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Biomedical subjects

W Harder

Publications and source records attributed to W Harder.

At least 91 records · Page 5Linked to original sources
PubMed1982

Strategies of mixed substrate utilization in microorganisms.

In natural and man-made environments microorganisms often grow in the presence of a diversity of functionally similar substrates. The pattern of utilization of these mixed substrates is generally dependent upon their concentration. When substrates are present in high (not growth-limiting) concentrations, sequential utilization and diauxic growth is often observed and the substrate that supports the highest growth rate is utilized preferentially from the mixture. When the substrate concentrations are growth-limiting, simultaneous utilization of the various compounds present in the mixture appears to be the general response. Recent studies on mixed substrate utilization in both batch and continuous cultures have thrown light on the strategies of the control mechanisms that, in microbes, govern the utilization of the various substrates. But perhaps more importantly these studies have indicated the possible significance of mixed substrate utilization in microbial competition in nutrient-limited natural ecosystems.

Bacteria↗
PubMed1981

Decreased rate of benzo(a)pyrene-induced sister chromatid exchange in fibroblast cultures from patients with lung cancer.

Fibroblast cultures from 29 lung cancer patients including eight familial cases and from 25 healthy controls were investigated. We studied the benzprene-induced sister chromatid exchange (SCE) following incubation in the presence of 150 nM benzo(a)pyrene and found spontaneously increased SCE values in the patients' cells but a reduced response to benzprene as compared to the control cultures. The benzpyrene-induced SCE was positively correlated with both benzpyrene metabolism and DNA adducts in normal control cells but not in the patients' cells. The ratio of benzypyrene DNA adducts and benzpyrene-induced rate of SCE calculated for the individual cell strains exhibited a bimodal distribution with patients' cells, but not with control cells. We speculate that the described differences between patients' and control fibroblasts might reflect a genetic predisposition to the development of lung cancer in about one half of the patients tested.

Benzopyrenes↗
PubMed1981

Arthrobacter P1, a fast growing versatile methylotroph with amine oxidase as a key enzyme in the metabolism of methylated amines.

A facultative methylotrophic bacterium was isolated from enrichment cultures containing methylamine as the sole carbon source. It was tentatively identified as an Arthrobacter species. Extracts of cells grown on methylamine or ethylamine contained high levels of amine oxidase (E.C. 1.4.3) activity. Glucose- or choline-grown cells lacked this enzyme. Oxidation of primary amines by the enzyme resulted in the formation of H2O2; as a consequence high levels of catalase were present in methylamine- and ethylamine-grown cells. The significance of catalase in vivo was demonstrated by addition of 20 mM aminotriazole (a catalase inhibitor) to exponentially growing cells. This completely blocked growth on methylamine whereas growth on glucose was hardly affected. Cytochemical studies showed that methylamine-dependent H2O2 production mainly occurred on invaginations of the cytoplasmic membrane. Assimilation of formaldehyde which is generated during methylamine oxidation was by the FBP variant of the RuMP cycle of formaldehyde fixation. The absence of NAD-dependent formaldehyde and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via hexulose phosphate synthase. Enzyme profiles of the organism grown on various substrates suggested that the synthesis of amine oxidase, catalase and the enzymes of the RuMP cycle is not under coordinate control.

Amine Oxidase (Copper-Containing)↗
PubMed1981

Substructure of crystalline peroxisomes in methanol-grown Hansenula polymorpha: evidence for an in vivo crystal of alcohol oxidase.

The substructural organization of completely crystalline peroxisomes present in Hansenula polymorpha cells grown under methanol limitation in a chemostat was investigated by different cytochemical and ultrastructural techniques. Time-dependent cytochemical staining experiments indicated that activities of the two main constituents of these organelles, namely, alcohol oxidase and catalase, were present throughout the crystalline matrix. Catalase was completely removed from isolated peroxisomes by osmotic shock treatment. After such treatment, the ultrastructure of the crystalline matrix of the organelles remained virtually intact. Because alcohol oxidase activity was still present in this matrix, it was concluded that alcohol oxidase protein is the only structural element of the peroxisomal crystalloids. The molecular architecture of the crystalloids was investigated in ultrathin cryosections which permitted recognition of individual molecules in the crystalline matrix. Depending on the plane of sectioning, different crystalline patterns were observed. Tilting experiments indicated that these images were caused by superposition of octameric alcohol oxidase molecules arranged in a tetragonal lattice. A three-dimensional model of the crystalloid is presented. The repeating unit of this structure is composed of four alcohol oxidase molecules. The crystalloid represents an open structure, which may explain the observed free mobility of catalase molecules.

Alcohol Oxidoreductases↗
PubMed1980

An electron microscopical study of the development of peroxisomes during formation and germination of ascospores in the methylotrophic yeast Hansenula polymorpha.

Ascospore formation was studied in liquid cultures of the yeast Hansenula polymorpha, previously grown under conditions in which the synthesis of alcohol oxidase was repressed (glucose as growth substrate) or derepressed (methanol, glycerol and dihydroxyacetone as growth substrates and after growth on malt agar plates). In ascospores obtained from repressed cells, generally one small peroxisome was present. The organelle probably originated from the small peroxisome, originally present in the vegetative cells. They had no crystalline inclusions and cytochemical experiments indicated the presence of catalase, urate oxidase and amino acid oxidase activities in these organelles. In ascospores obtained from derepressed cells, generally 1--3 crystalline peroxisomes were observed. These organelles also originated from the peroxisomes originally present in the vegetative cells by means of fragmentation or division. They contained, in addition to the enzymes characteristic for peroxisomes in spores from repressed cells, also alcohol oxidase. The latter enzyme is probably responsible for the crystalline substructure of these peroxisomes. Peroxisomes had no apparent physiological function in the process of ascosporogenesis. A glyoxysomal function of the organelles during germination of the ascospores was also not observed. Germination of mature ascospores in media containing different sources of carbon and nitrogen showed that the function of the peroxisomes present in ascospores of Hansenula polymorpha is probably identical to that in vegetative haploid cells. They are involved in the oxidative metabolism of different carbon and nitrogen sources. Their enzyme profile is a reflection of that peroxisomes of vegetative cells and their presence may enable the formation of cells which are optimally adapted to environmental conditions extant during spore germination.

Ascomycota↗
PubMed1980

Development of amine oxidase-containing peroxisomes in yeasts during growth on glucose in the presence of methylamine as the sole source of nitrogen.

The metabolism of methylamine as the nitrogen source for growth of the non-methylotrophic yeast Candida utilis and the methylotrophic yeast Hansenula polymorpha was investigated. Growth of both organisms in media with glucose and methylamine was associated with the presence of an amine oxidase in these cells. The enzyme catalyses the oxidation of methylamine by molecular oxygen into ammonia, formaldehyde and hydrogen peroxide and it is considered to be the key enzyme in methylamine metabolism in the organisms studied. In addition to synthesis of amine oxidase, derepression of catalase, formaldehyde and formate dehydrogenase was also observed upon transfer of cells of the two organisms from media containing ammonium ions into media containing methylamine as the nitrogen source. The synthesis of enzymes was paralleled by the development of a number of large microbodies in the cells. Cytochemical staining experiments indicated that the amine oxidase activity was located in the microbodies in both organisms. Catalase-activity was also demonstrated in these organelles, which can therefore be considered as peroxisomes. The present contribution is the first description of a peroxisomal amine oxidase.

Ascomycota↗
PubMed1980

Rate of sister chromatid exchanges in Bloom syndrome fibroblasts reduced by co-cultivation with normal fibroblasts.

Six strains of Bloom syndrome (BlS) fibroblasts responded to co-cultivation with normal fibroblasts at a 1:2 ratio by a reduced rate of sister chromatid exchanges (SCE's) from a mean of 67.5 (range = 59--78) to 28.4 (range = 21--35). The response was dose-dependent in one strain tested at 1:2, 1:1, and 2:1 ratios. In addition, quadriradial exchange figures and other signs of increased chromosomal instability were not found in BlS cells following co-cultivation with control cells. Control cells did not respond to BlS cells and maintained a normal rate of SCEs. Culture medium conditioned for 48 hrs by normal fibroblasts could also reduce the rate of SCEs in BlS fibroblasts, but less than in co-cultivation. We suggest that the reduced rate of SCEs and the lack of chromosomal instability in BlS cells following co-cultivation represent a corrective effect that is related to the basic defect and not dependent on cell-to-cell contact.

Adolescent↗
PubMed1978

Metabolic regulation in Pseudomonas oxalaticus OX1. Enzyme and coenzyme concentration changes during substrate transition experiments.

Metabolic control associated with diauxic growth of Pseudomonas oxalaticus in batch cultures on mixtures of formate and oxalate was investigated by measuring intracellular enzyme and coenzyme concentrations and QO2 values during transition experiments from oxalate to formate and vice versa. In transition from oxalate to formate oxalyl-CoA reductase concentration declined after the exhaustion of oxalate and ribulose-1,5-diphosphate carboxylase and 14CO2 fixation appeared upon addition of formate. In the reciprocal transition, ribulose-1,5-diphosphate carboxylase and 14CO2 fixation rate declined sharply after formate exhaustion, and oxalyl-CoA reductase appeared only after addition of oxalate. The intracellular NAD and NADP concentrations measured in the same experiments are reported. At substrate exhaustion the proportion of NAD in the reduced form fell from 15-20% to 2%. On addition of formate to an oxalate-starved culture there was an immediate increase in the proportion of NADH to 50%; such an increase was not observed in the reverse experiment.

Aldehyde Oxidoreductases↗
PubMed1978

Effect of environmental conditions on the production of two extracellular proteolytic enzymes by Vibrio SA1.

The production of two extracellular proteases, an endopeptidase and an aminopeptidase, by the marine bacterium Vibrio SA1 was studied in batch cultures. The production of the proteases was induced during growth of the organism in peptone media and by several amino acids during growth in minimal media. It was repressed by easily metabolisable carbon compounds such as glucose, lactate and succinate during growth in peptone media. During growth in a lactate basal medium, phenylalanine was one of the best inducers and this amino acid was therefore used in further experiments. That lactate did not repress the synthesis of the proteases during growth in the lactate basal medium supplement with 2mM phenylalanine as an inducer, appeared to be a consequence of the low iron content of this medium. Growth curves of Vibrio SA1 on such media showed a period of linear growth during which protease production was observed. When the iron concentration was made sufficiently high to prevent linear growth, the synthesis of the proteases remained repressed. Apparently by imposing an iron limitation on the organism, catabolite repression by lactate was relieved. Similarly, when growth was limited by very low values of the dissolved oxygen tension in the medium, a high rate of protease synthesis was found which was immediately repressed when the oxygen limitation was released. The results indicate that the growth rate and/or a factor associated with the energy metabolism play a role in the regulation of the synthesis of the enzymes.

Amino Acids↗
PubMed1978

A continuous culture study of the regulation of extracellular protease production in Vibrio SA1.

During growth of Vibrio SA1 in a lactate-limited chemostate in the presence of 2mM phenylalanine as an inducer, the rate of production of two proteolytic enzymes, namely an endopeptidase and an aminopeptidase, was dependent upon the dilution rate. An optimum in the rate of synthesis of both proteases was observed at a dilution rate of 0.23 h-1 and enzyme production only occurred between dilution rates of 0.06 and 0.45 h-1. Without inducer a low rate of aminopeptidase production was found with an optimum at 0.19 h-1, but only trace amounts of endopeptidase were detectable in the culture. In the presence of inducer the rate of enzyme production increased with increasing dilution rates over the range 0.06 to 0.23 h-1 which was explained by an increase in saturation of inducer sites. The progressive decrease in the rate of protease production at higher dilution rates was ascribed to an increasing effect of catabolite repression by the increasing concentration of the growth substrate. It was shown that 5 mM cyclic AMP could not relieve catabolite repression caused by glucose or lactate. Repression of protease production also occurred in the presence of higher concentrations (5 mM) phenylalanine and other amino acids and by ammonium ions. It is suggested that the energy-status of the cell may play an important role in the regulation of protease synthesis in Vibrio SA1.

Aminopeptidases↗
PubMed1978

Purification and some properties of two extracellular proteolytic enzymes produced by Vibrio SA1.

The purification and characterisation of an extracellular endo and amino-peptidase of the marine Vibrio SA1 is described. The endopeptidase was purified by ammonium sulphate precipitation, gel filtration and affinity chromatography. It had a molecular weight of approximately 31,000, a pH optimum at 7.8 and a temperature optimum at 50 C. The enzyme was rapidly inactivated at 65 C. The aminopeptidase was purified by ammonium sulphate precipitation, gel filtration and preparative polyacrylamide gel electrophoresis. This enzyme had a molecular weight of approximately 21,000, a pH optimum at 8.6 and a temperature optimum at 60 C. Both proteases were inactivated by EDTA while reactivation occurred by Ca2+, Zn2+ and Mg2+ ions. The endopeptidase hydrolysed several peptide bonds in the oxidized B-chain of insulin, particularly those involving amino groups of hydrophobic amino acid residues with bulky side chains. It was unable to hydrolyse synthetic dipeptides, but a number of tripeptides were hydrolysed at a low rate. The aminopeptidase hydrolysed leucinamide and di- and tripeptides containing hydrophobic bulky amino acids as the N-terminal residue. It was concluded that the endopeptidase and the aminopeptidase of Vibrio SA1 possess complementary specificities.

Aminopeptidases↗