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J Visser

Publications and source records attributed to J Visser.

At least 37 records · Page 2Linked to original sources
PubMed2002

Heterologous expression of the Aspergillus nidulans alcR-alcA system in Aspergillus niger.

The inducible and strongly expressed alcA gene encoding alcohol dehydrogenase I from Aspergillus nidulans was transferred together with the activator gene alcR, in the industrial fungus Aspergillus niger. This latter organism does not possess an inducible alc system but has an endogenously constitutive lowly expressed alcohol dehydrogenase activity. The overall induced expression of the alcA gene was of the same order in both fungi, as monitored by alcA transcription, alcohol dehydrogenase activity and heterologous expression of the reporter enzyme, beta-glucuronidase. However, important differences in the pattern of alcA regulation were observed between the two fungi. A high basal level of alcA transcription was observed in A. niger resulting in a lower ratio of alcA inducibility. This may be due to higher levels of the physiological inducer of the alc regulon, acetaldehyde, from general metabolism in A. niger which differs from that of A. nidulans.

Alcohol Dehydrogenase↗
PubMed2001

Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells.

We have synthesized three peptides from the mdm-2 binding domain of human p53, residues 12-26 (PPLSQETFSDLWKLL), residues 12-20, and 17-26. To enable transport of the peptides across the cell membrane and at the same time to maximize the active mdm-2 binding alpha-helical conformation for these peptides, each was attached at its carboxyl terminus to the penetratin sequence, KKWKMRRNQFWVKVQRG, that contains many positively charged residues that stabilize an alpha-helix when present on its carboxyl terminal end. All three peptides were cytotoxic to human cancer cells in culture, whereas a control, unrelated peptide attached to the same penetratin sequence had no effect on these cell lines. The same three cytotoxic peptides had no effect on the growth of normal cells, including human cord blood-derived stem cells. These peptides were as effective in causing cell death in p53-null cancer cells as in those having mutant or normal p53. Peptide-induced cell death is not accompanied by expression of apoptosis-associated proteins such as Bax and waf(p21). Based on these findings, we conclude that the antiproliferative effects of these p53-derived peptides are not completely dependent on p53 activity and may prove useful as general anticancer agents.

Amino Acid Sequence↗
PubMed2001

LPS-induced IL-10 production in whole blood cultures from chronic fatigue syndrome patients is increased but supersensitive to inhibition by dexamethasone.

Several causes have been held responsible for the chronic fatigue syndrome (CFS), including an altered hypothalamus-pituitary-adrenal gland (HPA)-axis activity, viral infections and a reduced Th1 activity. Therefore, it was investigated whether the regulation of IL-10 is different in CFS. LPS-induced cytokine secretion in whole blood cultures showed a significant increase in IL-10 and a trend towards a decrease in IL-12 as compared with healthy controls. In patients and controls, IL-12 secretion was equally sensitive to suppression by dexamethasone, whereas IL-10 secretion appeared more sensitive in CFS-patients. In controls, IL-10 and IL-12 secretion were inversely correlated with free serum cortisol (r=-0.492, p<0.02 and r=-0.434, p<0.05, respectively). In CFS, such an inverse correlation was found for IL-12 (r=-0.611, p<0.02) but not for IL-10 (r=-0.341, ns). These data are suggestive for a disturbed glucocorticoid regulation of IL-10 in CFS.

Adolescent↗
PubMed2001

Changing a single amino acid residue switches processive and non-processive behavior of Aspergillus niger endopolygalacturonase I and II.

Processivity, also known as multiple attack on a single chain, is a feature commonly encountered only in enzymes in which the substrate binds in a tunnel. However, of the seven Aspergillus niger endopolygalacturonases, which have an open substrate binding cleft, four enzymes show processive behavior, whereas the other endopolygalacturonases are randomly acting enzymes. In a previous study (Benen, J.A.E., Kester, H.C.M., and Visser, J. (1999) Eur. J. Biochem. 259, 577-585) we proposed that the high affinity for the substrate of subsite -5 of processive endopolygalacturonase I constitutes the origin of the multiple attack behavior. Based on primary sequence alignments of A. niger endopolygalacturonases and three-dimensional structure analysis of endopolygalacturonase II, an arginine residue was identified in the processive enzymes at a position commensurate with subsite -5, whereas a serine residue was present at this position in the non-processive enzymes. In endopolygalacturonase I mutation R95S was introduced, and in endopolygalacturonase II mutation S91R was introduced. Product progression analysis on polymer substrate and bond cleavage frequency studies using oligogalacturonides of defined chain length for the mutant enzymes revealed that processive/non-processive behavior is indeed interchangeable by one single amino acid substitution at subsite -5, Arg-->Ser or Ser-->Arg.

Amino Acid Sequence↗
PubMed2001

Prevention of meningococcal serogroup B infections in children: a protein-based vaccine induces immunologic memory.

Immunologic memory against meningococci was studied in 177 children (100 children were 10-11 years old and 77 were 5-6 years old) 2.5 years after vaccination with hexavalent meningococcal outer membrane vesicle (OMV) vaccine or hepatitis B (HepB) vaccine. Children were revaccinated with monovalent P1.7(h),4 meningococcal OMV vaccine. Serum bactericidal antibodies (SBAs) were measured before revaccination and after 4-6 weeks. A minimum 4-fold increase in SBAs against serosubtype P1.7(h),4 was detected in 48.5% of the children after hexavalent meningococcal vaccine and in 8.9% after HepB vaccine. Of the initial responders given hexavalent meningococcal vaccine, 78% had > or =4-fold increase in SBAs against strain P1.4. Thus, immunologic memory is present in toddlers and school-aged children previously given 3 hexavalent meningococcal vaccinations. Booster vaccination with monovalent P1.7(h),4 meningococcal OMV vaccine induces a significant increase in SBAs against serosubtype P1.7(h),4 and cross-reactivity against other serosubtypes in the hexavalent vaccine.

Antibodies, Bacterial↗
PubMed2001

Cytomegalovirus infection increases the expression and activity of ecto-ATPase (CD39) and ecto-5'nucleotidase (CD73) on endothelial cells.

We describe enhanced expression and enzymatic activity of ecto-ATPase and ecto-5'nucleotidase on CMV infected endothelial cells as compared to uninfected cells. These ectoenzymes play a major role in modulation of platelet activation and aggregation. Furthermore, adenosine has a modulatory effect upon inflammation. Addition of ATP, ADP or AMP to cultures of CMV infected or uninfected endothelial cells revealed increased turnover of AMP in CMV infected endothelial cells. In addition, the superoxide production by stimulated polymorphonuclear cells was inhibited in the presence of CMV infected endothelial cells as compared to uninfected cells, probably due to the enhanced activity of ecto-5'nucleotidase and associated to production of adenosine.

5'-Nucleotidase↗
PubMed2001

Botrytis cinerea endopolygalacturonase genes are differentially expressed in various plant tissues.

Botrytis cinerea, the causal agent of blight, rot, and gray mold on many plant species, secretes various endopolygalacturonases during all stages of infection. The expression pattern of the encoding genes (Bcpg 1-6) was studied on four hosts: tomato, broad bean, apple, and courgette (also known as zucchini). All gene family members are differentially expressed, depending on the stage of infection and the host. Bcpg1 is expressed in all tissues tested although differences in transcript levels occur. Bcpg2 expression is detected early in the infection of three of four plant tissues tested. Bcpg3 and Bcpg5 are expressed in apple fruit tissue, although probably as a result of different regulatory mechanisms. The expression patterns of Bcpg4 and 6 are in agreement with their inducibility by monogalacturonic acid. The pattern of Bcpg gene expression indicates that B. cinerea is equipped with a flexible enzymatic pectate degradation machinery. The studies pinpoint new targets for gene disruption studies.

Blotting, Northern↗
PubMed2001

Beta-glucosidase multiplicity from Aspergillus tubingensis CBS 643.92: purification and characterization of four beta-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition and acid tolerance.

From Aspergillus tubingensis CBS 643.92 four distinct beta-glucosidases (I-IV) were purified by a four-step purification procedure. SDS-PAGE revealed molecular masses of 131, 126, 54 and 54 kDa, respectively, and their isoelectric points were determined to be 4.2, 3.9, 3.7 and 3.6, respectively. The beta-glucosidases exhibited high diversity with respect to pH and temperature optima and stability, as well as to substrate specificity and glucose tolerance. The major beta-glucosidase (I) preferentially hydrolysed oligosaccharides. The acid-stable and heat-tolerant beta-glucosidase II hydrolysed aryl and terpenyl beta-D-glucosides as well as 1-O-trans-cinnamoyl beta-D-glucoside. In contrast to beta-glucosidases I and II, the minor beta-glucosidases III and IV were found to be glucose-tolerant; inhibition constants of 470 and 600 mM, respectively, were determined.

Amino Acid Sequence↗
PubMed2001

Isolation of UV-induced mutations in the areA nitrogen regulatory gene of Aspergillus niger, and construction of a disruption mutant.

The areA gene of filamentous fungi encodes a positive-acting transcriptional factor required for the expression of genes involved in the utilisation of nitrogen sources other than ammonium and glutamine. In Aspergillus niger we have isolated three UV-induced areA mutants and constructed a well-defined disruption allele of the areA gene. The areA gene was genetically localised on Linkage Group III, 3.6 map units (m.u.) from bioA1 and 4.5 m.u. from lysA7. Analysis of the expression of the nitrate reductase encoding gene and of nitrate reductase activities show that the mutated areA strains behave as loss-of-function mutants and can be classified as areAr type. In addition, growth tests were performed using several nitrogen sources in combination with glucose. The results suggest that, unlike the case in A. nidulans, in A. niger the AreA protein also plays a role in the presence of ammonium. Furthermore, the spectrum of protease activities secreted by A. niger differs from that produced by A. nidulans, as only A. niger is able to degrade elastin.

Aspergillus niger↗
PubMed2001

The Aspergillus niger D-xylulose kinase gene is co-expressed with genes encoding arabinan degrading enzymes, and is essential for growth on D-xylose and L-arabinose.

The Aspergillus niger D-xylulose kinase encoding gene has been cloned by complementation of a strain deficient in D-xylulose kinase activity. Expression of xkiA was observed in the presence of L-arabinose, L-arabitol and D-xylose. Expression of xkiA is not mediated by XLNR, the xylose-dependent positively-acting xylanolytic regulator. Although the expression of xkiA is subject to carbon catabolite repression, the wide domain regulator CREA is not directly involved. The A. niger D-xylulose kinase was purified to homogeneity, and the molecular mass determined using electrospray ionization mass spectrometry agreed with the calculated molecular mass of 62816.6 Da. The activity of XKIA is highly specific for D-xylulose. Kinetic parameters were determined as Km(D-xylulose) = 0.76 mM and Km(ATP) = 0.061 mM. Increased transcript levels of the genes encoding arabinan and xylan degrading enzymes, observed in the xylulose kinase deficient strain, correlate with increased accumulation of L-arabitol and xylitol, respectively. This result supports the suggestion that L-arabitol may be the specific low molecular mass inducer of the genes involved in arabinan degradation. It also suggests a possible role for xylitol in the induction of xylanolytic genes. Conversely, overproduction of XKIA did not reduce the size of the intracellular arabitol and xylitol pools, and therefore had no effect on expression of genes encoding xylan and arabinan degrading enzymes nor on the activity of the enzymes of the catabolic pathway.

Adenosine Diphosphate↗
PubMed2001

Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans.

In filamentous fungi, glycerol biosynthesis has been proposed to play an important role during conidiospore germination and in response to a hyperosmotic shock, but little is known about the genes involved. Here, we report on the characterization of the major Aspergillus nidulans glycerol 3-phosphate dehydrogenase (G3PDH)-encoding gene, gfdA. G3PDH is responsible for the conversion of dihydroxyacetone phosphate (DHAP) into glycerol 3-phosphate (G3P), which is subsequently converted into glycerol by an as yet uncharacterized phosphatase. Inactivation of gfdA does not abolish glycerol biosynthesis, showing that the other pathway from DHAP, via dihydroxyacetone (DHA), to glycerol is also functional in A. nidulans. The gfdA null mutant displays reduced G3P levels and an osmoremediable growth defect on various carbon sources except glycerol. This growth defect is associated with an abnormal hyphal morphology that is reminiscent of a cell wall defect. Furthermore, the growth defect at low osmolarity is enhanced in the presence of the chitin-interacting agent calcofluor and the membrane-destabilizing agent sodium dodecyl sulphate (SDS). As inactivation of gfdA has no impact on phospholipid biosynthesis or glycolytic intermediates levels, as might be expected from reduced G3P levels, a previously unsuspected link between G3P and cell wall integrity is proposed to occur in filamentous fungi.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation.

Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.

Amino Acid Sequence↗
PubMed2001

Combined molecular and biochemical approach identifies Aspergillus japonicus and Aspergillus aculeatus as two species.

We examined nine Aspergillus japonicus isolates and 10 Aspergillus aculeatus isolates by using molecular and biochemical markers, including DNA sequences of the ITS1-5.8S rRNA gene-ITS2 region, restriction fragment length polymorphisms (RFLP), and secondary-metabolite profiles. The DNA sequence of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene could not be used to distinguish between A. japonicus and A. aculeatus but did show that these two taxa are more closely related to each other than to other species of black aspergilli. Aspergillus niger pyruvate kinase (pkiA) and pectin lyase A (pelA) and Agaricus bisporus 28S rRNA genes, which were used as probes in the RFLP analysis, revealed clear polymorphism between these two taxa. The A. niger pkiA and pelA probes placed six strains in an A. japonicus group and 12 isolates in an A. aculeatus group, which exhibited intraspecific variation when they were probed with the pelA gene. The secondary-metabolite profiles supported division of the isolates into the two species and differed from those of other black aspergilli. The strains classified as A. japonicus produced indole alkaloids and a polar metabolite, while the A. aculeatus isolates produced neoxaline, okaramins, paraherquamidelike compounds, and secalonic acid. A. aculeatus CBS 114.80 showed specific RFLP patterns for all loci examined. The secondary-metabolite profile of strain CBS 114.80 also differed from those of A. japonicus and A. aculeatus. Therefore, this strain probably represents a third taxon. This study provides unambiguous criteria for establishing the taxonomic positions of isolates of black aspergilli, which are important in relation to industrial use and legal protection of these organisms.

Aspergillus↗
PubMed2001

Purification and characterization of two different alpha-L-rhamnosidases, RhaA and RhaB, from Aspergillus aculeatus.

Two proteins exhibiting alpha-L-rhamnosidase activity, RhaA and RhaB, were identified upon fractionation and purification of a culture filtrate from Aspergillus aculeatus grown on hesperidin. Both proteins were shown to be N glycosylated and had molecular masses of 92 and 85 kDa, of which approximately 24 and 15%, respectively, were contributed by carbohydrate. RhaA and RhaB, optimally active at pH 4.5 to 5, showed K(m) and V(max) values of 2.8 mM and 24 U/mg (RhaA) and 0.30 mM and 14 U/mg (RhaB) when tested for p-nitrophenyl-alpha-L-rhamnopyranoside. Both enzymes were able to hydrolyze alpha-1,2 and alpha-1,6 linkages to beta-D-glucosides. Using polyclonal antibodies, the corresponding cDNA of both alpha-L-rhamnosidases, rhaA and rhaB, was cloned. On the basis of the amino acid sequences derived from the cDNA clones, both proteins are highly homologous (60% identity).

Amino Acid Sequence↗
PubMed2001

Aspergillus enzymes involved in degradation of plant cell wall polysaccharides.

Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.

Aspergillus↗
PubMed2001

Increased sensitivity to glucocorticoids in peripheral blood mononuclear cells of chronic fatigue syndrome patients, without evidence for altered density or affinity of glucocorticoid receptors.

BACKGROUND: In this study we tested the hypothesis that the increased sensitivity to glucocorticoids in chronic fatigue syndrome (CFS)-patients can be attributed to an altered functioning of their glucocorticoid receptors (GR). METHODS: For this purpose, affinity and distribution of the GR were studied in purified, peripheral blood mononuclear cells (PBMC) of 10 CFS patients and 14 controls along with the responsiveness of these cells to glucocorticoids in vitro. RESULTS: Affinity (Kd) and number of GR was not different in PBMC of CFS patients when compared with the controls (Kd, 12.9 +/- 8.9 nmol vs 18.8 +/- 16.2 nmol and GR number, 4,839 +/- 2,824/ cell vs 4,906 +/- 1,646/cell). Moreover, RT-PCR revealed no differences in GR messenger RNA expression. Nevertheless, PBMC from CFS patients showed an increased sensitivity to glucocorticoids in vitro. In CFS patients 0.01 micromol dexamethasone suppressed PBMC proliferation by 37%, whereas the controls were only suppressed by 17% (P < 0.01). Addition of phorbol 12-myristate 13-acetate to the cultures rendered the cells resistant to dexamethasone with regard to proliferation and IL-10 and IFN-gamma production, but not to IL-2 and TNF-alpha production in both patients and controls. No difference between patients and controls was observed in this respect CONCLUSIONS: In conclusion, PBMC of CFS patients display an increased sensitivity to glucocorticoids, which cannot be explained by number or affinity of the GR but should rather be attributed to molecular processes beyond the actual binding of the ligand to the GR.

Adolescent↗