The complementation of yeast with human or Plasmodium falciparum Hsp90 confers differential inhibitor sensitivities

Developing novel drugs against the unicellular parasite Plasmodium is complicated by the paucity of simple screening systems. Heat-shock proteins are an essential class of proteins for the parasite's cyclical life style between different cellular milieus and temperatures. The molecular chaperone Hsp90 assists a large variety of proteins, but its supporting functions for many proteins that are important for cancer have made it into a well-studied drug target. With a better understanding of the differences between Hsp90 of the malarial parasite and Hsp90 of its human host, new therapeutic options might become available. We have generated a set of isogenic strains of the budding yeast Saccharomyces cerevisiae where the essential yeast Hsp90 proteins have been replaced with either of the two human cytosolic isoforms Hsp90 alpha or Hsp90 beta, or with Hsp90 from Plasmodium falciparum (Pf). All strains express large amounts of the Flag-tagged Hsp90 proteins and are viable. Even though the strain with Pf Hsp90 grows more poorly, it provides a tool to reconstitute additional aspects of the parasite Hsp90 complex and its interactions with substrates in yeast as a living test tube. Upon exposure of the set of Hsp90 test strains to the two Hsp90 inhibitors radicicol (Rd) and geldanamycin (GA), we found that the strain with Pf Hsp90 is relatively more sensitive to GA than to Rd compared to the strains with human Hsp90's. This indicates that this set of yeast strains could be used to screen for new Pf Hsp90 inhibitors with a wider therapeutic window.

Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalentlv associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hGG alpha and hCG beta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Go-expression of both subunits in the same cell resulted in secretion of heterodimeric hGG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hGG. The le level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermenter. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hGG and is suitable for structure-function studies of the hormone.

Peculiar cytological behaviour of a distillery yeast

The question whether so-called ‘pure’ strains of yeast are cytologically pure ought to receive the earnest attention of those engaged in the study of the genetics of yeasts. The classification of yeasts is purely arbitrary, and the only reliable method of obtaining any particular species is to get a sample of the original culture. But even if the original culture is available one is not sure that it is cytologically pure, for proportion changes might have occurred in it since isolation. In rapidly growing organisms like the yeasts this is but natural. Investigations on higher plants indicate that polyploids usually mutate to dwarfness as a survival-measure and hence the random size relationships between the diploids and the polyploids offer no morphological criterion for differentiation into types.

Some cytoplasmic details of yeast revealed by the electron microscope

Abstract is not available.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a modelantigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection.Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.

Triacylglycerol lipases of yeast and plants: more than just hydrolases

In the yeast, mobilization of triacylglycerols (TAG) is facilitated by TGL3, TGL4 and TGL5 gene products. Interestingly, experiments using [32P] orthophosphate as a precursor for complex glycerophospholipids revealed that tgl mutants had a lower steady-state level of these membrane lipids. To understand a possible link between TAG lipolysis and phospholipid metabolism, we performed overexpression studies with Tgl3p and Tgl5p which clearly demonstrated that these two enzymes enhanced the level of phospholipids. Domains and motifs search analyses indicated that yeast TAG hydrolases posses a GXSXG lipase motif but also a HX4D acyltransferase motif. Purified Tgl3p and Tgl5p did not only exhibit TAG lipase activity but also catalyzed acyl-CoA dependent acylation of lyso-phosphatidylethanolamine and lyso-phosphatidic acid (LPA), respectively. Search for lipase/hydrolase homologues in the Arabidopsis thaliana genome led to the identification of At4g24160 which possess three motifs that are conserved across the plant species such as GXSXG motif, a HX4D motif and a probable lipid binding motif V(X)3HGF. Characterization of At4g24160 expressed in bacteria revealed that the presence of an acyl-CoA dependent LPA acyltransferase activity. In addition, the purified recombinant At4g24160 protein hydrolyzed both TAG and phosphatidylcholine. We hypothesize that the plant enzyme may be involved in membrane repair. In summary, our results indicate that these TAG lipases play a dual role and thereby contribute to both anabolic and catabolic processes in yeast and plants.

Nature of induction of tryptophan pyrrolase in cold exposure

The activity of hepatic tryptophan pyrrolase in rats exposed to cold increased rapidly and reached a maximum of three-fold at 8 h. On continued exposure up to 48 h stress, the activity partly decreased but remained at a level higher than the initial. Withdrawal from the cold stress reversed the change. Adrenalectomy or treatment with inhibitors of protein synthesis abolished the increase in the enzyme activity during cold stress indicating a possible involvement of corticosteroids and de novo protein synthesis. Treatment with drugs known to block autonomic nervous system failed to inhibit the cold-mediated increase in enzyme activity. The results suggest that the increase in enzyme activity obtained on cold exposure is mediated by corticosteroids and not by either indoleaklylamines or autonomic nervous system. The changes in the enzyme obtained under cold stress with respect to the overshoot phenomenon, relationship to the degree of stress and reversibility on withdrawal from the stress indicate the "adaptate" nature of the response.

On the roles of the northeast cold surge, the Borneo vortex, the Madden-Julian Oscillation, and the Indian Ocean Dipole during the extreme 2006/2007 flood in southern Peninsular Malaysia

The mid-December 2006 to late January 2007 flood in southern Peninsular Malaysia was the worst flood in a century and was caused by three extreme precipitation episodes. These extreme precipitation events were mainly associated with strong northeasterly winds over the South China Sea. In all cases, the northeasterlies penetrated anomalously far south and followed almost a straight trajectory. The elevated terrain over Sumatra and southern Peninsular Malaysia caused low-level convergence. The strong easterly winds near Java associated with the Rossby wave-type response to Madden-Julian Oscillation (MJO) inhibited the counter-clockwise turning of the northeasterlies and the formation of the Borneo vortex, which, in turn, enhanced the low-level convergence over the region. The abrupt termination of the Indian Ocean Dipole (IOD) in December 2006 played a secondary role as warmer equatorial Indian Ocean helped in the MJO formation.

Effect of cold exposure on the metabolism of ubiquinone in the rat

1. Accumulation of ubiquinone in the livers of rats exposed to a cold environment was shown to be due to both decreased catabolism during the entire experimental period and increased synthesis during an intermediate stage (10–20 days). 2. The increased endogenous synthesis in the cold-exposed rats was eliminated when ubiquinone accumulated in the liver after exposure for 40 days (coinciding with cclimatization), or by absorption of the exogenous dietary supply, possibly by the mechanism of end-product regulation.

Characteristics of yeasts isolated from phenol- and catechol-adapted activated sludges

Phenol- and catechol-adapted sludges contained large numbers of the yeasts, Candida tropicalis and Trichosporon cutaneum. Both were able to grow on a variety of aromatic compounds and utilized phenol and catechol at a high rate. This property was inducible. The feasibility of using these yeasts for removing phenols from waste waters is suggested.

Differential fluorescence of the chromocenters and nucleolar equivalents of the yeast nucleus in acridine orange

The nucleus with its limiting membrane and organelles was visible in the majority of the yeast cells stained vitally with the fluorochrome, acridine orange, at a dilution of 1 in 40,000. The intra-nuclear structures could be distinguished by their differential fluorescence. The chromocenters were green while the nucleolar equivalents were orange. The vacuole showed no fluorescence.

Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae

Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast triacylglycerol (TAG) lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction K. Athenstaedt and G. Daum, J. Biol. Chem. 278 (2003) 23317-23323]. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out metabolic labeling experiments with wild type and deletion strains bearing defects in the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Using H-3]inositol. P-32]orthophosphate, 3H]palmitate and C-14]acetate as precursors for complex lipids we demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Activity and expression level of fatty acid elongases, Elo1p and Elo2p were enhanced as a consequence of reduced TAG lipolysis. Finally, the pattern of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine molecular species was altered in tgl deletion strain underlining the important role of TAG turnover in maintaining the pool size of these compounds and the remodeling of complex membrane lipids. (C) 2010 Elsevier B.V. All rights reserved.