl-CoA and octanoyl-CoA were the best primers for the elongation system. When decanoyl-CoA was used as the primer, the major product was found to be a lauroyl derivative (probably lauroyl-CoA). Evidence was obtained suggesting that acyl-CoA dehydrogenase, catalyzing the first step of beta-oxidation, was not functional in the elongation system.
|
Acetyl-CoA-dependent elongation of fatty acids in Mycobacterium smegmatis. An enzyme system of Mycobacterium smegmatis catalyzing the elongation of medium-chain fatty acids with acetyl-CoA was obtained free from de novo fatty acid synthetase by ammonium sulfate fractionation. The system was resolved by gel filtration and DEAE-cellulose chromatography into three fractions, all of which were required for reconstitution of the elongation activity. The three fractions were highly purified enoyl-CoA hydratase, highly purified 3-hydroxyacyl-CoA dehydrogenase, and a fraction containing both enoyl-CoA reductase and thiolase. The reconstituted system was avidin-insenstive, required NADH as a sole hydrogen donor, and was sensitive to pCMB, but not to N-ethylmaleimide or monoiodoacetate. Decanoyl-CoA and octanoyl-CoA were the best primers for the elongation system. When decanoyl-CoA was used as the primer, the major product was found to be a lauroyl derivative (probably lauroyl-CoA). Evidence was obtained suggesting that acyl-CoA dehydrogenase, catalyzing the first step of beta-oxidation, was not functional in the elongation system.
|
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] |
PMID:21178
|
Isolation and characterization of malic enzyme from the pupa of Bombyx mori.
|
Malic enzyme, which requires NADP+ as a coenzyme, was isolated and purified from pupae of the silkworm, Bombyx mori. The purified enzyme appeared homogeneous and had a molecular weight of 195,000 on polyacrylamide gel electrophoresis. The optimum pH for the oxidative decarboxylation of malate, measured in terms of the increase of NADPH (MH activity) and CO2 (MC activity), was pH 7.5, while that for the decarboxylation of oxaloacetate measured in terms of the increase of CO2 (OC activity) was pH 4.6. Several differences between MH and OC activity were investigated.
|
Isolation and characterization of malic enzyme from the pupa of Bombyx mori. Malic enzyme, which requires NADP+ as a coenzyme, was isolated and purified from pupae of the silkworm, Bombyx mori. The purified enzyme appeared homogeneous and had a molecular weight of 195,000 on polyacrylamide gel electrophoresis. The optimum pH for the oxidative decarboxylation of malate, measured in terms of the increase of NADPH (MH activity) and CO2 (MC activity), was pH 7.5, while that for the decarboxylation of oxaloacetate measured in terms of the increase of CO2 (OC activity) was pH 4.6. Several differences between MH and OC activity were investigated.
|
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] |
PMID:21179
|
The structure and function of ribonuclease T1 XXIV. Preparation and properties of a stable water-insoluble polyacrylamide derivative of ribonuclease T1.
|
Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme.
|
The structure and function of ribonuclease T1 XXIV. Preparation and properties of a stable water-insoluble polyacrylamide derivative of ribonuclease T1. Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme.
|
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] |
PMID:21180
|
Reactivity of ferrous myoglobin at low pH.
|
The rates of reaction of myoglobin with carbon monoxide at low pH are reported. The pH versus rate profile of these kinetics resembles that found for heme model compounds, revealing an increase in combination rate at low pH. These facts suggest that CO binding by myoglobin changes from a mechanism of "direct ligant association" at pH 5 to a mechanism, similar to that proposed for heme model compounds, which assumes a tetracoordinated intermediate as a result of the protonation of the proximal imidazole.
|
Reactivity of ferrous myoglobin at low pH. The rates of reaction of myoglobin with carbon monoxide at low pH are reported. The pH versus rate profile of these kinetics resembles that found for heme model compounds, revealing an increase in combination rate at low pH. These facts suggest that CO binding by myoglobin changes from a mechanism of "direct ligant association" at pH 5 to a mechanism, similar to that proposed for heme model compounds, which assumes a tetracoordinated intermediate as a result of the protonation of the proximal imidazole.
|
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] |
PMID:21182
|
Endogenous activating factor for guanylate cyclase in synaptosomal-soluble fraction of rat brain.
|
When the crude mitochondrial fraction of rat brain was homogenized with distilled water and centrifuged, most of guanylate cyclase activity was detected in the soluble fraction. The total guanylate cyclase activity recovered in the soluble fraction was 5- to 8-fold higher than that of the crude mitochondrial fraction. The greater recovery of guanylate cyclase activity was found to be due to a release of an endogenous activating factor for guanylate cyclase. The activating factor was partially purified by acid extraction followed by a gel filtration and ion exchange resin columns. The factor was a dialyzable small molecule. The molecular weight was estimated to be between 300 and 600 by a Sephadex G-15 column and Diaflo ultrafilter membranes. It was stable in dilute acids, but labile in alkaline solution. It was readily soluble in water, but insoluble in organic solvents. Treatment with various enzymes, so far as tested, failed to abolish the activity. The activating factor stimulated the initial velocity of the reaction. It altered neither the Km value for GTP nor the dependency of the enzyme on divalent metals. The activation by the factor was due to an increase in the Vmax of the reaction. The activation was prevented by lysolecithin, Lubrol PX, hydroxylamine, methylhydroxylamine, or hemoglobin.
|
Endogenous activating factor for guanylate cyclase in synaptosomal-soluble fraction of rat brain. When the crude mitochondrial fraction of rat brain was homogenized with distilled water and centrifuged, most of guanylate cyclase activity was detected in the soluble fraction. The total guanylate cyclase activity recovered in the soluble fraction was 5- to 8-fold higher than that of the crude mitochondrial fraction. The greater recovery of guanylate cyclase activity was found to be due to a release of an endogenous activating factor for guanylate cyclase. The activating factor was partially purified by acid extraction followed by a gel filtration and ion exchange resin columns. The factor was a dialyzable small molecule. The molecular weight was estimated to be between 300 and 600 by a Sephadex G-15 column and Diaflo ultrafilter membranes. It was stable in dilute acids, but labile in alkaline solution. It was readily soluble in water, but insoluble in organic solvents. Treatment with various enzymes, so far as tested, failed to abolish the activity. The activating factor stimulated the initial velocity of the reaction. It altered neither the Km value for GTP nor the dependency of the enzyme on divalent metals. The activation by the factor was due to an increase in the Vmax of the reaction. The activation was prevented by lysolecithin, Lubrol PX, hydroxylamine, methylhydroxylamine, or hemoglobin.
|
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] |
PMID:21183
|
Studies of the beta-galactoside transporter in inverted membrane vesicles of Escherichia coli. I. Symmetrical facilitated diffusion and proton gradient-coupled transport.
|
Facilitated diffusion of [14C]lactose into inverted membrane vesicles of Escherichia coli was measured using HgCl2 as a stopping reagent and polylysine to flocculate the vesicles for filtration. Equilibration of lactose between the internal and external volumes required expression of the y gene of the lac operon and was inhibited by thiodigalactoside or by prior incubation with N-ethylmaleimde or HgCl2. The initial rate of uptake was saturable, with a Kt of 0.95 mM. Counterflow of [14C]lactose was demonstrated in either direction. ATP hydrolysis or respiration drove the efflux of internal lactose. The effect of ATP required addition of F1 coupling factor (ATPase) from E. coli when lactose transport was studied in F1-deficient inverted vesicles. Accumulation of lactose against a concentration gradient was achieved by forming an artificial electrochemical proton gradient consisting of a membrane potential negative inside or a pH gradient basic inside. Addition of ATP inhibited this proton driven uptake showing that it occurred in inverted vesicles. It was concluded that the lactose-proton co-transport protein (M protein) is qualitatively symmetrical with respect to the facilitated diffusion of lactose and the coupling of proton and lactose transport.
|
Studies of the beta-galactoside transporter in inverted membrane vesicles of Escherichia coli. I. Symmetrical facilitated diffusion and proton gradient-coupled transport. Facilitated diffusion of [14C]lactose into inverted membrane vesicles of Escherichia coli was measured using HgCl2 as a stopping reagent and polylysine to flocculate the vesicles for filtration. Equilibration of lactose between the internal and external volumes required expression of the y gene of the lac operon and was inhibited by thiodigalactoside or by prior incubation with N-ethylmaleimde or HgCl2. The initial rate of uptake was saturable, with a Kt of 0.95 mM. Counterflow of [14C]lactose was demonstrated in either direction. ATP hydrolysis or respiration drove the efflux of internal lactose. The effect of ATP required addition of F1 coupling factor (ATPase) from E. coli when lactose transport was studied in F1-deficient inverted vesicles. Accumulation of lactose against a concentration gradient was achieved by forming an artificial electrochemical proton gradient consisting of a membrane potential negative inside or a pH gradient basic inside. Addition of ATP inhibited this proton driven uptake showing that it occurred in inverted vesicles. It was concluded that the lactose-proton co-transport protein (M protein) is qualitatively symmetrical with respect to the facilitated diffusion of lactose and the coupling of proton and lactose transport.
|
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] |
PMID:21184
|
Nucleus-dependent regulation of tyrosine aminotransferase degradation in hepatoma tissue culture cells.
|
Upon removal of the nucleus from rat hepatoma tissue culture cells, levels of the enzyme tyrosine aminotransferase no longer change in response to withdrawal of glucocorticoids. The rate of tyrosine aminotransferase degradation is drastically reduced in rat hepatoma tissue culture cytoplasts leading to stabilization of pre-existing levels of tyrosine aminotransferase. Moreover, the rate of synthesis of the enzyme in cytoplasts is very low near that observed in uninduced whole cells. These effects of enucleation occur very rapidly and appear to be specific for tyrosine aminotransferase and a small number of other unstable hepatoma proteins. A nuclear effect is thus directly involved in the control of tyrosine aminotransferase degradation and synthesis.
|
Nucleus-dependent regulation of tyrosine aminotransferase degradation in hepatoma tissue culture cells. Upon removal of the nucleus from rat hepatoma tissue culture cells, levels of the enzyme tyrosine aminotransferase no longer change in response to withdrawal of glucocorticoids. The rate of tyrosine aminotransferase degradation is drastically reduced in rat hepatoma tissue culture cytoplasts leading to stabilization of pre-existing levels of tyrosine aminotransferase. Moreover, the rate of synthesis of the enzyme in cytoplasts is very low near that observed in uninduced whole cells. These effects of enucleation occur very rapidly and appear to be specific for tyrosine aminotransferase and a small number of other unstable hepatoma proteins. A nuclear effect is thus directly involved in the control of tyrosine aminotransferase degradation and synthesis.
|
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] |
PMID:21188
|
Stabilization of adenylate energy charge and its relation to human sperm motility.
|
The adenylate energy charge of human ejaculated spermatozoa was studied when the sperm motility was perturbed by varying pH, prolonged incubation, and caffeine. Between pH 8 and 9, which was optimal for the sperm motility, the energy charge was in the physiological range of 0.8 to 0.9. Above pH 9, the mobility, ATP content, and adenine nucleotide pool declined rapidly but the energy charge was maintained slightly below 0.8. Below pH 8, the motility also dropped drastically, but the ATP, nucleotide pool, and energy charge fell only slightly. Prolonged incubations of the spermatozoa decreased the motility, ATP, and nucleotide pool. However, the energy charge would remain above 0.6. Caffeine stimulation of the motility caused a rapid fall of ATP and the reduction of the physiological energy charge by 0.2 unit, unless glucose was added. Imidazole which reduced the caffeine-stimulated motility did not alter the physiological energy charge of the spermatozoa. The study showed that the spermatozoa could maintain the energy charge above 0.6 under stress.
|
Stabilization of adenylate energy charge and its relation to human sperm motility. The adenylate energy charge of human ejaculated spermatozoa was studied when the sperm motility was perturbed by varying pH, prolonged incubation, and caffeine. Between pH 8 and 9, which was optimal for the sperm motility, the energy charge was in the physiological range of 0.8 to 0.9. Above pH 9, the mobility, ATP content, and adenine nucleotide pool declined rapidly but the energy charge was maintained slightly below 0.8. Below pH 8, the motility also dropped drastically, but the ATP, nucleotide pool, and energy charge fell only slightly. Prolonged incubations of the spermatozoa decreased the motility, ATP, and nucleotide pool. However, the energy charge would remain above 0.6. Caffeine stimulation of the motility caused a rapid fall of ATP and the reduction of the physiological energy charge by 0.2 unit, unless glucose was added. Imidazole which reduced the caffeine-stimulated motility did not alter the physiological energy charge of the spermatozoa. The study showed that the spermatozoa could maintain the energy charge above 0.6 under stress.
|
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] |
PMID:21189
|
Hydrogen exchange study of membrane-bound rhodopsin. I. Protein structure.
|
Structural parameters of rhodopsin in disc membrane preparations from frog and cattle were studied by hydrogen exchange methods. The method measures the exchange of protein amide hydrogens with water and can distinguish protons which are internally bonded from those which are hydrogen-bonded to water. The results show that about 70% of rhodopsin's peptide group protons are exposed to water. The identification of these groups as free peptides was made initially on the usual basis of the identity of their exchange rate with the well characterized free peptide rate; other experiments specifically excluded contributions from lipids, protein side chains, adventitious mucopolysaccharides, and intradisc water. In contrast to rhodopsin, other proteins generally have only 20 to 40% free peptide groups. Apparently rhodopsin has some unusual structural feature. Our results together with available information on rhodopsin suggest that a considerable length of its polypeptide chain is arranged at the surface of a channel of water penetrating into the membrane. Physicochemical considerations indicate that such a channel would have to be quite wide, 10 to 12 A or more, to explain the hydrogen exchange results.
|
Hydrogen exchange study of membrane-bound rhodopsin. I. Protein structure. Structural parameters of rhodopsin in disc membrane preparations from frog and cattle were studied by hydrogen exchange methods. The method measures the exchange of protein amide hydrogens with water and can distinguish protons which are internally bonded from those which are hydrogen-bonded to water. The results show that about 70% of rhodopsin's peptide group protons are exposed to water. The identification of these groups as free peptides was made initially on the usual basis of the identity of their exchange rate with the well characterized free peptide rate; other experiments specifically excluded contributions from lipids, protein side chains, adventitious mucopolysaccharides, and intradisc water. In contrast to rhodopsin, other proteins generally have only 20 to 40% free peptide groups. Apparently rhodopsin has some unusual structural feature. Our results together with available information on rhodopsin suggest that a considerable length of its polypeptide chain is arranged at the surface of a channel of water penetrating into the membrane. Physicochemical considerations indicate that such a channel would have to be quite wide, 10 to 12 A or more, to explain the hydrogen exchange results.
|
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] |
PMID:21190
|
Hydrogen exchange study of membrane-bound rhodopsin. II. Light-induced protein structure change.
|
Hydrogen exchange studies of rhodopsin in disc membranes demonstrated that photolysis induces changes in the protein itself. Two different altered forms were detected. A late photointermediate in the bleaching sequence, which can be identified with metarhodopsin II, displays accelerated exchange. Subsequently, at the stage of fully bleached opsin, exchange becomes even slower than in rhodopsin. These changes involve only a small fraction of the protein's internally hydrogen-bonded peptide groups. The unusually large fraction of exposed peptide hydrogens observed previously for rhodopsin is unaltered in the photolyzed forms.
|
Hydrogen exchange study of membrane-bound rhodopsin. II. Light-induced protein structure change. Hydrogen exchange studies of rhodopsin in disc membranes demonstrated that photolysis induces changes in the protein itself. Two different altered forms were detected. A late photointermediate in the bleaching sequence, which can be identified with metarhodopsin II, displays accelerated exchange. Subsequently, at the stage of fully bleached opsin, exchange becomes even slower than in rhodopsin. These changes involve only a small fraction of the protein's internally hydrogen-bonded peptide groups. The unusually large fraction of exposed peptide hydrogens observed previously for rhodopsin is unaltered in the photolyzed forms.
|
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] |
PMID:21191
|
Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. IV. The COOH-terminal 669 residues of the peptide chain; comparison with other glutamate dehydrogenases.
|
A sequence is presented for the COOH-terminal 669 residues of the NAD-specific glutamate dehydrogenase of Neurospora crassa. Comparison of this sequence with those of the vertebrate glutamate dehydrogenases of chicken and bovine liver and with the NADP-specific enzyme of Neurospora shows some similarities in sequences around residues previously identified as important for the function of these enzymes. These are: (a) the reactive lysine residue of low pK in the NADP and the vertebrate enzymes; (b) the tyrosine residue of the NADP enzyme that is readily nitrated by tetranitromethane with inactivation, a residue protected by NADP or by NMN; and (c) the arginine residue of the NADP-enzyme that is reactive with 1,2-cyclohexanedione with inactivation. Despite these similarities, comparison of the sequence of the NAD-enzyme with those of the other glutamate dehydrogenases of known sequences revealed relatively little overall homology as determined by computer analysis.
|
Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora. IV. The COOH-terminal 669 residues of the peptide chain; comparison with other glutamate dehydrogenases. A sequence is presented for the COOH-terminal 669 residues of the NAD-specific glutamate dehydrogenase of Neurospora crassa. Comparison of this sequence with those of the vertebrate glutamate dehydrogenases of chicken and bovine liver and with the NADP-specific enzyme of Neurospora shows some similarities in sequences around residues previously identified as important for the function of these enzymes. These are: (a) the reactive lysine residue of low pK in the NADP and the vertebrate enzymes; (b) the tyrosine residue of the NADP enzyme that is readily nitrated by tetranitromethane with inactivation, a residue protected by NADP or by NMN; and (c) the arginine residue of the NADP-enzyme that is reactive with 1,2-cyclohexanedione with inactivation. Despite these similarities, comparison of the sequence of the NAD-enzyme with those of the other glutamate dehydrogenases of known sequences revealed relatively little overall homology as determined by computer analysis.
|
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] |
PMID:21192
|
Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells.
|
HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.
|
Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells. HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.
|
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] |
PMID:21193
|
Patterns of plasminogen activator production in cultured normal embryonic cells.
|
Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor.
|
Patterns of plasminogen activator production in cultured normal embryonic cells. Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor.
|
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] |
PMID:21194
|
Complementary use of the reversed-phase and anion-exchange modes of high-pressure liquid chromatography for studies of reduced nicotinamide adenine dinucleotide.
|
The reversed-phase and anion-exchange modes of high-pressure liquid chromatography were used to separate breakdown products and impurities in solutions of the reduced form of nicotinamide adenine dinucleotide (NADH). The two chromatographic modes are compared for studies of NADH. Their use in following the course of acidic breakdown of NADH is described.
|
Complementary use of the reversed-phase and anion-exchange modes of high-pressure liquid chromatography for studies of reduced nicotinamide adenine dinucleotide. The reversed-phase and anion-exchange modes of high-pressure liquid chromatography were used to separate breakdown products and impurities in solutions of the reduced form of nicotinamide adenine dinucleotide (NADH). The two chromatographic modes are compared for studies of NADH. Their use in following the course of acidic breakdown of NADH is described.
|
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] |
PMID:21195
|
Fractionation and characterization of acidic ogligosaccharides and glycopeptides from normal and pathological urines.
|
A general procedure is described for the isolation of urinary acidic oligosaccharides and glycopeptides resulting from catabolism of glycoproteins. This procedure has been applied to normal urine and to urine from patients with diseases of the metabolism, including mucolipidosis and fucosidosis.
|
Fractionation and characterization of acidic ogligosaccharides and glycopeptides from normal and pathological urines. A general procedure is described for the isolation of urinary acidic oligosaccharides and glycopeptides resulting from catabolism of glycoproteins. This procedure has been applied to normal urine and to urine from patients with diseases of the metabolism, including mucolipidosis and fucosidosis.
|
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0.0342831164598465,
0.00036244653165340424,
0.012668156065046787,
0.016317786648869514,
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] |
PMID:21196
|
Bromothymol blue and carbohydrate-sensitive plating media.
|
A new plating medium using bromothymol blue (BTB) indicator is described and compared with eosin-methylene blue (EMB), MacConkey, and Endo media. These media were tested with L-arabinose by plating fermenting and nonfermenting mutant strains of Escherichia coli. The minimum concentrations of L-arabinose that permitted differentiation of these strains were determined. Different concentrations were required for differentiating confluent patches of cells, isolated colonies, and closely spaced or adjacent colonies. L-Arabinose, L-rhamnose, D-lactose, and D-galactose were tested with modified enteric media and with BTB medium, again to determine minimum usable concentrations. BTB media and reformulated conventional media allowed detection of acidification, aerobically, at one-fifth to one-hundredth the (1%, wt/vol) concentration of carbohydrate used in standard indicator plates.
|
Bromothymol blue and carbohydrate-sensitive plating media. A new plating medium using bromothymol blue (BTB) indicator is described and compared with eosin-methylene blue (EMB), MacConkey, and Endo media. These media were tested with L-arabinose by plating fermenting and nonfermenting mutant strains of Escherichia coli. The minimum concentrations of L-arabinose that permitted differentiation of these strains were determined. Different concentrations were required for differentiating confluent patches of cells, isolated colonies, and closely spaced or adjacent colonies. L-Arabinose, L-rhamnose, D-lactose, and D-galactose were tested with modified enteric media and with BTB medium, again to determine minimum usable concentrations. BTB media and reformulated conventional media allowed detection of acidification, aerobically, at one-fifth to one-hundredth the (1%, wt/vol) concentration of carbohydrate used in standard indicator plates.
|
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] |
PMID:21197
|
Effect of aldosterone on the coupling between H+ transport and glucose oxidation.
|
The mode of action of aldosterone on the energetics of H+ transport in the turtle bladder was examined with the rate of glucose oxidation as an index of the metabolic activity of the epithelium (we show that H+ transport is not coupled to fatty acid oxidation). Within 6 h of addition of aldosterone H+, transport increased; so did glucose oxidation. The amount of H+ transport per mole of 14CO2 produced from glucose oxidation was 15.6 eq-mol-1 in the control hemi-bladder, while in the aldosterone-treated bladder it was 13.6, delta = 2.0+/-4.0 (n = 6). However, in bladders exposed to aldosterone for 20 h, the relation of transport to glucose oxidation was significantly altered: control 10.8, aldosterone 16.4, delta = 4.5+/-2.5, P less than 0.02, n = 7. The slope of H+ transport on the applied electrochemical gradient was steeper during both short- and long-term incubations. However, the maximum gradient necessary to nullify the net rate of secretion was unaltered in both experiments. Evidence is presented that aldosterone does not alter the passive backflux into the cell. In five additional experiments where aldosterone produced no significant stimulation of H+ transport, no change was noted in any of the metabolic or transport characteristics measured, suggesting that the alterations discussed above are dependent on the stimulation of H+ transport by the hormone. These results, along with some thermodynamic considerations, suggest that the effect of aldosterone is primarily exerted on the transport process rather than on metabolism. Further, it appears that prolonged stimulation of transport work leads to secondary alterations in the metabolic pathways reminiscent of the changes that occur in skeletal muscles of athletes undergoing physical conditioning.
|
Effect of aldosterone on the coupling between H+ transport and glucose oxidation. The mode of action of aldosterone on the energetics of H+ transport in the turtle bladder was examined with the rate of glucose oxidation as an index of the metabolic activity of the epithelium (we show that H+ transport is not coupled to fatty acid oxidation). Within 6 h of addition of aldosterone H+, transport increased; so did glucose oxidation. The amount of H+ transport per mole of 14CO2 produced from glucose oxidation was 15.6 eq-mol-1 in the control hemi-bladder, while in the aldosterone-treated bladder it was 13.6, delta = 2.0+/-4.0 (n = 6). However, in bladders exposed to aldosterone for 20 h, the relation of transport to glucose oxidation was significantly altered: control 10.8, aldosterone 16.4, delta = 4.5+/-2.5, P less than 0.02, n = 7. The slope of H+ transport on the applied electrochemical gradient was steeper during both short- and long-term incubations. However, the maximum gradient necessary to nullify the net rate of secretion was unaltered in both experiments. Evidence is presented that aldosterone does not alter the passive backflux into the cell. In five additional experiments where aldosterone produced no significant stimulation of H+ transport, no change was noted in any of the metabolic or transport characteristics measured, suggesting that the alterations discussed above are dependent on the stimulation of H+ transport by the hormone. These results, along with some thermodynamic considerations, suggest that the effect of aldosterone is primarily exerted on the transport process rather than on metabolism. Further, it appears that prolonged stimulation of transport work leads to secondary alterations in the metabolic pathways reminiscent of the changes that occur in skeletal muscles of athletes undergoing physical conditioning.
|
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] |
PMID:21198
|
The maladaptive renal response to secondary hypocapnia during chronic HCl acidosis in the dog.
|
It has generally been thought that homeostatic mechanisms of renal origin are responsible for minimizing the alkalemia produced by chronic hypocapnia. Recent observations from this laboratory have demonstrated, however, that the decrement in [HCO(-) (3)], which "protects" extracellular pH in normal dogs, is simply the by-product of a nonspecific effect of Paco(2) on renal hydrogen ion secretion; chronic primary hypocapnia produces virtually the same decrement in plasma [HCO(-) (3)] in dogs with chronic HCl acidosis as in normal dogs (Delta[HCO(-) (3)]/DeltaPaco(2) = 0.5), with the result that plasma [H(+)] in animals with severe acidosis rises rather than falls during superimposed forced hyperventilation. This observation raised the possibility that the secondary hypocapnia which normally accompanies metabolic acidosis, if persistent, might induce an analogous renal response and thereby contribute to the steady-state decrement in plasma [HCO(-) (3)] observed during HCl feeding. We reasoned that if sustained secondary hypocapnia provoked the kidney to depress renal bicarbonate reabsorption, the acute salutary effect of hypocapnia on plasma acidity might be seriously undermined. To isolate the possible effects of secondary hypocapnia from those of the hydrogen ion load, per se, animals were maintained in an atmosphere of 2.6% CO(2) during an initial 8-day period of acid feeding (7 mmol/kg per day); this maneuver allowed Paco(2) to be held constant at the control level of 36 mm Hg despite the hyperventilation induced by the acidemia. Steady-state bicarbonate concentration during the period of eucapnia fell from 20.8 to 16.0 meq/liter, while [H(+)] rose from 42 to 55 neq/liter. During the second phase of the study, acid feeding was continued but CO(2) was removed from the inspired air, permitting Paco(2) to fall by 6 mm Hg. In response to this secondary hypocapnia, bicarbonate concentration fell by an additional 3.0 meq/liter to a new steady-state level of 13.0 meq/liter. This reduction in bicarbonate was of sufficient magnitude to more than offset the acute salutary effect of the hypocapnia on plasma hydrogen ion concentration; in fact, steady-state [H(+)] rose as a function of the adaptive fall in Paco(2), Delta[H(+)]/Delta Paco(2) = -0.44. That the fall in bicarbonate observed in response to chronic secondary hypocapnia was the result of the change in Paco(2) was confirmed by the observation that plasma bicarbonate returned to its eucapnic level in a subgroup of animals re-exposed to 2.6% CO(2). These data indicate that the decrement in plasma [HCO(-) (3)] seen in chronic HCl acidosis is a composite function of (a) the acid load itself and (b) the renal response to the associated hyperventilation. We conclude that this renal response is maladaptive because it clearly diminishes the degree to which plasma acidity is protected by secondary hypocapnia acutely. Moreover, under some circumstances, this maladaptation actually results in more severe acidemia than would occur in the complete absence of secondary hypocapnia.
|
The maladaptive renal response to secondary hypocapnia during chronic HCl acidosis in the dog. It has generally been thought that homeostatic mechanisms of renal origin are responsible for minimizing the alkalemia produced by chronic hypocapnia. Recent observations from this laboratory have demonstrated, however, that the decrement in [HCO(-) (3)], which "protects" extracellular pH in normal dogs, is simply the by-product of a nonspecific effect of Paco(2) on renal hydrogen ion secretion; chronic primary hypocapnia produces virtually the same decrement in plasma [HCO(-) (3)] in dogs with chronic HCl acidosis as in normal dogs (Delta[HCO(-) (3)]/DeltaPaco(2) = 0.5), with the result that plasma [H(+)] in animals with severe acidosis rises rather than falls during superimposed forced hyperventilation. This observation raised the possibility that the secondary hypocapnia which normally accompanies metabolic acidosis, if persistent, might induce an analogous renal response and thereby contribute to the steady-state decrement in plasma [HCO(-) (3)] observed during HCl feeding. We reasoned that if sustained secondary hypocapnia provoked the kidney to depress renal bicarbonate reabsorption, the acute salutary effect of hypocapnia on plasma acidity might be seriously undermined. To isolate the possible effects of secondary hypocapnia from those of the hydrogen ion load, per se, animals were maintained in an atmosphere of 2.6% CO(2) during an initial 8-day period of acid feeding (7 mmol/kg per day); this maneuver allowed Paco(2) to be held constant at the control level of 36 mm Hg despite the hyperventilation induced by the acidemia. Steady-state bicarbonate concentration during the period of eucapnia fell from 20.8 to 16.0 meq/liter, while [H(+)] rose from 42 to 55 neq/liter. During the second phase of the study, acid feeding was continued but CO(2) was removed from the inspired air, permitting Paco(2) to fall by 6 mm Hg. In response to this secondary hypocapnia, bicarbonate concentration fell by an additional 3.0 meq/liter to a new steady-state level of 13.0 meq/liter. This reduction in bicarbonate was of sufficient magnitude to more than offset the acute salutary effect of the hypocapnia on plasma hydrogen ion concentration; in fact, steady-state [H(+)] rose as a function of the adaptive fall in Paco(2), Delta[H(+)]/Delta Paco(2) = -0.44. That the fall in bicarbonate observed in response to chronic secondary hypocapnia was the result of the change in Paco(2) was confirmed by the observation that plasma bicarbonate returned to its eucapnic level in a subgroup of animals re-exposed to 2.6% CO(2). These data indicate that the decrement in plasma [HCO(-) (3)] seen in chronic HCl acidosis is a composite function of (a) the acid load itself and (b) the renal response to the associated hyperventilation. We conclude that this renal response is maladaptive because it clearly diminishes the degree to which plasma acidity is protected by secondary hypocapnia acutely. Moreover, under some circumstances, this maladaptation actually results in more severe acidemia than would occur in the complete absence of secondary hypocapnia.
|
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] |
PMID:21200
|
Solubility and heat stability of whey protein concentrates.
|
Rennet whey protein concentrates have excellent nutritional properties, but their use in fluid food systems is impaired by the poor heat stability of the protein. Heating whey protein concentrated solutions at neutral pH caused up to 70% losses in solubility. In the absence of added calcium, protein coagulation occurred. near the iso-electric zone whereas in the presence of .03 M calcium chloride, similar protein coagulation occurred in the whole pH range (pH 2 to pH 12). Tryptic hydrolysis of the protein increased the heat stability of whey protein concentrates considerably.
|
Solubility and heat stability of whey protein concentrates. Rennet whey protein concentrates have excellent nutritional properties, but their use in fluid food systems is impaired by the poor heat stability of the protein. Heating whey protein concentrated solutions at neutral pH caused up to 70% losses in solubility. In the absence of added calcium, protein coagulation occurred. near the iso-electric zone whereas in the presence of .03 M calcium chloride, similar protein coagulation occurred in the whole pH range (pH 2 to pH 12). Tryptic hydrolysis of the protein increased the heat stability of whey protein concentrates considerably.
|
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] |
PMID:21201
|
Lymphocyte subpopulations of atopic children and the effect of therapy upon them.
|
Recent reports note decreased T cell function in association with certain atopic conditions in man. This study was performed to determine whether numbers of circulating T cells are decreased in atopic children and adolescents in comparison with nonatopic age-matched control subjects. The subjects were not selected on the basis of a particular atopic diagnosis, but relatively more had allergic rhinitis and/or asthma (52) than had atopic eczema (7). Numbers of circulating T cells were not found to be significantly different in allergic children aged 2 to 10 yr than in control subjects. Atopic children and adolescents over age 10 yr had significantly fewer T cells in relative percentages (p less than 0.05), but when absolute numbers were considered significance was lost. Atopic children aged 2 to 10 years had significantly more B cells in both relative percentages and absolute numbers than did control subjects (p less than 0.02 and p less than 0.05, respectively). When those subjects treated with corticosteroids were separated from the total atopic group, there were no significant differences between the atopic and control subjects. The effects of corticosteroids, bronchodilators, antihistamines, and immunotherapy were considered and could be shown to produce no consistent effect on lymphocyte subpopulations.
|
Lymphocyte subpopulations of atopic children and the effect of therapy upon them. Recent reports note decreased T cell function in association with certain atopic conditions in man. This study was performed to determine whether numbers of circulating T cells are decreased in atopic children and adolescents in comparison with nonatopic age-matched control subjects. The subjects were not selected on the basis of a particular atopic diagnosis, but relatively more had allergic rhinitis and/or asthma (52) than had atopic eczema (7). Numbers of circulating T cells were not found to be significantly different in allergic children aged 2 to 10 yr than in control subjects. Atopic children and adolescents over age 10 yr had significantly fewer T cells in relative percentages (p less than 0.05), but when absolute numbers were considered significance was lost. Atopic children aged 2 to 10 years had significantly more B cells in both relative percentages and absolute numbers than did control subjects (p less than 0.02 and p less than 0.05, respectively). When those subjects treated with corticosteroids were separated from the total atopic group, there were no significant differences between the atopic and control subjects. The effects of corticosteroids, bronchodilators, antihistamines, and immunotherapy were considered and could be shown to produce no consistent effect on lymphocyte subpopulations.
|
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] |
PMID:21202
|
[Cervical mucus. III. Physiological roles].
|
The first part of this large review on the bibliography of cervical mucus was concerned both with the mechanism and the determinism ensuring its secretion. A second paper reviewed the present knowledge on chemical, physical ultrastructural characteristics of cervical mucus. This paper, the last part of the review, will be concerned with the different functions of the mucus ensuring synchronisation and encounter of the germ cells. From a physiological point of view, the following properties may be ascribed to the cervical secretion: - protection of sperm from the hostile environment of the vagina, and from being phagocytized along the cervical canal ; - cyclic receptivity to sperm penetration at or near ovulation and interference with entry at other periods ; - filtering effect on sperm cells; - possible intracervical sperm storage ; - supplementing the energy requirements of sperm ; - protection of the uterine cavity against bacteria.
|
[Cervical mucus. III. Physiological roles]. The first part of this large review on the bibliography of cervical mucus was concerned both with the mechanism and the determinism ensuring its secretion. A second paper reviewed the present knowledge on chemical, physical ultrastructural characteristics of cervical mucus. This paper, the last part of the review, will be concerned with the different functions of the mucus ensuring synchronisation and encounter of the germ cells. From a physiological point of view, the following properties may be ascribed to the cervical secretion: - protection of sperm from the hostile environment of the vagina, and from being phagocytized along the cervical canal ; - cyclic receptivity to sperm penetration at or near ovulation and interference with entry at other periods ; - filtering effect on sperm cells; - possible intracervical sperm storage ; - supplementing the energy requirements of sperm ; - protection of the uterine cavity against bacteria.
|
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] |
PMID:21203
|
[Neuroleptic analgesia in the management of labor].
|
Neuroleptic analgesia, which is used for the major part of the conduct of labour, has the advantage of abolishing the pain of uterine contractions without altering the mother's state of consciousness. A study of the effects of droperidol together with phenoperidine on the mother and the child has been carried out. Clinical results, the parameters of the intra-uterine pressure curves and the fetal heart rates as well as of the acid base balance and the pO2 of the mother and infant during dilatation of the cervix and the two first hours of life have been monitored. The conclusion is that neuroleptic analgesia does not cause neonatal depression and can be used as a method for conducting labour so long as very strict monitoring conditions are applied.
|
[Neuroleptic analgesia in the management of labor]. Neuroleptic analgesia, which is used for the major part of the conduct of labour, has the advantage of abolishing the pain of uterine contractions without altering the mother's state of consciousness. A study of the effects of droperidol together with phenoperidine on the mother and the child has been carried out. Clinical results, the parameters of the intra-uterine pressure curves and the fetal heart rates as well as of the acid base balance and the pO2 of the mother and infant during dilatation of the cervix and the two first hours of life have been monitored. The conclusion is that neuroleptic analgesia does not cause neonatal depression and can be used as a method for conducting labour so long as very strict monitoring conditions are applied.
|
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] |
PMID:21204
|
The supply of physicians and physicians' incomes: some projections.
|
This study identifies trends that will lead to a dramatic increase in the number of active physicians in the United States during the next decade. The supply of active medical doctors (MDs) and doctors of osteopathy (DOs) as well as active post-graduate MDs and DOs in the U.S. is projected to increase by approximately 50 percent in the decade ending in 1985. The number of active physicians per 100,000 population is similarly expected to increase by approximately one-third. The production of surgical specialists, in particular, appears to be excessive. In response, the average length of physician graduate training programs is anticipated to be shortened as more MD and DO graduates enter shorter, general practice residencies. The authors expect that the effects of this projected increase in the supply of physicians may relieve geographic disparities in physician distribution, rationalize the organization of medical practice, and reduce physicians' incomes relative to other professional groups and possibly in absolute terms. The projected increases in the supply of physicians will give the federal government much more flexibility and bargaining power should it choose to implement a national health insurance program with salaried physicians.
|
The supply of physicians and physicians' incomes: some projections. This study identifies trends that will lead to a dramatic increase in the number of active physicians in the United States during the next decade. The supply of active medical doctors (MDs) and doctors of osteopathy (DOs) as well as active post-graduate MDs and DOs in the U.S. is projected to increase by approximately 50 percent in the decade ending in 1985. The number of active physicians per 100,000 population is similarly expected to increase by approximately one-third. The production of surgical specialists, in particular, appears to be excessive. In response, the average length of physician graduate training programs is anticipated to be shortened as more MD and DO graduates enter shorter, general practice residencies. The authors expect that the effects of this projected increase in the supply of physicians may relieve geographic disparities in physician distribution, rationalize the organization of medical practice, and reduce physicians' incomes relative to other professional groups and possibly in absolute terms. The projected increases in the supply of physicians will give the federal government much more flexibility and bargaining power should it choose to implement a national health insurance program with salaried physicians.
|
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] |
PMID:21217
|
Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.
|
A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.
|
Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application. A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.
|
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] |
PMID:21220
|
Enumeration of t rosette-forming cell population in rabbit peripheral blood using human, sheep, guinea-pig, rat, mouse and chicken red blood cells under different experimental conditions.
|
Attempts were made to estimate peripheral T-lymphocytes in rabbits by their ability to form rosettes with sheep, human, guinea-pig, rat, mouse and chicken RBC. The reaction was studied under different conditions of pH and temperature. The test was also carried out in the presence or absence of glutaraldehyde. With sheep RBC, lymphocytes from 2 of the 5 rabbits studied formed rosettes to the extent of 1.5% in the system containing glutaraldehyde. These results however were not reproducible when studied after a week. The RBCs from other species, under similar experimental conditions, did not give rise to rosettes. The test carried out at pH 6.8 and pH 7.2 showed up to 4 and 8% rosettes respectively with rat RBCs, while RBCs of no other species showed rosettes. When rabbit lymphocytes preincubated at 37 degrees C for 1 h were used for rosette test with guinea-pig RBC, a maximum of 14.6% rosettes were obtained. Here, again the percentage of rosettes formed in the 5 animals, over a period of one month, varied widely. The reasons for the variations of the percentage of rosettes formed are discussed.
|
Enumeration of t rosette-forming cell population in rabbit peripheral blood using human, sheep, guinea-pig, rat, mouse and chicken red blood cells under different experimental conditions. Attempts were made to estimate peripheral T-lymphocytes in rabbits by their ability to form rosettes with sheep, human, guinea-pig, rat, mouse and chicken RBC. The reaction was studied under different conditions of pH and temperature. The test was also carried out in the presence or absence of glutaraldehyde. With sheep RBC, lymphocytes from 2 of the 5 rabbits studied formed rosettes to the extent of 1.5% in the system containing glutaraldehyde. These results however were not reproducible when studied after a week. The RBCs from other species, under similar experimental conditions, did not give rise to rosettes. The test carried out at pH 6.8 and pH 7.2 showed up to 4 and 8% rosettes respectively with rat RBCs, while RBCs of no other species showed rosettes. When rabbit lymphocytes preincubated at 37 degrees C for 1 h were used for rosette test with guinea-pig RBC, a maximum of 14.6% rosettes were obtained. Here, again the percentage of rosettes formed in the 5 animals, over a period of one month, varied widely. The reasons for the variations of the percentage of rosettes formed are discussed.
|
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] |
PMID:21221
|
[Multiple forms of NADPH-dependent glutathione reductase in serum. Studies on the NADPH-dependent glutathione-reductase in serum II. (author's transl)].
|
Sera with elevated activities of glutathione reductase were investigated by gel electrophoresis in agar or polyacrylamide, and by gel filtration. In both separation methods the glutathione reductase activity in individual samples was resolved, showing up to three fractions differing in rate of migration and molecular size. The fractions with the lowest and highest molecular weight, corresponded respectively to the slowest and fastest migrating bands in agar gel electrophoresis. Preincubation of the serum samples with FAD or neuraminidase had no effect on the rate of migration of the three fractions. After the addition of beta-mercaptoethanol to the serum, gel electrophoresis and gel filtration showed only the enzyme fraction with the slowest rate of migration and the lowest molecular weight (140,000). The other two fractions reappeared after removal of the thiol from the serum. Further studies on the isolated (agar gel electrophoresis) fractions showed the existence of oligomeric forms of the enzyme, which are reversibly interconvertible.
|
[Multiple forms of NADPH-dependent glutathione reductase in serum. Studies on the NADPH-dependent glutathione-reductase in serum II. (author's transl)]. Sera with elevated activities of glutathione reductase were investigated by gel electrophoresis in agar or polyacrylamide, and by gel filtration. In both separation methods the glutathione reductase activity in individual samples was resolved, showing up to three fractions differing in rate of migration and molecular size. The fractions with the lowest and highest molecular weight, corresponded respectively to the slowest and fastest migrating bands in agar gel electrophoresis. Preincubation of the serum samples with FAD or neuraminidase had no effect on the rate of migration of the three fractions. After the addition of beta-mercaptoethanol to the serum, gel electrophoresis and gel filtration showed only the enzyme fraction with the slowest rate of migration and the lowest molecular weight (140,000). The other two fractions reappeared after removal of the thiol from the serum. Further studies on the isolated (agar gel electrophoresis) fractions showed the existence of oligomeric forms of the enzyme, which are reversibly interconvertible.
|
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] |
PMID:21223
|
Liquid and solid-state Cl- -sensitive microelectrodes. Characteristics and application to intracellular Cl- activity in Balanus photoreceptor.
|
When intracellular chloride activity (aiCl) was monitored with chloride-sensitive liquid ion exchanges (CLIX) microelectrodes in Balanus photoreceptors, replacement of extracellular chloride (Cl0) by methanesulfonate or glutamate was followed by a rapid but incomplete loss of aiCl. When propionate was used as the extracellular anion substitute, CLIX electrodes detected an apparent gain in aiCl, while a newly designed Ag-AgCl wire-in glass microelectrode showed a loss of aiCl under the same conditions. This discrepancy in Cl- washout when propionate replaced Cl0 is explained by the differences in selectivity of CLIX and Ag-AgCl electrodes for native intracellular anions and for the extracellular anion substitute which also replaces Cli and interferes in the determination of aiCl. Both electrodes indicate that ECl approximately Em when the cells are bathed in normal barnacle saline, and both electrodes showed the rate of Cl washout (tau approximately 5 min) to be independent of Cli when Cl0 was replaced by glutamate. Details of Ag-AgCl microelectrode construction are presented. These electrodes were tested and found to be insensitive to the organic anion substitutes used in this study. Selectivity data of CLIX electrodes for several anions of biological interest are described.
|
Liquid and solid-state Cl- -sensitive microelectrodes. Characteristics and application to intracellular Cl- activity in Balanus photoreceptor. When intracellular chloride activity (aiCl) was monitored with chloride-sensitive liquid ion exchanges (CLIX) microelectrodes in Balanus photoreceptors, replacement of extracellular chloride (Cl0) by methanesulfonate or glutamate was followed by a rapid but incomplete loss of aiCl. When propionate was used as the extracellular anion substitute, CLIX electrodes detected an apparent gain in aiCl, while a newly designed Ag-AgCl wire-in glass microelectrode showed a loss of aiCl under the same conditions. This discrepancy in Cl- washout when propionate replaced Cl0 is explained by the differences in selectivity of CLIX and Ag-AgCl electrodes for native intracellular anions and for the extracellular anion substitute which also replaces Cli and interferes in the determination of aiCl. Both electrodes indicate that ECl approximately Em when the cells are bathed in normal barnacle saline, and both electrodes showed the rate of Cl washout (tau approximately 5 min) to be independent of Cli when Cl0 was replaced by glutamate. Details of Ag-AgCl microelectrode construction are presented. These electrodes were tested and found to be insensitive to the organic anion substitutes used in this study. Selectivity data of CLIX electrodes for several anions of biological interest are described.
|
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] |
PMID:21224
|
Exopolysaccharide production by Pseudomonas NCIB11264 grown in batch culture.
|
Fermentation studies using batch culture indicated that exopolysaccharide production by Pseudomonas NCIBI1264 in a chemically defined medium increased under conditions of nitrogen limitation and excess carbon substrate at pH values above 6. The polysaccharide was formed from a variety of carbon substrates and its composition was not affected by the nature of the carbohydrate source. Polysacharide formation did not increase in media containing small amounts of phosphate, and, as in secondary metabolite production, it started late in the exponential growth phase continuing maximally after growth had ceased. The efficiency of glucose conversion into exopolysaccharide was low. Colorimetric, viscometric, and total carbon estimation techniques are described for determining exopolysaccharide levels in cell-free culture supernatants.
|
Exopolysaccharide production by Pseudomonas NCIB11264 grown in batch culture. Fermentation studies using batch culture indicated that exopolysaccharide production by Pseudomonas NCIBI1264 in a chemically defined medium increased under conditions of nitrogen limitation and excess carbon substrate at pH values above 6. The polysaccharide was formed from a variety of carbon substrates and its composition was not affected by the nature of the carbohydrate source. Polysacharide formation did not increase in media containing small amounts of phosphate, and, as in secondary metabolite production, it started late in the exponential growth phase continuing maximally after growth had ceased. The efficiency of glucose conversion into exopolysaccharide was low. Colorimetric, viscometric, and total carbon estimation techniques are described for determining exopolysaccharide levels in cell-free culture supernatants.
|
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] |
PMID:21225
|
Ribonucleic acid polymerase activity associated with purified calf rotavirus.
|
The presence of an RNA-dependent RNA polymerase is demonstrated in purified rotavirus particles. Optimum polymerase activity was found between 45 to 50 degrees C, at pH 8, and in the presence of 10 mM-magnesium ions. The polymerase product was highly sensitive to pancreatic RNase (97%) in low or high salt concentration. The enzyme was activated by EDTA treatment of intact particles or heat shock. The similarities between reovirus, blue-tongue virus and rotavirus polymerases are discussed.
|
Ribonucleic acid polymerase activity associated with purified calf rotavirus. The presence of an RNA-dependent RNA polymerase is demonstrated in purified rotavirus particles. Optimum polymerase activity was found between 45 to 50 degrees C, at pH 8, and in the presence of 10 mM-magnesium ions. The polymerase product was highly sensitive to pancreatic RNase (97%) in low or high salt concentration. The enzyme was activated by EDTA treatment of intact particles or heat shock. The similarities between reovirus, blue-tongue virus and rotavirus polymerases are discussed.
|
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