Biomedical subjects

Z Petek Cakar

Publications and source records attributed to Z Petek Cakar.

5 recordsLinked to original sources

PubMed2005

Accumulation of polyhydroxyalkanoates by Microlunatus phosphovorus under various growth conditions.

Microlunatus phosphovorus is an activated-sludge bacterium with high levels of phosphorus-accumulating activity and phosphate uptake and release activities. Thus, it is an interesting model organism to study biological phosphorus removal. However, there are no studies demonstrating the polyhydroxyalkanoate (PHA) storage capability of M. phosphovorus, which is surprising for a polyphosphate-accumulating organism. This study investigates in detail the PHA storage behavior of M. phosphovorus under different growth conditions and using different carbon sources. Pure culture studies in batch-growth systems were conducted in shake-flasks and in a bioreactor, using chemically defined growth media with glucose as the sole carbon source. A batch-growth system with anaerobic-aerobic cycles and varying concentrations of glucose or acetate as the sole carbon source, similar to enhanced biological phosphorus removal processes, was also employed. The results of this study demonstrate for the first time that M. phosphovorus produces significant amounts of PHAs under various growth conditions and with different carbon sources. When the PHA productions of all cultivations were compared, poly(3-hydroxybutyrate) (PHB), the major PHA polymer, was produced at about 20-30% of the cellular dry weight. The highest PHB production was observed as 1,421 mg/l in batch-growth systems with anaerobic-aerobic cycles and at 4 g/l initial glucose concentration. In light of these key results regarding the growth physiology and PHA-production capability of M. phosphovorus, it can be concluded that this organism could be a good candidate for microbial PHA production because of its advantages of easy growth, high biomass and PHB yield on substrate and no significant production of fermentative byproducts.

Acetic Acid↗

PubMed2005

Evolutionary engineering of multiple-stress resistant Saccharomyces cerevisiae.

Various selection procedures in chemostats and batch cultures were systematically tested for their efficiency to select for a multiple-stress resistance phenotype in Saccharomyces cerevisiae. To determine the relative stress resistance phenotypes, mutant populations harvested at different time points and randomly chosen clones from selected populations were grown in batch cultures and exposed to oxidative, freezing-thawing, high-temperature and ethanol stress. For this purpose, we developed a high-throughput procedure in 96-well plates combined with a most-probable-number assay. Among all chemostat and batch selection strategies tested, the best selection strategy to obtain highly improved multiple-stress-resistant yeast was found to be batch selection for freezing-thawing stress. The final mutant populations selected for this particular stress were not only significantly improved in freezing-thawing stress resistance, but also in other stress resistances. The best isolated clone from these populations exhibited 102-, 89-, 62-, and 1429-fold increased resistance to freezing-thawing, temperature, ethanol, and oxidative stress, respectively. General selection guidelines for improving multiple-stress resistance in S. cerevisiae are presented and discussed.

Culture Media↗

PubMed2004

LDL transcytosis by protein membrane diffusion.

Endothelial cell (EC) cultures of different, selected vascular beds and/or organs were screened for receptor-mediated transport of proteins with a semipermeable filter assay. In SVEC4-10 cells, a mouse lymphoid endothelial cell line, orosomucoid, albumin, insulin and LDL were transcytosed from the apical (luminal) to basal (abluminal) side by a receptor-mediated pathway. Specific LDL transcytosis involved transport of intact LDL. A pathway of degradation of LDL and basal release involved vesicles in transport to lysosomes and amino acid merocrine secretion. This newly described transcellular passage of LDL via lysosomes, as well as the standard pathway, were reduced to 70% by PEG(50)-cholesterol (PEG-Chol). Combined results of temperature-dependence analysis and PEG(50)-cholesterol sensitivity show that two pathways contribute to general LDL transcellular passage. We suggest a mechanism of domain hopping by protein membrane diffusion of receptors as the pathway for intact LDL delivery. Based on theoretical considerations we propose that active transport by protein membrane diffusion can be facilitated by an organizational structure of lipid microdomains and polar cellular organization.

Animals↗

PubMed2004

Caveolae: biochemical analysis.

Caveolae appear in a multitude of processes encompassing growth regulation and trafficking. We demonstrate the abundant presence of ESA/reggie-1/flotillin-2, ATP synthase beta subunit and annexin V in endothelial caveolae by immunopurification of caveolae from vascular endothelial membrane. Five proteins are abundant in a caveolin-1 protein complex, analyzed by sucrose gradient velocity sedimentation following octyl-beta-D-glucopyranoside extraction. Caveolin-1 alpha interacts with caveolin-1beta, caveolin-2, actin, the microsomal form of NADH cytochrome B5 reductase and ESA/reggie-1/flotillin-2 as shown by co-immunoprecipitation. We propose the concept that ATP biosynthesis in caveolae regulates mechanosignaling and is induced by membrane depolarization and a proton gradient. Pressure stimuli and metabolic changes may trigger gene regulation in endothelial cells, involving a nuclear conformer of caveolin-1, shown here with an epitope-specific caveolin-1 antibody, and immediate response of ion channel activity, regulated by ESA/reggie-1/flotillin-2.

Animals↗

PubMed2003

Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis.

The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.

Amino Acids↗