A comprehensive method for the quantification of the non-oxidative pentose phosphate pathway intermediates in Saccharomyces cerevisiae by GC-IDMS.

A gas chromatography isotope dilution mass spectrometry (GC-IDMS) method was developed for the quantification of the metabolites of the non-oxidative part of pentose phosphate pathway (PPP). A mid-polar GC column (Zebron ZB-AAA, 10m, film composition 50% phenyl 50% dimethyl polysiloxane) was used for the chromatographic separation of the intermediates. The optimized GC-MS procedure resulted in improved separation performances and higher sensitivities compared to previous methods. Furthermore, the use of (13)C-labeled cell extracts as internal standards improved the data quality and eliminated the need to perform a recovery check for each metabolite. The applicability of the new method was demonstrated by analyzing intracellular metabolite levels in samples derived from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae at steady state as well as following a short-term glucose pulse. The major achievements of the proposed quantitative method are the independent quantification of the epimers ribulose-5-phosphate and xylulose-5-posphate and the measurement of compounds present at very low concentrations in biological samples such as erythrose-4-phosphate and glyceraldehyde-3-phosphate.

Detection and determination of glyceraldehyde-derived pyridinium-type advanced glycation end product in streptozotocin-induced diabetic rats.

GLAP, glyceraldehyde-derived pyridinium-type advanced glycation end product (AGE), formed by glyceraldehyde-related glycation, was identified in the plasma protein and the tail tendon collagen of streptozotocin (STZ)-induced diabetic rats. It was detected in the plasma protein and the collagen in diabetic rats by LC-MS and LC-MS/MS analysis, but was not detected in normal rats. In addition, GLAP was formed from glyceraldehyde-3-phosphate (GA3P) with lysine as well as glyceraldehyde (GLA) with lysine in vitro. Accordingly, it is suggested that an increase in the GLAP level reflects an increase in the GLA level and the GA3P level. GLAP might be a biomarker for reduced activity of the glyceraldehyde-related enzymes in the metabolic diseases such as diabetic complications.

A potential role of the cytoskeleton of Saccharomyces cerevisiae in a functional organization of glycolytic enzymes.

Numerous individual enzymes participate in a given synthetic or degradative pathway in which the product of one reaction becomes the substrate for the subsequent enzyme. This raises the question of whether the product of one 'soluble' enzyme diffuses freely through the available cell volume, where it accidentally collides with the subsequent 'soluble' enzyme. Alternatively, enzymes acting in a given pathway may be organized in ordered structures, metabolons. Certain glycolytic enzymes have been shown to co-localize with the cytoskeleton in mammalian cells. We deleted genes coding for proteins associated with the cytoskeleton of Saccharomyces cerevisiae: TPM1 coding for tropomyosin, SAC6 for fimbrin and CIN1 for a microtubule-associated protein. Single deletions or deletions of two such genes had no effect on the specific activities of glycolytic enzymes, or on the rates of glucose consumption and ethanol production. However, the concentrations of glycolytic metabolites during a switch from a gluconeogenic mode of metabolism, growth on an ethanol medium, to glycolysis after glucose addition showed transient deviations from the normal change in metabolite concentrations, as observed in wild type cells. However, all metabolites in mutant strains reached wild-type levels within 2-4 h after the shift. Only ATP levels remained low in all but the tmp1-Delta-sac6-Delta double mutant strains. These observations can be interpreted to mean that metabolic reorganization from a gluconeogenic to a glycolytic metabolism is facilitated by an intact cytoskeleton in yeast.

Sampling tube device for monitoring intracellular metabolite dynamics.

Continuous sampling of microorganisms from a controlled bioreactor with rapid inactivation of metabolism and extraction of metabolites using precooled -40 degrees C perchloric acid solution (35%) was achieved with a sampling tube, thus fixing fast dynamic reactions at a certain position in the tube. After sampling was stopped (200 s) the tube was frozen at -80 degrees C and divided into identical parts and the extracted metabolites were analyzed enzymatically. A high resolution in time was achieved due to the axial dispersion of the metabolites in the sampling tube: The events of 1 s in the cells of the reactor were represented by 15 parts of the sampling tube. Axial dispersion was determined quantitatively with tracer measurements. The performance of the sampling tube device was evaluated with dynamic investigations on glucose-metabolism of Zymomonas mobilis. The dynamics of intracellular glucose 6-phosphate, glyceraldehyde 3-phosphate, and 3-phosphoglycerate concentrations were monitored after adding a glucose pulse to a glucose-limited steady-state culture.

Studies on the mechanism of early onset macular degeneration in cynomolgus (Macaca fascicularis) monkeys. I. Abnormal concentrations of two proteins in the retina.

Cynomolgus (Macaca fascicularis) monkeys from three families, which showed symptoms of early onset macular degeneration was studied. Two proteins, albumin and glyceraldehyde 3-phosphate dehydrogenase, were found to have markedly altered concentrations in whole retina of the monkeys with early onset macular degeneration, compared with normal controls. SDS-polyacrylamide gel patterns detected a 40-70% increase in the concentration of albumin and about 65% decrease in the concentration of glyceraldehyde 3-phosphate dehydrogenase in these affected retinas. There was however no significant difference in the relative concentrations of albumin in the plasma samples of affected and normal monkeys belonging to the three families studied and to an unrelated family. These initial findings suggest that degradative as well as antioxidant enzymes might be involved in the mechanisms leading to macular degeneration. In addition, the results also correlate with a possible role of these two proteins in H2O2 toxicity and appear to indicate that oxidative stress is significant in the etiology of early onset macular degeneration.

Riboprobe expression cassettes for measuring IGF-I, beta-actin and glyceraldehyde 3-phosphate dehydrogenase transcripts.

The development of riboprobe expression cassettes for phosphorimager-based quantitation of steady-state transcripts for three different genes using solution hybridization, RNase protection assays is described. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin genes are widely used as reporter genes to estimate the amount and integrity of RNA as well as for comparing gene expression among different tissues. To directly compare expression of these two genes in lymphoid tissue and liver, cDNA fragments of beta-actin and GAPDH from both mice and rats were generated by RT-PCR and cloned together into pGEM1 under control of the T7 RNA polymerase promoter. Antisense transcripts from this fusion construct protected the appropriate-sized fragments of beta-actin (115 nt) and GAPDH (214 nt) in RNA isolated from rat spleen, thymus and liver. Expression of GAPDH transcripts was less variable across tissues because this mRNA was only two-fold lower in liver as compared to either thymus or spleen, whereas expression of beta-actin transcripts was eight-fold lower in liver than in these tissues. Two other riboprobe expression cassettes (IGF-I/actin) were constructed by ligating a cDNA fragment of mouse or rat beta-actin that would protect 115 nt to either a mouse or rat IGF-I genomic DNA fragment containing 182 bp of exon 4. These mouse and rat IGF-I/actin riboprobes were used to conclusively demonstrate that rat CSF-1-derived bone marrow macrophages, mouse elicited peritoneal macrophages and the murine PU5-1R macrophage cell line synthesize abundant transcripts for both IGF-I and beta-actin. However, the mouse M1 progenitor myeloid cell line does not express RNA for IGF-I, as demonstrated by the absence of protected transcripts for IGF-I in the presence of abundant protected transcripts for beta-actin. Phosphorimager scanning of the gels revealed that macrophages of both mice and rats express IGF-I transcripts at a level of 60-100% of those found in liver. These data show that a single riboprobe can be developed to generate multigene antisense RNAs that can then be used to quantitatively compare IGF-I transcripts in macrophages and other tissues to an internal standard, with GAPDH transcripts being less variable among tissues than those for beta-actin. This approach should be broadly applicable for measuring a variety of markers of cellular activation.