Fate and role of peroxisomes during the life cycle of the yeast Saccharomyces cerevisiae: inheritance of peroxisomes during meiosis

 Sporulation in the yeast Saccharomyces cerevisiae is a meiotic developmental process that occurs in MAT a/MATα heterozygotes in response to nutrient deprivation. Here, the fate and role of peroxisomes during sporulation and germination has been examined by a combination of immunoelectron microscopy and the use of pex mutants defective in peroxisomal functions. Using a green fluorescent protein probe targeted to peroxisomes we show that peroxisomes are inherited through meiosis and that they do not increase in number either during sporulation or spore germination. In addition, there is no requirement for peroxisome degradation prior to spore packaging. Unlike the situation in filamentous fungi, peroxisomes do not proliferate during the yeast life cycle. Functional peroxisomes are dispensable for efficient meiotic development on acetate medium since homozygous Δpex6 diploids sporulated well and produced mature spores that were resistant to diethyl ether. Like haploids, diploid cells can proliferate their peroxisomes in response to oleate as sole carbon source in liquid medium, but under these conditions they do not sporulate. On solid oleate medium, homozygous pex5,Δpex6, and pex7 cells were unable to sporulate efficiently, whereas the wild type was. The results presented here are discussed in terms of the transmission of organelles to progeny cells. Accepted: 19 December 1997     

Quantum-dye labeled proteins for glycobiology: a viable nonradioactive alternative tracer

Quantum dye (QD), a macrocyclic europium-chelate, developed as a cytological marker, has never been used for quantitative applications. It would be ideal, however, if the same tracer can be used for both qualitative and quantitative purposes. We have labeled some lectins and neoglycoproteins with QD for the purpose of quantitative analyses in glycobiology, and tested its suitability in three different areas in glycobiology: (1) glycosyltransferase, (2) an animal lectin - mannose- binding protein, and (3) the Gal/GalNAc receptor of rat liver membrane. Usefulness of QD-labeled lectins was amply demonstrated by the quantification of galactosyltransferase activity using QD-soybean agglutinin and QD-RCA120 ( Ricinus communis agglutinin). We also showed that QD-labeled neoglycoproteins, QD-Man-BSA and QD-Gal-BSA, can replace radioiodinated counterparts in the binding assays of animal lectins (serum mannose binding protein and hepatic Gal/GalNAc receptor.) The advantage of QD and other europium labels is that it does not decay as radioiodides do. The long shelf-life results in more consistent results from repeated experiments.      

Evidence for the existence of independent chloromethane- and S-adenosylmethionine-utilizing systems for methylation in Phanerochaete chrysosporium.

O methylation of acetovanillone at 4 position by C2H3Cl and S-adenosyl[methyl-2H3]methionine was monitored in whole mycelia of Phanerochaete chrysosporium in the presence and absence of S-adenosylhomocysteine. Both the amount of the methylation product, 3,4-dimethoxyacetophenone, and the percent C2H3 incorporation into the 4-methoxyl group of the compound were determined. The results strongly suggest the presence of biochemically distinct systems for O methylation of acetovanillone utilizing S-adenosylmethionine and chloromethane, respectively, as the methyl donor. The S-adenosylmethionine-dependent enzyme is induced early in the growth cycle, with activity attaining an initial maximum after 55 h of incubation. Methylation by this enzyme is totally suppressed by 1 mM S-adenosylhomocysteine over almost the entire growth cycle. S-Adenosylmethionine-dependent O-methyltransferase activity is detectable in cell extracts, and the purification and characterization of the enzyme are described elsewhere (C. Coulter, J. T. Kennedy, W. C. McRoberts, and D. B. Harper, Appl. Environ. Microbiol. 59:706-711, 1993). The chloromethane-utilizing methylation system is absent in early growth but attains peak activity in the mid-growth phase after 72 h of incubation. The system is not significantly inhibited by S-adenosylhomocysteine at any stage of growth. No chloromethane-dependent O-methyltransferase activity is detectable in cell extract, suggesting that the enzyme is membrane bound and/or part of a multienzyme complex. Although the biochemical role of the chloromethane-dependent methylation system in metabolism is not known, one possible function could be the regeneration of veratryl alcohol degraded by the attack of lignin peroxidase.     

Effects of wetland connectivity on overwintering and movement behaviours of Australian freshwater turtles

Freshwater turtles are important consumers in Australian freshwater ecosystems. They serve as scavengers, nutrient regulators, and as food sources and Totems for Traditional Owners throughout Australia. Despite their importance, most Australian freshwater turtle species are declining. The impact of winter wetland drying on turtle populations remains unknown, and winter exposure of hibernating turtles may be an important additional source of mortality. We aimed to examine turtle responses to seasonal and episodic wetland drying in wetlands using acoustic telemetry and active trapping. Wetlands were chosen that spanned a range of hydrological connectivity to the adjacent Edward/Kolety-Wakool River. We found that tagged Emydura macquarii typically exit wetlands disconnected from the adjacent permanent river prior to winter, and overwinter in the river. Female E. macquarii rapidly re-entered ‘home’ wetlands (wetlands in which they were initially tagged) the following spring, whereas males tended to leave the study area, returning occasionally. Although we were not able to evaluate a winter drying event, one of the wetlands experienced partial summer drying. All three local turtle species (E. macquarii, Chelodina expansa, C. longicollis) exited the wetland long before winter drying would have become a potential threat. Our results suggest that turtles in this system may be protected from winter wetland drying because they move to the adjacent permanent river prior to winter. Spending the winter in the river channel reduces the risks of being trapped in a drying wetland as temperatures drop in winter.     

Column-switching high-performance liquid chromatographic method for the determination of a thymidylate synthase inhibitor, LY231514, an investigational agent for the treatment of solid tumors, in human plasma

A reversed-phase, column-switching high-performance liquid chromatographic (HPLC) method is described for the determination of a new thymidylate synthase inhibitor in human plasma. The compound and an internal standard are extracted from plasma using a Certify II solid-phase cartridge. Extracts are evaporated to dryness and the residue is reconstituted with mobile phase buffer. The analytes are separated from polar interferences and buffer salts originating from the elution step on a 4-mm YMC Basic pre-column. The fraction containing the analytes is further separated on a 25-cm YMC Basic column. The analytes are detected by their absorbance at 250 nm. The limit of quantitation is 10 ng/ml. The method is linear from 10 ng/ml to 80 μg/ml using three standard curve ranges. Validation studies for all three ranges show the method to be reproducible. The method has been successfully used to support pharmacokinetic studies.     

A Method for Combined C-Banding and Silver Staining

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.     

Isolation and characterization of porcine parathyroid cathepsin B.

Cathepsin B was isolated from porcine parathyroid tissue and from liver by a procedure involving acetone precipitation, gel filtration, and carboxymethylcellulose chromatography. The final preparations of each migrated as single bands upon sodium dodecyl sulfate polyacrylamide gels but exhibited several minor active variants upon isoelectric focusing. The parathyroid and liver enzymes were similar to each other and also resembled cathepsin B from other sources. The molecular weights for the porcine enzymes were estimated as 25,000, and the isoelectric point was at pH 4.8. The parathyroid enzyme cleaved benzyloxycarbonyl-Val-Lys-Lys-Arg-(4-methoxy)-2-naphthylamide at pH 5.8 and 37 degrees C with a Km of 0.14 mM and a kcat of 68 s-1. The pH optimum for this reaction was pH 6 to 7. The enzyme was unstable above pH 7.5 and below pH 4.5. It was strongly inhibited by HgCl2, ZnSO4, iodoacetate, iodoacetamide, and N-ethylmaleimide which indicated that it is a thiol protease, and by leupeptin, a strong inhibitor of cathepsin B from other sources. Antibodies to the parathyroid enzyme were elicited in rabbits. The antisera formed single precipitin bands upon double diffusion in agar gels against both the parathyroid and liver enzymes. Precipitin bands were formed at both pH 6 and pH 8.5 which indicated that the antisera recognized both native and denatured forms of the enzymes.     

M?ssbauer studies of cytochrome c' from Rhodospirillum rubrum.

Cytochrome c' from Rhodospirillum rubrum has been investigated in the ferric form with M?ssbauer and EPR spectroscopy. In the pH range from 6 to 9.5, three species are observed which belong to two pH-dependent equilibria with pK values near 6 and 8.5. The pK = 6 transition is resolved only with high-field M?ssbauer spectroscopy. For the three species we have determined the zero-field splitting parameters and the hyperfine coupling constants. The data were fitted to a spin Hamiltonian which takes into account a weak mixing of excited S = 3/2 states into the sextet ground manifold. The low temperature spectra clearly show that the quadruple coupling constant deltaEQ is positive for ferricytochrome c' and thus in accord with all other high-spin ferric heme proteins.