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J Visser

Publications and source records attributed to J Visser.

At least 163 records · Page 9Linked to original sources
PubMed1993

Arabinan degrading enzymes from Aspergillus nidulans: induction and purification.

The presence in Aspergillus nidulans of two enzymes related to the Aspergillus niger endo-arabinase and alpha-L-arabinofuranosidase B has been established using antibodies against the purified A. niger enzymes. Moreover, the absence of an equivalent in A. nidulans to the alpha-L-arabinofuranosidase A of A. niger has been confirmed both at the protein and at the DNA level. Both A. nidulans arabinases have been purified and physico-chemically and kinetically characterized. They have a much higher temperature optimum than the corresponding A. niger enzymes. The pattern of induction has been studied on media containing different carbon sources showing an important role of L-arabitol in the induction of these enzymes.

Aspergillus nidulans↗
PubMed1993

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei.

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.

Amino Acid Sequence↗
PubMed1993

Effect of different positively charged amino acids, C-terminally of the signal peptidase cleavage site, on the translocation kinetics of a precursor protein in Escherichia coli K-12.

Introduction of positively charged amino acids immediately downstream of the signal sequence in prokaryotic precursor proteins is known to affect the export process. However, it is not clear whether different positively charged amino acids affect the export process similarly. To investigate this, the glutamate at position +2 of outer membrane protein PhoE was substituted by arginine, lysine or histidine. Pulse-chase experiments revealed that the Lys and Arg residues at position +2 caused a reduced processing rate, and that the effect was markedly more severe in the case of the Arg residue. Trypsin accessibility experiments revealed that the accumulated precursors were present in the cytoplasm. Since the degree of the inhibitory effect corresponded to the pKa of the different positively charged amino acids, this suggests that the positively charged residues must be deprotonated during the secretory process.

Amino Acids↗
PubMed1993

Molecular cloning, expression and structure of the endo-1,5-alpha-L-arabinase gene of Aspergillus niger.

Secretion of endo-1,5-alpha-L-arabinase A (ABN A) by an Aspergillus niger xylulose kinase mutant upon mycelium transfer to medium containing L-arabitol was immunochemically followed with time to monitor its induction profile. A cDNA expression library was made from polyA+ RNA isolated from the induced mycelium. This library was immunochemically screened and one ABN A specific clone emerged. The corresponding abnA gene was isolated from an A. niger genomic library. Upon Southern blot analysis, a 3.1-kb HindIII fragment was identified and subcloned to result in plasmid pIM950. By means of co-transformation using the A. niger pyrA gene as selection marker, the gene was introduced in both A. niger and A. nidulans uridine auxotrophic mutants. Prototrophic A. niger and A. nidulans transformants overproduced A. niger ABN A upon growth in medium containing sugar beet pulp as the sole carbon source, thereby establishing the identity and functionality of the cloned gene. The DNA sequence of the complete HindIII fragment was determined and the structure of the abnA gene as well as of its deduced gene product were analysed. Gene abnA contains three introns within its structural region and codes for a protein of 321 amino acids. Signal peptide processing results in a mature protein of 302 amino acids with a deduced molecular mass of 32.5 kDa. A. niger abnA is the first gene encoding an ABN to be isolated and characterized.

Amino Acid Sequence↗
PubMed1993

Cloning of the Aspergillus niger gene encoding alpha-L-arabinofuranosidase A.

Using L-arabitol as an inducer, simple induction conditions were established that resulted in high-level expression of alpha-L-arabinofuranosidase A by an Aspergillus niger D-xylulose kinase mutant strain. These conditions were adapted to construct a cDNA expression library from which an alpha-L-arabinofuranosidase A cDNA clone was isolated using specific antiserum. The corresponding gene encoding alpha-L-arabinofuranosidase A (abfA) was isolated from a genomic library and cloned into a high copy plasmid vector. By co-transformation of uridine auxotrophic mutants lacking orotidine-5-phosphate decarboxylase activity, the afbA gene was introduced both in A. niger and A. nidulans, using the A. niger pyrA gene as selection marker. The identity of the abfA gene was confirmed by overexpression of the gene product by A. niger and A. nidulans transformants, upon growth using sugar beet pulp as the carbon source.

Aspergillus nidulans↗
PubMed1993

Induction of extracellular arabinases on monomeric substrates in Aspergillus niger.

The induction of extracellular arabinases by pentose sugars and polyols generated by the metabolic pathway of L-arabinose and D-xylose catabolism in Aspergillus niger was investigated. Induction occurred with L-arabinose and L-arabitol but not with D-xylose or xylitol. L-arabitol in particular was found to be a good inducer for alpha-L-arabinofuranosidase and endo-arabinase activities. Western blotting analysis showed both alpha-L-arabinofuranosidase A and B to be present. No induction was observed using D-arabitol. Unlike the wild type A. niger N402 strain, the A. niger xylulose kinase negative mutant N572 also showed induction of alpha-L-arabinofuranosidases A and B and endo-arabinase activity on D-xylose and xylitol. This is due to metabolic conversion of these compounds leading to the accumulation of both xylitol and L-arabitol in this mutant, the latter of which then acts as inducer. The induction of the two alpha-L-arabinofuranosidases and endo-arabinase is under the control of two regulatory systems namely pathway specific induction and carbon catabolite repression. Under derepressing conditions in the wild type only alpha-L-arabinofuranosidase B could be detected by Western blotting analysis. This indicates that alpha-L-arabinofuranosidase B is of importance in the initiation of specific induction of the various arabinose activities in A. niger grown on arabinose containing structural polysaccharides.

Arabinofuranosyluracil↗
PubMed1993

Cloning and characterization of the abfB gene coding for the major alpha-L-arabinofuranosidase (ABF B) of Aspergillus niger.

Based on amino-acid sequence data from Aspergillus niger alpha-L-arabinofuranosidase B (ABF B), and cyanogen bromide fragments derived thereof, deoxyoligonucleotide mixtures were designed to be employed as primers in a polymerase chain reaction (PCR) on A. niger genomic DNA. This resulted in amplification of three related PCR products. The abfB gene encoding ABF B was isolated from a genomic library using such an amplification product as a probe. A 5.1-kb BamHI fragment was subcloned to result in plasmid pIM991. Upon introduction by co-transformation into both A. niger and A. nidulans uridine auxotrophic strains, pIM991 was shown to contain the functional gene since prototrophic transformants overproduced ABF B upon growth on the inducing carbon source sugar beet pulp. A plate assay was developed enabling quick selection of ABF B-overproducing transformants. The sequence of a 4122-bp long BamHI/SstI fragment was determined. The abfB gene does not contain introns and codes for a protein of 499 amino acids. The mature ABF B, 481 amino acids in length, has a deduced molecular weight of 50.7 kDa. A. niger abfB is the first eukaryotic gene encoding an ABF to be characterized.

Amino Acid Sequence↗
PubMed1993

Induction of glucose oxidase, catalase, and lactonase in Aspergillus niger.

The induction of glucose oxidase, catalase, and lactonase activities was studied both in wild-type and in glucose oxidase regulatory and structural mutants of Aspergillus niger. The structural gene for glucose oxidase was isolated and used for Northern analysis and in transformation experiments using various gox mutations. Wild-type phenotype could be restored in the glucose oxidase-negative mutant (goxC) by transformation with the structural gene. We conclude, therefore, that the goxC marker which is located on chromosome 2 represents the structural gene of glucose oxidase. Glucose and a high oxygen level are necessary for the induction of all three enzyme activities in the wild-type strain and it was shown that both glucose and oxygen effects reflect regulation at the transcriptional level. The goxB mutation results in constitutive expression of all three activities although modulated to some extent by the carbon source. The goxE mutation only has an effect on lactonase and glucose oxidase expression and does not relieve the necessity for a high oxygen level. Catalase and lactonase could not be induced in the glucose oxidase-negative strain (goxC). Addition of H2O2 resulted in the induction of all three enzymes in the wild-type without glucose being present. The H2O2 induction is probably mediated by the goxB product. Besides the H2O2 induction there is still an effect of the carbon source on the induction. A model for induction of glucose oxidase, catalase, and lactonase in A. niger is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspergillus↗
PubMed1993

Topology of PhoE porin: the 'eyelet' region.

A model for the topology of the PhoE porin has been proposed according to which the polypeptide traverses the outer membrane sixteen times mostly as amphipathic beta-sheets, thereby exposing eight loops at the cell surface. Until now, no evidence has been obtained for the surface exposure of the third loop. Recently, the structure of porin of Rhodobacter capsulatus has been determined. The proposed model of PhoE is very similar to the structure of the R. capsulatus porin, which has an 'eyelet' region, extending into the interior of the pore. The proposed third external loop of PhoE might form a similar 'eyelet' region. To determine the location of the predicted third external loop of PhoE, multiple copies of an oligonucleotide linker encoding an antigenic determinant of VP1 protein of foot-and-mouth disease virus (FMDV) were inserted. All hybrid proteins were properly inserted in the outer membrane. The monoclonal antibody MA11, directed against the linear FMDV epitope, was able to bind only to intact cells expressing a hybrid PhoE protein with at least three copies of the FMDV epitope present. Antibiotic sensitivity tests and single-channel conductance measurements revealed that the insertions influenced the channel size. These results are consistent with a location of the third loop of PhoE within the pore channel.

Amino Acid Sequence↗
PubMed1992

Clearance of indomethacin occurs predominantly by renal glucuronidation.

In this report we describe the conditions of collection, storage and handling of urine samples, collected after oral dosing with indomethacin in man, in order to maintain the integrity of the labile glucuronide formed. We found that the body clearance occurs predominantly by renal metabolism, due to glucuronidation in the human kidney. These glucuronides may be converted to isomeric glucuronides and/or the parent compound indomethacin during the residence time in the bladder.

Adult↗
PubMed1992

The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes.

Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases I and II (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lambda EMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergillus nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A. nidulans transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pgaC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intron/exon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A. niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to represent a binding site for a regulatory protein as it shows a high similarity to the yeast CYC1 upstream activation site recognized by the HAP2/3/4 activation complex.

Amino Acid Sequence↗
PubMed1992

Simultaneous analysis of azidothymidine and its mono-, di- and triphosphate derivatives in biological fluids, tissue and cultured cells by a rapid high-performance liquid chromatographic method.

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of the antiviral drug azidothymidine (AZT), AZT monophosphate, AZT diphosphate and AZT triphosphate, with ultraviolet detection in the nanomolar range, is described. Determination of these compounds in vitro in the human MT-4 lymphocyte cell line did not require a prior extraction, and AZT and its phosphorylated derivatives could be accurately analysed in one HPLC run. However, plasma, bile, liver homogenate and urine samples could not be injected directly into the chromatograph. Therefore, a solid-phase extraction procedure was developed, using azidodideoxyinosine as internal standard. The extractions of the compounds of interest from all but urine samples were reproducible, with recoveries between 65% (AZT triphosphate from plasma) and 100% (AZT from plasma).

Animals↗
PubMed1992

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression.

The nucleotide sequence of pelB, a member of the Aspergillus niger pectin lyase multigene family, has been determined. The pelB gene product, PLB, shares 65% amino acid identity with pectin lyase A (PLA) and 60% with pectin lyase D (PLD). Although growth of pelB multicopy transformants on pectin-containing media results in elevated pelB mRNA levels, pectin lyase B (PLB) is barely detectable. This is probably due to degradation of PLB by acid proteases, since multicopy transformants grown on pectin medium with a high concentration of phosphate, leading to a less rapid decline in pH, secrete detectable amounts of PLB. To produce PLB in high amounts under conditions where few other extracellular enzymes are present, we tried two strategies. Firstly, heterologous expression of the pelB gene in A. nidulans, and secondly, expression of the pelB gene under control of the constitutive A. niger pki promoter.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of the Aspergillus niger pyruvate kinase gene.

The Aspergillus niger gene encoding pyruvate kinase was cloned by heterologous hybridization using a fragment from the corresponding yeast gene as a probe. The primary structure of the gene, including 5' and 3' flanking sequences, was determined. The structural part of the A. niger pkiA gene is 2054 bp long and is interrupted by seven putative introns. Splicing of the intron sequences results in an open reading frame of 1578 bp, encoding a protein of 526 amino-acid residues and a molecular weight of 58,130 Da. Extensive homology is found with pyruvate kinase from A. nidulans; only 33 amino acids are different between both proteins. Transformation experiments using the pyrA gene as a selection marker and the subcloned pkiA gene as a co-transforming marker led to increased levels of pyruvate kinase. Analysis of the transformants showed that in none of the transformants integration had occurred at the pkiA locus. Predominantly co-integration of the pyrA- and the pkiA-containing plasmids was found in the cases examined.

Amino Acid Sequence↗
PubMed1992

Oral alimentary tract cyst.

A cyst in the floor of the mouth of a 2-yr-old boy was characterized by smooth muscle tissue with an overlying epithelial lining of varying morphology including gastric mucosa. The combination of smooth muscle with a variety of epithelial linings argues against the "misplaced anlage" histogenesis.

Child, Preschool↗
PubMed1992

Localization of Glucose Oxidase and Catalase Activities in Aspergillus niger.

The subcellular localization of glucose oxidase (EC 1.1.3.4) in Aspergillus niger N400 (CBS 120.49) was investigated by (immuno)cytochemical methods. By these methods, the bulk of the enzyme was found to be localized in the cell wall. In addition, four different catalases (EC 1.11.1.6) were demonstrated by nondenaturing polyacrylamide gel electrophoresis of crude extracts of induced and noninduced cells. Comparison of both protoplast and mycelial extracts indicated that, of two constitutive catalases, one is located outside the cell membrane whereas the other is intracellular. Parallel with the induction of glucose oxidase, two other catalases are also induced, one located intracellularly and one located extracellularly. Furthermore, lactonase (EC 3.1.1.17) activity, catalyzing the hydrolysis of glucono-delta-lactone to gluconic acid, was found to be exclusively located outside the cell membrane, indicating that gluconate formation in A. niger occurs extracellularly.

Journal Article↗
PubMed1992

The effect of decrease in body temperature with Nembutal on meiosis and ovulation after induction by gonadotrophin-releasing hormone and luteinizing hormone in adult rats.

Sodium pentobarbital (Nembutal) is often used to block the pro-oestrous luteinizing hormone (LH) surge in rats. Nembutal is also known to lower body temperature. This study was designed to investigate whether Nembutal affected the time course of meiosis and timing of ovulation induced by exogenous hormones, and whether the possible effects of Nembutal on these processes were related to temperature. Gonadotrophin-releasing hormone (GnRH), the GnRH-analogue Ovalyse, or rat luteinizing hormone (LH) were administered to trigger resumption of meiosis and ovulation; Nembutal (35 mg kg-1 body weight) or saline was given 10 or 60 min later. Plasma profiles of LH were measured and Graafian follicles were studied histologically for meiotic progress and ovulation. Nembutal suppressed the spontaneous surge of LH at pro-oestrus and caused a long-lasting decrease in body temperature. If 1000 ng GnRH was given 2 h before the pro-oestrous LH surge, most of the oocytes had extruded a polar body 10 h later and most follicles had ovulated 14 h later. Nembutal given 1 h after GnRH delayed extrusion of the polar body and ovulation by about 2 h. Nembutal caused a similar delay in ovulation when it was administered after 100 ng of Ovalyse, and it also delayed meiosis when given after 1000 ng of LH. This effect of Nembutal was prevented if body temperature was maintained at 37 degrees C. The delaying effect of Nembutal on meiosis and ovulation induced by exogenous GnRH or LH is related to a long-lasting decrease in body temperature.

Animals↗