Effect of pH on sodium dependent and independent glycine uptake in rat small intestine.
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Animals↗
Biomedical subjects
M Kapoor
Publications and source records attributed to M Kapoor.
Heat shock induces peroxidase activity in Neurospora crassa and confers tolerance toward oxidative stress.
Heat shock treatment of 14-h-old Neurospora crassa mycelium, for 1 h at 48 degrees C, led to the induction of high levels of peroxidase (EC. 1.11.1.7) activity. No significant change was observed in the superoxide dismutase content. Colonies formed by plating conidial suspensions on sorbose-medium also exhibited high peroxidase activity following exposure to hyperthermia and were found to be resistant to normally toxic doses of H2O2. Thus one of the heat shock proteins of N. crassa has the function of protection against oxidative stress.
Hot Temperature↗
Establishment of thermotolerance in maize by exposure to stresses other than a heat shock does not require heat shock protein synthesis.
Maize (Zea mays) seedlings were pretreated prior to heat shock with either a progressive water stress of -0.25 megapascal PEG/hour from 0 to -1.25 megapascal over a 6-hour time period, or various concentrations of copper, cadmium, or zinc for 4 days. When the subsequent heat shock of 40 or 45 degrees C was administered for 3 hours, the seedlings showed an induced thermotolerance to these temperatures, which were otherwise lethal to control (water grown) seedlings. Thermotolerance was exhibited by both the root and the shoot of pretreated seedlings, even though the water and heavy metal stresses were applied only to the roots. Neither of these pretreatments had induced the synthesis of detectable levels of heat shock proteins (Hsps) at the time of heat shock. Pretreatment of seedlings with a progressive heat shock of 2 degrees C/hour from 26 to 36 degrees C, which did induce Hsps 18, 70, and 84, resulted in tolerance of a severe water stress of -1.5, -1.75, or -2.0 megapascal for 24 hours. But these seedlings producing Hsps were no better protected against water stress than those pretreated with a progressive water stress which did not produce Hsps. Hsps appear not to act as general stress proteins and their presence is not always required for the establishment of thermotolerance.
Journal Article↗
Antibiotic-induced derepression of the NAD-specific glutamate dehydrogenase of Neurospora crassa.
The catabolic, NAD-specific glutamate dehydrogenase (NAD-GDH) of Neurospora crassa is under carbon catabolite repression. Cells grown on a glycolytic carbon source, such as sucrose, have low basal levels of enzyme activity. Treatment of repressed cells with either polymyxin B or amphotericin B resulted in derepression of NAD-GDH. Derepression at the transcriptional level occurred very rapidly (within 30 min) in response to polymyxin B addition but reached a plateau within 2 h. Amphotericin B-induced derepression initiated more slowly but continued for at least 6 h, resulting in a specific activity comparable to that seen with cells transferred to glutamate as the sole carbon source. These antibiotics had no significant effect upon the activities of two constitutive enzymes, pyruvate kinase and malate dehydrogenase. Curiously, only polymyxin B treatment derepressed invertase, another catabolite-repressed enzyme. The addition of 100 mM KCl to the growth medium blocked derepression by both antibiotics, but the addition of 50 mM MgCl2 only annulled derepression by polymyxin B. The ergosterol-deficient erg-1 mutant, which is resistant to amphotericin B, did not derepress NAD-GDH when treated with this drug. These results are consistent with derepression resulting from interactions of these antibiotics with the plasma membrane.
Amphotericin B↗
Some aspects of the regulation of pyruvate kinase levels in Neurospora crassa.
Pyruvate kinase levels were monitored in Neurospora crassa mycelium (grown on different carbon sources for varying time intervals) by immunoprecipitation using polyclonal antibodies raised against a purified enzyme preparation. Pyruvate kinase specific mRNA was demonstrated by hybridization of Northern and dot blots of total RNA with a N. crassa pyruvate kinase gene fragment. Two pyruvate kinase specific mRNA species were detected in mycelia of all ages examined. An age-dependent and carbon source dependent variation in the pyruvate kinase protein and mRNA levels was encountered: both registered an increase for up to about 20 h and a subsequent decline; growth on acetate and sucrose resulted in significantly higher yields of both, relative to that on medium containing ethanol and alanine. Stress caused by heat shock depressed the pyruvate kinase mRNA levels.
DNA Restriction Enzymes↗
A study of the messenger RNA encoding pyruvate kinase of Neurospora crassa.
In Neurospora crassa, there is a single pyruvate kinase (PK) consisting of four identical subunits of approximately 60k daltons. Northern and dot blot hybridization studies, using most of the yeast pyruvate kinase gene as a probe, suggest the presence of two distinct mRNA species for pyruvate kinase, separable on the basis of the length of their polyadenylated tails, by oligo(dT)cellulose chromatography. These messages are present in polysomes, immuno-precipitated by anti-PK antibodies, indicating probable translation in vivo. Fractions containing both messages were translated in vitro in the heterologous systems as well as in a homologous N. crassa lysate, the newly-synthesized PK being detected by immunoadsorption. Protection studies using S1-nuclease suggest no major structural differences in the 5'-untranslated and most of the coding regions of the two messages.
Animals↗
Effects of high doses of celiprolol in asthmatic patients.
Sixteen normotensive asthmatic patients received single doses of 400 and 600 mg of celiprolol and 100 mg of atenolol in this placebo-controlled, double-blind crossover study. Pulmonary function was assessed by spirometry. Changes in forced one-second expiratory volume (FEV1) and mid-maximal expiratory flow (MMEF) following both doses of celiprolol were indistinguishable from the effects of placebo, whereas atenolol caused a significant reduction in both measurements of pulmonary function. Graded doses of albuterol, a beta 2-selective sympathomimetic administered at 15-min intervals starting 3 h after each treatment, as expected, caused bronchodilation. The overall bronchodilatory effects of this combined, beta-blocker plus albuterol treatment on pulmonary function were greatest after celiprolol 600 mg, followed in order by celiprolol 400 mg, placebo, and atenolol. Celiprolol 400 mg and 600 mg neither caused bronchoconstriction nor antagonized albuterol in this acute study. Because of its bronchosparing properties, even at high doses, celiprolol may offer an advantage over other, similar agents in the treatment of hypertension and angina in asthmatics.
Adult↗
A study of derepression of NAD-specific glutamate dehydrogenase of Neurospora crassa.
Transfer of Neurospora crassa mycelium from a 1% (w/v) sucrose medium to carbon-free or 1% (w/v) glutamate medium results in the onset of derepression of the catabolic NAD-specific glutamate dehydrogenase (NAD-GDH), within 30 min of the shift. Immunoprecipitation of in vivo pulse-labelled NAD-GDH demonstrated that this enzyme was synthesized de novo, correlating with increasing enzyme activity in shifted cells. Derepression was shown to be under transcriptional control by using the RNA synthesis inhibitor, picolinic acid, and by immunoprecipitation of the in vitro translation products of poly(A)-containing mRNA from repressed and derepressed cells. A brief (5 min) shift to derepression medium followed by a return to 1% (w/v) sucrose medium was sufficient to trigger synthesis of abundant NAD-GDH transcripts and low levels of the active enzyme. A secondary level of translational control is proposed to account for the discrepancy between the detectable levels of NAD-GDH transcripts and protein, following transient derepression.
Autoradiography↗
Preparation of a cell-free translation system from a wild-type strain of Neurospora crassa and translation of pyruvate kinase messenger RNA.
A cell-free in vitro translation system exhibiting high activity has been developed from wild-type Neurospora crassa mycelium. The isolation is simple and fast, and the homogenization does not appear to affect the activity of mycelial proteases and nucleases. This system is capable of supporting efficient translation of exogenously added homologous RNA as demonstrated by the experiments with PK-specific mRNA. In addition, it translates heterologous RNA efficiently, shown by the translation of globin mRNA. We did not examine the Neurospora lysate for post-translational modification activity. The procedure used for the preparation of Neurospora cell-free extracts should be readily applicable to the other filamentous fungi.
Cell-Free System↗
A two-dimensional immunoelectrophoretic analysis of the heat-shock response exhibited by Neurospora crassa cells.
The heat-shock (HS) response of Neurospora crassa was studied by two-dimensional (2-D) immunoelectrophoresis, in conjunction with in vivo labelling of proteins with [35S]methionine. Antisera against extracts of normally grown and shocked cells were tested with both extracts as antigens. The resolution of normal cell proteins by interaction with homologous antisera yielded at least 35 immunoprecipitates. Using antisera to shocked cells with normal and shocked cell extracts resolved four heat-shock proteins (nhsps): new polypeptides synthesized upon HS and those with an enhanced production. Extracts of partially recovered cells showed proteins typical of normal cells, as well as those specific to the shocked state. Immunogenicity of some proteins appeared altered after heat shock. Following adsorption of HS cell extracts by normal antisera, three unique proteins were recognized in shocked cell extracts, along with a fourth displaying a markedly enhanced level. Stained bands corresponding to two abundant hsps (relative mass 68 000 and 81 000 daltons; referred to as nhsp 70 and nhsp 80) were excised from slab gels and the eluted proteins were employed as immunogens in rabbits. The resulting antibodies were used to identify corresponding proteins in autoradiographs of 2-D immunoelectrophoretograms.
Heat-Shock Proteins↗
Selenium suppresses the metabolism of benzo[a]pyrene by rat-liver extracts, and exerts a dual effect on its mutagenicity.
Liver homogenates from rats injected with 3-methylcholanthrene were employed for metabolism of benzo[a]pyrene (BP) and in assays of aryl hydrocarbon hydroxylase (AHH) activity in vitro. Sodium selenite inhibited AHH activity to a maximum of approximately 70%. It suppressed the overall metabolism of benzo[a]pyrene; a distinct reduction in the products was evident on h.p.l.c. analysis. Sodium thiosulphate also inhibited AHH activity by approximately 47%. Inclusion of S2O3(2-) and SeO3(2-), in combination, led to a cumulative inhibition of 87%. The mutagenicity of BP in the Salmonella auxotroph reversion system (Ames test) was enhanced by SeO3(2-) at concentrations below 0.2 mM. Above this level a significant antimutagenic effect was observed.
Animals↗
Studies on the induction of aryl hydrocarbon(benzo[a]pyrene) hydroxylase in Neurospora crassa, and its suppression by sodium selenite.
Six fungal species were grown in the presence of benzo[a]pyrene (BP); four showed benzo[a]pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) activity. Penicillium sp. and Neurospora crassa metabolized BP to a limited extent. N. crassa AHH activity was induced by BP, the major product of metabolism being 3-hydroxy-BP. Both induction of AHH activity and metabolism of BP were suppressed by sodium selenite in the growth medium. Two polypeptides, unique to BP-grown cells, were revealed by two-dimensional electrophoretic separation of [35S]methionine-labelled proteins in N. crassa cell extracts. In selenium-grown cells the synthesis of BP-specific polypeptides was severely inhibited.
Aryl Hydrocarbon Hydroxylases↗
A study of the properties of pyruvate kinase isolated from a mutant of Neurospora crassa: a comparison with the parental enzyme.
1. A mutant of Neurospora crassa has been isolated whose pyruvate kinase is twice as active as the wild type enzyme. 2. The purified mutant and the wild type enzymes exhibit similar immunological properties, pI values (6.4) and Arrhenius activation energy (11.2 kcal/mol). 3. Both the enzymes show hyperbolic saturation kinetics with ADP and sigmoidal kinetics with PEP. 4. The mutant enzyme displays a higher affinity for PEP and a greater extent of cooperativity in binding than the wild type. 5. Conformational alterations in the mutant enzyme are inferred on the basis of electrophoretic analyses and denaturation by urea, SDS and heat.
Hot Temperature↗
A study of the heat-shock response in Neurospora crassa.
1. Neurospora crassa was grown at 28 degrees C for 12 hr and transferred to higher temperatures for 2 hr. 2. Cultures labelled with [35S]methionine showed the synthesis of several new proteins in response to heat-shock at 46 to 48 degrees C. 3. Major polypeptides of approximate Mr 105,000, 99,000, 78,000, 43,000 and 23,000 were detectable in one-dimensional SDS-polyacrylamide slab gel electropherograms. 4. 2-D analysis using isoelectric-focussing in the first dimension and electrophoresis in SDS-polyacrylamide gels in the second led to the resolution of some of the heat-induced polypeptide into multiple spots differing in pI values. 5. mRNA from heat-shocked cells was translated poorly in Wheat Germ extract and rabbit reticulocyte lysate in vitro translation systems.
Adaptation, Physiological↗
An immunological study of the interaction of ligands with pyruvate kinase of Neurospora crassa.
Antibodies against pyruvate kinase of Neurospora crassa, induced in rabbits, were used to monitor the interaction of ligands with this enzyme. The technique of microcomplement fixation was employed to probe for conformational alterations elicited by binding of substrates (phosphoenolpyruvate (PEP) and adenosine diphosphate), the allosteric activator (fructose 1,6-diphosphate), and the inhibitor (valine). On binding of PEP and valine to pyruvate kinase a pronounced reduction in the extent of complement fixation was observed. The second substrate, ADP, had no effect while FDP elicited a moderate suppression of complement fixation. These results suggest that as a consequence of conformational changes induced by PEP and valine, some antigenic determinants on the surface of pyruvate kinase are rendered inaccessible to the antibodies.
Adenosine Diphosphate↗
Negative cooperativity in the interaction of fructose 1,6-diphosphate with pyruvate kinase of Neurospora crassa.
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Adenosine Diphosphate↗
Studies on the structure-function relationships of Neurospora crassa pyruvate kinase: refolding and reactivation following denaturation in guanidine hydrochloride.
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Adenosine Diphosphate↗
Studies of the structure-function relationships of Neurospora pyruvate kinase: denaturation by urea and the effect of ligands on the refolding and renaturation process.
1. The renaturation of Neurospora pyruvate kinase following denaturation with urea was investigated. 2. The substrates, phosphoenol pyruvate and adenosine diphosphate, were observed to stabilize the enzyme against urea-induced structural disorganization. 3. It was demonstrated that for refolding/reactivation of the denatured protein the allosteric activator, fructose-1,6-diphosphate and a sulphydryl protectant are required. 4. The enzyme recovered following renaturation showed complete immunological identity with the native enzyme in Ouchterlony double diffusion tests.
Adenosine Diphosphate↗