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Biomedical subjects

W Knogge

Publications and source records attributed to W Knogge.

11 recordsLinked to original sources
PubMed2001

Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress.

The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans, Pichia pastoris and Pichia anomala, as well as in the six fungal species Sordaria macrospora, Pyrenophora teres, Ustilago maydis, Acremonium chrysogenum, Penicillium olsonii and Rhynchosporium secalis. Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism. Sterol glycosides were detected in P. pastoris strain GS115, U. maydis, S. macrospora and R. secalis. This glycolipid occurred in both yeast and filamentous forms of U. maydis but in neither form of C. albicans. This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms. Cerebrosides and sterol glycosides from P. pastoris and R. secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The cerebrosides are beta-glucosyl ceramides consisting of a saturated alpha-hydroxy or non-hydroxy fatty acid and a Delta4,8-diunsaturated, C9-methyl-branched sphingobase. Sterol glycoside from P. pastoris was identified as ergosterol-beta-D-glucopyranoside, whereas the sterol glucosides from R. secalis contain two derivatives of ergosterol. The biosynthesis of sterol glucoside in P. pastoris CBS7435 and GS115 depended on the culture conditions. The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations. From these data we suggest that, in addition to Saccharomyces cerevisiae, new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells. This suggestion is based on the ubiquitous and frequent occurrence of cerebrosides and sterol glycosides, both of which are rarely detected in S. cerevisiae. We suggest P. pastoris and two plant pathogenic fungi to be selected for this approach.

Cerebrosides↗
PubMed1999

Heterologous expression of the avirulence gene product, NIP1, from the barley pathogen Rhynchosporium secalis.

NIP1, the product of the avirulence gene AvrRrs1 from Rhynchosporium secalis, a fungal pathogen of barley, is a small secreted cysteine-rich protein. This protein is essential for the specific recognition of the fungus by host plants carrying the complementary resistance gene Rrs1. Different heterologous expression systems were tested to produce sufficient quantities of NIP1 to allow its utilization in receptor identification and isolation. In addition, protein amounts higher than those produced in fungal cultures are required to determine its 3D structure and to analyze its interaction with a receptor. The most efficient method, the synthesis of a His-tag fusion protein in Escherichia coli combined with a refolding procedure, yielded up to 3 mg of recombinant NIP1 from a 1-liter bacterial culture. After removal of the His-tag, the recombinant protein showed the same physicochemical characteristics as the native NIP1 and, most importantly, full biological activity.

Animals↗
PubMed1998

Fungal pathogenicity.

Successful penetration of living plant tissue by fungal pathogens is preceded by an exchange of signals between both organisms. Recent mutational approaches revealed the importance of cAMP-dependent signalling pathways for fungal development and virulence on their hosts.

Fungi↗
PubMed1996

Transformation of the plant pathogenic fungus, Rhynchosporium secalis.

The barley leaf scald fungus, Rhynchosporium secalis, was transformed to hygromycin-B and phleomycin resistance using the hph gene from E. coli and the ble gene from Streptoalloteichus hindustanus under the control of Aspergillus nidulans promoter and terminator sequences. Plasmid DNA was introduced into fungal protoplasts by PEG/CaCl2 treatment. Transformation frequencies varied from 59 to 493 transformants per 10 microg of DNA and 5 x 10(7) protoplasts. The antibiotic-resistant phenotype appeared to be stable under selective, as well as under non-selective, conditions for several generations. Co-transformation using the E. coli uidA gene under the control of A. nidulans promoter and terminator sequences on a non-selectable plasmid occurred at frequencies of up to 66%.

Actinomycetales↗
PubMed1995

The race-specific elicitor, NIP1, from the barley pathogen, Rhynchosporium secalis, determines avirulence on host plants of the Rrs1 resistance genotype.

NIP1, a small phytotoxic protein secreted by the barley pathogen Rhynchosporium secalis, is a race-specific elicitor of defense responses in barley cultivars carrying the resistance gene, Rrs1. Co-inoculation employing spores from a virulent fungal race together with the NIP1 protein converted the phenotype of the interaction from compatible to incompatible only on Rrs1-containing plants. In addition, transformation of a virulent fungal race with the nip1 gene yielded avirulent transformants. This demonstrated that the protein is the product of a fungal avirulence gene. The fungal genome was found to contain a single copy of the nip1 gene. Sequence analysis of nip1 cDNA and genomic clones revealed that the gene consists of two exons and one intron. The derived amino acid sequence comprised a secretory signal peptide of 22 amino acids and a cysteine-rich mature protein of 60 amino acids. All fungal races that were avirulent on barley cultivars of the Rrs1 resistance genotype carry and express the nip1 gene and secrete an elicitor-active NIP1 polypeptide. In contrast, races lacking this gene were virulent. In addition, single nucleotide exchanges were detected in the coding region of the nip1 alleles in one virulent fungal race and in a race whose interaction with barley is not controlled by the Rrs1 gene. The resulting exchanges of single amino acids render the gene products elicitor-inactive. Thus, the R.secalis-barley interaction provides the first example of a pathosystem conforming to the gene-for-gene hypothesis in which a plant with a particular resistance gene recognizes a pathogen by a virulence factor, i.e. one of its offensive weapons. On the fungal side, in turn, recognition by the host plant is eluded by either deletion of the encoding gene or alteration of the primary structure of the gene product.

Amino Acid Sequence↗
PubMed1986

The role of chalcone synthase in the regulation of flavonoid biosynthesis in developing oat primary leaves.

The role of chalcone synthase in the regulation of flavonoid biosynthesis during organogenesis of oat primary leaves has been investigated at the level of enzyme activity and mRNA translation in vitro. Chalcone synthase was purified about 500-fold. The apparent Km values were 1.5 and 6.3 microM for 4-coumaroyl-CoA and malonyl-CoA, respectively. The end products of oat flavonoid biosynthesis, three C-glucosylflavones, did not inhibit the reaction at concentrations as measured up to 60 microM each. Apigenin (4',5,7-trihydroxyflavone), a stable structural analog of the reaction product, 2',4,4',6'-tetrahydroxychalcone, was found to be a strong competitive inhibitor of 4-coumaroyl-CoA binding and a strong noncompetitive inhibitor of malonyl-CoA binding. Although apigenin is not supposed to be an intermediate of C-glucosylflavone biosynthesis, this compound might be a valuable tool for future kinetic studies. To date, there is no indication of chalcone synthase regulation by feedback or similar mechanisms which modulate enzyme activity. Mathematical correlation of chalcone synthase activity and flavonoid accumulation during leaf development, however, indicates that chalcone synthase is the rate-limiting enzyme of the pathway. By in vitro translation studies using preparations of total RNA from different leaf stages, we could demonstrate for the first time that the translational activity of chalcone synthase mRNA undergoes marked daily changes. The high values found at the end of the dark phase suggest that light does not exert direct influence on flavonoid biosynthesis but probably functions by controlling the basic diurnal rhythm.

Acyltransferases↗
PubMed1984

Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine: vitexin 2"-O-rhamnoside 7-O-methyltransferase of Avena sativa L.

An O-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the A-ring 7-hydroxyl group of vitexin 2"-O-rhamnoside has been isolated from oat primary leaves and purified 180-fold by protein fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose and S-adenosyl-L-homocysteine-sepharose. Km values for S-adenosyl-L-methionine and the flavonoid substrate were 1.6 microM and 15 microM, respectively. The lack of methyltransfer to biosynthetic intermediates suggests that the reaction is the last step in the biosynthetic pathway to the oat flavonoid 7-O-methylvitexin 2"-O-rhamnoside. Based on results obtained from kinetic inhibition studies and affinity chromatography a mono-iso Theorell-Chance mechanism is proposed with the nucleotide substrate binding before the flavonoid.

Catalysis↗
PubMedDate not supplied

Cultivar-specific elicitation of barley defense reactions by the phytotoxic peptide NIP1 from Rhynchosporium secalis.

Resistance of barley to the phytopathogenic fungus, Rhynhosporium secalis race US238.1, was found to be controlled by resistance gene Rrs1, which segregated in a manner characteristics for a codominant gene. PRHv-1, a thaumatin-like pathogenesis-related protein, was shown to be encoded by a gene family on chromosome 1. As part of the barley defense response, significant accumulation of PRHv-1 and peroxidase transcripts was induced early during pathogenesis in two Rrs1 cultivars but not or to a lower level in a near-isogenic, susceptible rrs1 cultivar or a cultivar lacking known resistance genes. R. secalis secretes a small group of necrosis-inducing peptides. One of these, NIP1, which was detected in culture filtrates only of fungal race US238.1, was found to elicit the accumulation of PRHv-1 and peroxidase mRNAs in Rrs1 cultivars with a time course similar to that upon fungal infection. Therefore, NIP1 is a candidate for the product of fungal avirulence gene avrRrs1, which, together with barley resistance gene Rrs1, determines incompatibility of the interaction.

Amino Acid Sequence↗
PubMedDate not supplied

Application of liquid chromatography to a study on 4-coumarate: coenzyme A ligase activity.

This report describes the separation of components from a 4-coumarate:CoA ligase assay by means of liquid chromatography. With the aid of polyamide column chromatography it is possible to enrich and isolate chromatographically and UV spectroscopically pure p-coumaroyl-CoA using as a solvent 0.01% NH4OH in methanol subsequent to water and methanol alone. High performance liquid chromatography on octadecylsilane-bonded silica stationary phase allows a discontinuous determination of ligase activity. All components - ATP, Coenzyme A, p-coumaric acid, and the products AMP and p-coumaroyl-CoA - can be separated and accurately quantified within 20 min using a water-acetonitrile gradient, containing 1% phosphoric acid. The presented HPLC method may be used to affirm the accuracy of optical tests.

Chromatography, High Pressure Liquid↗