The Ginkgo biloba extract EGb761 reduces stress sensitivity, ROS accumulation and expression of catalase and glutathione S-transferase 4 in Caenorhabditis elegans.

The standardised extract EGb761 from the leaves of Ginkgo biloba is a popular herbal dietary supplement and it is used as a phytopharmacon for the therapy of diverse cerebral insufficiencies. The beneficial impact of EGb761 is believed to be conferred by diverse biological actions under physiological conditions as well as in response to stress. In this study we examined effects of EGb761 in the model organism Caenorhabditis elegans. EGb761 reduced the body size but did not affect the reproduction of C. elegans. In fluorescence-based assays performed in microtiter plates we demonstrated the protective action of EGb761 by the increase of resistance to thermal stress and the attenuation of ROS accumulation under conditions of thermal stress in single living worms. Under normal conditions the lifespan of the worms was extended by the EGb761 supporting the beneficial effects found under stress conditions. In a reporter gene approach using individual living worms the expression of the stress-inducible glutathione S-transferase 4 was shown to be reduced by EGb761 under physiological conditions as well as under oxidative stress. EGb761 also led to a decrease in transcription of the stress-inducible catalase genes. These results suggest that the beneficial impact of EGb791 on resistance to thermal stress and lifespan in C. elegans is at least partially due to its ability to relieve oxidative stress.

Morphogenesis, adhesive properties, and antifungal resistance depend on the Pmt6 protein mannosyltransferase in the fungal pathogen candida albicans.

Protein mannosyltransferases (Pmt proteins) initiate O glycosylation of secreted proteins in fungi. We have characterized PMT6, which encodes the second Pmt protein of the fungal pathogen Candida albicans. The residues of Pmt6p are 21 and 42% identical to those of C. albicans Pmt1p and S. cerevisiae Pmt6p, respectively. Mutants lacking one or two PMT6 alleles grow normally and contain normal Pmt enzymatic activities in cell extracts but show phenotypes including a partial block of hyphal formation (dimorphism) and a supersensitivity to hygromycin B. The morphogenetic defect can be suppressed by overproduction of known components of signaling pathways, including Cek1p, Cph1p, Tpk2p, and Efg1p, suggesting a specific Pmt6p target protein upstream of these components. Mutants lacking both PMT1 and PMT6 are viable and show pmt1 mutant phenotypes and an additional sensitivity to the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The lack of Pmt6p significantly reduces adherence to endothelial cells and overall virulence in a mouse model of systemic infection. The results suggest that Pmt6p regulates a more narrow subclass of proteins in C. albicans than Pmt1p, including secreted proteins responsible for morphogenesis and antifungal sensitivities.

Multiple functions of Pmt1p-mediated protein O-mannosylation in the fungal pathogen Candida albicans.

Protein mannosylation by Pmt proteins initiates O-glycosylation in fungi. We have identified the PMT1 gene and analyzed the function of Pmt1p in the fungal human pathogen Candida albicans. Mutants defective in PMT1 alleles lacked Pmt in vitro enzymatic activity, showed reduced growth rates, and tended to form cellular aggregates. In addition, multiple specific deficiencies not known in Saccharomyces cerevisiae (including defective hyphal morphogenesis; supersensitivity to the antifungal agents hygromycin B, G418, clotrimazole, and calcofluor white; and reduced adherence to Caco-2 epithelial cells) were observed in pmt1 mutants. PMT1 deficiency also led to faster electrophoretic mobility of the Als1p cell wall protein and to elevated extracellular activities of chitinase. Homozygous pmt1 mutants were avirulent in a mouse model of systemic infection, while heterozygous PMT1/pmt1 strains showed reduced virulence. The results indicate that protein O-mannosylation by Pmt proteins occurs in different fungal species, where PMT1 deficiency can lead to defects in multiple cellular functions.

A yeast phosphofructokinase insensitive to the allosteric activator fructose 2,6-bisphosphate. Glycolysis/metabolic regulation/allosteric control.

In this work we used in vitro mutagenesis to modify the allosteric properties of the heterooctameric yeast phosphofructokinase. Specifically, we identified two amino acids involved in the binding of the most potent allosteric activator fructose 2,6-bisphosphate. Thus, Ser724 was replaced by an aspartate and His859 by a serine in each of the enzyme subunits. Whereas the substitutions had no drastic effects when introduced only in one of the two types of subunits, kinetic parameters were modified when both subunits carried the mutation. Thus, the enzyme with His859 --> Ser showed an increase in Ka for binding of the activator, whereas the one with Ser724 --> Asp failed to react to the addition of fructose 2, 6-bisphosphate, at all. The enzymes still responded to other allosteric activators, such as AMP. Stabilities of the mutant subunits were not significantly altered in vivo, as judged from Western blot analysis. Phenotypically, strains expressing the mutant PFK genes showed a pronounced effect on the level of intermediary metabolites after growth on glucose. Mutants not responding to the activator at all (Ser724 --> Asp) also displayed higher generation times on glucose medium. This could be suppressed by increasing the gene dosage of the mutant alleles. These results indicate that fructose 2,6-bisphosphate through its activation of phosphofructokinase plays an important role in regulation of the glycolytic flux.