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J Visser

Publications and source records attributed to J Visser.

At least 145 records · Page 8Linked to original sources

Molecular cloning and sequence of the cytoplasmic ribosomal protein S15a gene from Agaricus bisporus.

We have isolated an Agaricus bisporus cDNA which encodes an open reading frame of 130 amino acids. A comparison with the Genbank database shows that the deduced amino acid sequence of this open reading frame is highly homologous to the small subunit ribosomal proteins S15a of Brassica napus and Drosophila melanogaster and to the small subunit ribosomal proteins S24 of Strongylocentrotus purpuratus and Saccharomyces cerevisiae.

Agaricus↗

Purification and characterization of NADP-dependent glutamate dehydrogenase from the commercial mushroom Agaricus bisporus.

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent glutamate dehydrogenase (NADP-GDH) of Agaricus bisporus, a key enzyme in ammonia assimilation, was purified to apparent electrophoretic homogeneity with 27% recovery of the initial activity. The molecular weight of the native enzyme was 330 kDa. The enzyme is probably a hexamer, composed of identical subunits of 48 kDa. The isoelectric point of the enzyme was found at pH 4.8. The N-terminus appeared to be blocked. The enzyme was specific for NADP(H). The Km-values were 2.1, 3.2, 0.074, 27.0, and 0.117 mM for ammonia, 2-oxoglutarate, NADPH, L-glutamate, and NADP respectively. The pH optima for the amination and deamination reactions were found to be 7.6 and 9.0, respectively. The temperature optimum was 33 degrees C. The effect of several metabolites on the enzyme's activity was tested. Pyruvate, oxaloacetate, ADP, and ATP showed some inhibitory effect. Divalent cations slightly stimulated the aminating reaction. Antibodies raised against the purified enzyme were able to precipitate NADP-GDH activity from a cell-free extract in an anticatalytic immunoprecipitation test. Analysis of a Western blot showed the antibodies to be specific for NADP-GDH.

Adenine Nucleotides↗

New protease mutants in Aspergillus niger result in strongly reduced in vitro degradation of target proteins; genetical and biochemical characterization of seven complementation groups.

Several mutants of Aspergillus niger, deficient in extracellular protease expression, have been isolated and characterized both genetically and biochemically. The mutant strains, obtained after in vivo UV-mutagenesis of conidiospores and selected by haloscreening on a new dual-substrate plate assay, belong to at least seven different complementation groups. These seven prt loci were assigned to linkage groups using master strains with marked chromosomes. One prt locus (prtC) could be assigned to linkage group I, three (prtB, prtE and prtG) to linkage group III, one (prtF) to linkage group V and the two remaining loci (prtA and prtD) to linkage group VIII. Extracellular proteolytic activities varied from 2 to 3% up to 80% of the protease activity of the parental strain. Assigning the different prt mutants to structural or regulatory genes is difficult since only one structural gene, pepA, has been mapped unambiguously on linkage group I but is not identical to prtC. All prt mutants except for prtC are likely to be regulatory mutants or else belong to a proteolytic cascade because residual activities showed that more proteolytic activities were affected simultaneously. Double mutants were constructed both by recombination and by a second round of mutagenesis. In both cases mutants with further reduced extracellular proteolytic activities were isolated. A sensitive in vitro degradation assay, based on the homologous pectin lyase B (PELB) protein to analyze proteolytic degradation in A. niger, was developed and used to show extremely reduced proteolytic PELB degradation in the culture media of some of these mutants.

Aspergillus niger↗

Cloning, sequence and expression of the gene coding for rhamnogalacturonase of Aspergillus aculeatus; a novel pectinolytic enzyme.

Rhamnogalacturonase was purified from culture filtrate of Aspergillus aculeatus after growth in medium with sugar-beet pulp as carbon source. Purified protein was used to raise antibodies in mice and with the antiserum obtained a gene coding for rhamnogalacturonase (rhgA) was isolated from a lambda cDNA expression library. The cloned rhgA gene has an open-reading frame of 1320 base pairs encoding a protein of 440 amino acids with a predicted molecular mass of 45 962 Da. The protein contains a potential signal peptidase cleavage site behind Gly-18 and three potential sites for N-glycosylation. Limited homology with A. niger polygalacturonase amino acid sequences is found. A genomic clone of rhgA was isolated from a recombinant phage lambda genomic library. Comparison of the genomic and cDNA sequences revealed that the coding region of the gene is interrupted by three introns. Furthermore, amino acid sequences of four different peptides, derived from purified A. aculeatus rhamnogalacturonase, were also found in the deduced amino acid sequence of rhgA. A. aculeatus strains overexpressing rhamnogalacturonase were obtained by cotransformation using either the A. niger pyrA gene or the A. aculeatus pyrA gene as selection marker. For expression of rhamnogalacturonase in A. awamori the A. awamori pyrA gene was used as selection marker. Degradation patterns of modified hairy regions, determined by HPLC, show the recombinant rhamnogalacturonase to be active, and the enzyme was found to have a positive effect in the apple hot-mash liquefaction process.

Amino Acid Sequence↗

Complete degradation of tetrachloroethene by combining anaerobic dechlorinating and aerobic methanotrophic enrichment cultures.

Degradation of tetrachloroethene (perchloroethylene, PCE) was investigated by combining the metabolic abilities of anaerobic bacteria, capable of reductive dechlorination of PCE, with those of aerobic methanotrophic bacteria, capable of co-metabolic degradation of the less-chlorinated ethenes formed by reductive dechlorination of PCE. Anaerobic communities reductively dechlorinating PCE, trichloroethene (TCE) and dichloroethenes were enriched from various sources. The maximum rates of dechlorination observed for various chloroethenes in these batch enrichments were: PCE to TCE (341 mumol l-1 day-1), TCE to cis-dichloroethene (159 mumol l-1 day-1), cis-dichloroethene to chloroethene (99 mumol l-1 day-1) and trans-dichloroethene to chloroethene (22 mumol l-1 day-1). A mixture of these enrichments was inoculated into an anoxic fixed-bed upflow column. In this column PCE was converted mainly into cis-1,2-dichloroethene, small amounts of TCE and chloroethene, and chloride. Enrichments of aerobic methanotrophic bacteria were grown in an oxic fixed-bed downflow column. Less-chlorinated ethenes, formed in the anoxic column, were further metabolized in this oxic methanotrophic column. On the basis of analysis of chloride production and the disappearance of chlorinated ethenes it was demonstrated that complete degradation of PCE was possible by combining these two columns. Operation of the two-column system under various process conditions indicated that the sensitivity of the methanotrophic bacteria to chlorinated intermediates represented the bottle-neck in the sequential anoxic/oxic degradation process of PCE.

Bacteria, Aerobic↗

An extreme creA mutation in Aspergillus nidulans has severe effects on D-glucose utilization.

Aspergillus nidulans wild-type and the extreme carbon catabolite derepressed mutant creAd-30 were characterized with respect to enzyme activities, metabolite concentrations and polyol pools all related to glycolysis, after growth on D-glucose. In the creAd-30 strain the enzymes hexokinase and fructose-6-phosphate reductase showed a two- and threefold increase in activity, respectively, whereas phosphofructokinase and pyruvate kinase activity decreased two- and threefold, respectively, in comparison with the wild-type strain. The most notable changes in metabolite concentrations were that fructose 2,6-bisphosphate and fructose 1,6-bisphosphate showed a 2.5-fold increase, whereas both pyruvate and citrate decreased in the creAd-30. Striking differences were found for the polyol concentrations measured for the two strains tested. Intracellular glycerol and arabitol concentrations were 10-fold higher and erythritol fivefold higher in creAd-30, whereas intracellular trehalose and mannitol were both decreased. The total internal polyol concentration appears to be constant at approximately 700 mumol (g dry wt)-1. All polyols were also detected in high amounts in the culture filtrate of the creAd-30 mutant strain but no extracellular trehalose was found. The overall production of polyols in this strain was therefore much higher than in the wild-type. The high level of polyols produced and the changes in metabolite concentrations in the creAd-30 strain suggest that the differences in enzyme activities result in an altered flow through glycolysis leading to a more rapid formation of polyols which are subsequently secreted.

Aspergillus nidulans↗

Cloning, characterization and expression of pepF, a gene encoding a serine carboxypeptidase from Aspergillus niger.

We have cloned a gene (pepF) encoding a serine carboxypeptidase, proteinase F (PEPF), from Aspergillus niger. The sequences were identified in a phage lambda genomic DNA library using a synthetic probe based on the N-terminal sequence of PEPF. Nucleotide sequence data from pepF genomic and cDNA clones reveals that it is composed of four exons of 199, 283, 227 and 881 bp, interrupted by three introns of 53, 69 and 59 bp. The sequence of pepF codes for a polypeptide of 530 amino acids (aa), of which the first 52 aa are not present in the mature PEPF. This region may represent a prepro sequence that is removed by proteolytic cleavage as a monobasic cleavage site (Lys52). Northern blot analysis of total cellular RNA extracted from A. niger cells indicates that pepF is transcribed as a single 1.8-kb mRNA, which is regulated by nitrogen and carbon repression, specific induction and the pH of the culture medium.

Amino Acid Sequence↗

Crystallization and preliminary crystallographic characterization of endo-polygalacturonase II from Aspergillus niger.

The endo-polygalacturonase II from Aspergillus niger has been crystallized from an ammonium sulfate solution by the hanging drop method. The crystals belong to the monoclinic space group P2(1), with cell dimensions a = 69.6 A, b = 152.6 A, c = 74.0 A and beta = 91.2 degrees with four molecules per asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for X-ray analysis.

Aspergillus niger↗

Characterization of an Aspergillus nidulans L-arabitol dehydrogenase mutant.

The degradation pathway for L-arabinose, which consists of a sequence of alternating reduction and oxidation reactions prior to ultimate phosphorylation, was studied in Aspergillus nidulans wild-type as well as in an L-arabinose non-utilizing mutant. The inability of the mutant to use L-arabinose was caused by the absence of L-arabitol dehydrogenase activity. The effect of the mutation on polyol accumulation patterns was studied upon growth on various carbon sources. The presence of L-arabinose resulted in intracellular accumulation of arabitol in this mutant. Moreover, the mutant secreted arabitol under these conditions and, in contrast to the wild-type, featured enhanced expression of enzymes involved in L-arabinose catabolism as well as of extracellular glycosyl hydrolases involved in degradation of the plant cell wall polysaccharide L-arabinan.

Arabinose↗

Extracellular arabinases in Aspergillus nidulans: the effect of different cre mutations on enzyme levels.

The regulation of the syntheses of two arabinan-degrading extracellular enzymes and several intracellular L-arabinose catabolic enzymes was examined in wild-type and carbon catabolite derepressed mutants of Aspergillus nidulans. alpha-L-Arabinofuranosidase B, endoarabinase, L-arabinose reductase, L-arabitol dehydrogenase, xylitol dehydrogenase, and L-xylulose reductase were all inducible to varying degrees by L-arabinose and L-arabitol and subject to carbon catabolite repression by D-glucose. With the exception of L-xylulose reductase, all were clearly under the control of creA, a negative-acting wide domain regulatory gene mediating carbon catabolite repression. Measurements of intracellular enzyme activities and of intracellular concentrations of arabitol and xylitol in mycelia grown on D-glucose in the presence of inducer indicated that carbon catabolite repression diminishes, but does not prevent uptake of inducer. Mutations in creA resulted in an apparently, in some instances very marked, elevated inducibility, perhaps reflecting an element of "self" catabolite repression by the inducing substrate. creA mutations also resulted in carbon catabolite derepression to varying degrees. The regulatory effects of a mutation in creB and in creC, two genes whose roles are unclear, but likely to be indirect, were, when observable, more modest. As with previous data showing the effect of creA mutations on structural gene expression, there were striking instances of phenotypic variation amongst creA mutant alleles and this variation followed no discernible pattern, i.e. it was non-hierarchical. This further supports molecular data obtained elsewhere, indicating a direct role for creA in regulating structural gene expression, and extends the range of activities under creA control.

Arabinose↗

Arabinase gene expression in Aspergillus niger: indications for coordinated regulation.

Aspergillus niger secretes three glycosylated glycosyl hydrolases which are involved in degradation of the plant cell wall polysaccharide L-arabinan: alpha-L-arabinofuranosidases (ABF) A and B, and endo-1,5-alpha-L-arabinase (ABN) A. The nucleotide sequence of the previously cloned gene encoding ABF A (abfA) from A. niger was determined. The coding region contains seven introns. Mature ABF A comprises 603 amino acids with a molecular mass of 65.4 kDa as deduced from the nucleotide sequence. The secreted enzyme is N-glycosylated. The primary structures of the three A. niger arabinases characterized lack similarity. Regulation of arabinase expression upon induction by sugar beet pulp and by L-arabitol was studied as a function of time. This was done in wild-type A. niger as well as in transformants carrying multiple copies of either one of the ABF-encoding genes. Each arabinase gene responded differently upon a mycelial transfer to L-arabitol-containing medium. Extra copies of abfA or abfB led to a decreased expression level of ABN A, though the repression elicited by abfB is stronger and more persistent than that effected by abfA. Multiple copies of both abf genes influence expression of the other ABF similarly, but to a far less pronounced degree than they affect ABN A synthesis. Four putative promoter elements, shared by all three arabinase genes, could be involved in coordination of L-arabinan degradation by A. niger.

Amino Acid Sequence↗

Regulation of the xylanase-encoding xlnA gene of Aspergillus tubigensis.

A gene encoding an endo-1,4-beta-xylanase from Aspergillus tubigensis was cloned by oligonucleotide screening using oligonucleotides derived from amino acid sequence data obtained from the purified protein. The isolated gene was functional as it could be expressed in the very closely related fungus Aspergillus niger. The xylanase encoded by this gene is synthesized as a protein of 211 amino acids. After cleavage of the presumed prepropeptide this results in a mature protein of 184 amino acids with a molecular weight of 19 kDa and an isoelectric point of 3.6. The regulatory region of the xlnA gene was studied with respect to the response to xylan induction and carbon catabolite repression. By deletion analysis of the 5' upstream region of the gene a 158 bp region involved in the xylan specific induction was identified. To study this regulatory element a reporter system for transcriptional activating sequences was developed that is based on the A. niger glucose oxidase-encoding gene. From the results with this reporter system it is concluded that this 158 bp fragment not only contains the information required for induction of transcription but that it also plays a role in carbon catabolite repression of the xlnA gene. The region directly upstream of this fragment contains four potential CREA target sites; deletion of this region leads to an increase in the level of transcription. These results suggest that carbon catabolite repression of the xlnA gene is controlled at two levels, directly by repression of xlnA gene transcription and indirectly by repression of the expression of a transcriptional activator. This type of mechanism would be similar to the double lock mechanism for the regulation of gene expression of alcA in Aspergillus nidulans. The reporter system was also used to study the regulation of expression via the functions located on this fragment in A. niger and in A. nidulans. Essentially the same pattern of regulation was found in both of these hosts. Therefore, regulation of xylanase gene expression is basically conserved in all three aspergilli.

Amino Acid Sequence↗

Upregulation of antithrombotic ectonucleotidases by aspirin in human endothelial cells in-vitro.

Ecto ATP-diphosphohydrolase (apyrase) activity of human endothelial cells following aspirin treatment has been studied in-vitro. It was shown by HPLC analysis of supernatant samples that pre-incubation of the cultures with aspirin resulted in a significantly increased turnover of supplemented ATP into its degradation products (ADP and AMP). Enhanced expression of ectoenzyme after aspirin treatment could be observed as demonstrated by immunofluorescence-staining with monoclonal anti-apyrase antibodies. This suggests enhancement of endothelial ATP-diphosphohydrolase activity induced by aspirin. The present data obtained in human vascular cells in-vitro are in line with results from previous animal studies in-vivo, suggesting a novel cyclooxygenase-independent antithrombotic activity of aspirin.

Adenosine Diphosphate↗

Enzymes of glucose and methanol metabolism in the actinomycete Amycolatopsis methanolica.

The actinomycete Amycolatopsis methanolica was found to employ the normal bacterial set of glycolytic and pentose phosphate pathway enzymes, except for the presence of a PPi-dependent phosphofructokinase (PPi-PFK) and a 3-phosphoglycerate mutase that is stimulated by 2,3-bisphosphoglycerate. Screening of a number of actinomycetes revealed PPi-PFK activity only in members of the family Pseudonocardiaceae. The A. methanolica PPi-PFK and 3-phosphoglycerate mutase enzymes were purified to homogeneity. PPi-PFK appeared to be insensitive to the typical effectors of ATP-dependent PFK enzymes. Nevertheless, strong N-terminal amino acid sequence homology was found with ATP-PFK enzymes from other bacteria. The A. methanolica pyruvate kinase was purified over 250-fold and characterized as an allosteric enzyme, sensitive to inhibition by P(i) and ATP but stimulated by AMP. By using mutants, evidence was obtained for the presence of transketolase isoenzymes functioning in the pentose phosphate pathway and ribulose monophosphate cycle during growth on glucose and methanol, respectively.

Actinobacteria↗

Carbon metabolism.

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Aspergillus nidulans↗

Does aminophylline benefit adults admitted to the hospital for an acute exacerbation of asthma?

OBJECTIVE: To determine the effect of adding intravenous theophylline (administered as aminophylline) to nebulizations of albuterol and intravenous methylprednisolone in adults hospitalized for acute asthma. DESIGN: Randomized, placebo-controlled, double-blind study. SETTING: Inpatient service of a tertiary-care, university teaching hospital. PATIENTS: 21 adults (22 to 48 years old)--10 in the aminophylline group and 11 in the placebo group. INTERVENTIONS: Nebulized albuterol, 2.5 or 5.0 mg every 0.5 to 4 hours; intravenous methylprednisolone, 60 mg every 6 hours; and either individualized doses of aminophylline or placebo for 48 hours. MEASUREMENTS: Forced expiratory volume in 1 second (FEV1), the number of "as needed" albuterol nebulizations and total dose, asthma symptom scores, and adverse effects. RESULTS: At admission from the emergency department, the mean +/- SD baseline FEV1 was 49% +/- 19% of the predicted value in the aminophylline group and 43% +/- 13% of the predicted value in the placebo group. The improvement in FEV1 at 3 hours was greater in the aminophylline group (29% +/- 23% compared with 10% +/- 10% in the placebo group; mean difference, 19 percentage points; 95% CI, 3 to 35 percentage points; P = 0.023). At 48 hours, FEV1 was 75% +/- 19% of the predicted value in the aminophylline group and 58% +/- 15% of the predicted value in the placebo group (mean difference, 17 percentage points; CI, 0.2 to 34.8 percentage points; P = 0.048). Aminophylline-treated patients required fewer nebulizations of albuterol (10.3 +/- 3.8 compared with 16.4 +/- 5.3; mean difference, -6.1; CI, -10.3 to -1.8) and less total dosage (34 +/- 16 mg compared with 70 +/- 34 mg; mean difference, -36 mg; CI, -60.6 to -11.3 mg P = 0.02). No statistical differences were observed in asthma symptom scores or frequency of adverse effects. CONCLUSIONS: Individualized doses of intravenous theophylline added to frequent nebulizations of albuterol and intravenous methylprednisolone appear to benefit adults admitted to the hospital with acute asthma and are well tolerated when serum concentrations are maintained in the therapeutic range.

Acute Disease↗