For the investigation of the metabolic regulation of Saccharomyces cerevisiae under different culture conditions, the proteins of cell utilizing various carbon sources were separated by two-dimensional electrophoresis with immobilized pH gradients as the first dimension and SDS-PAGE as the second. Samples were taken in the log phase of batch culture using glucose or lactic acid as carbon source, while another sample was taken from the broth when glucose was consumed up and ethanol accumulated in the previous phase was further metabolized. After electrophoresis, the protein spots were detected by silver-stain in a Hoefer Automated Gel Stainer with a protein silver staining kit. Silver-stained gels were scanned and digitized to create computer images. About 500 protein spots were detected by employing the 2D proteome image analysis system Image Master 2D Elite and SWISS-2DPAGE proteome database. Most of the protein expressed and involved in the glycolysis, pentose phosphate (PP) pathway, anaplerotic pathway, as well as TCA cycle were analyzed. The metabolism regulation of protein level for Saccharomyces cerevisiae under various carbon sources, as well as during different phase of growth, was studied. The expression of several glycolytic enzymes (glk, pgi, pgk, eno, pyk) was up-regulated while the expression of enzymes in oxidative pentose phosphate pathway (zwf, gnd) was down-regulated when ethanol and lactic acid were taken as carbon source. Simultaneously, frucotose 1,6-biphosphatase was found to be up-regulated due to the gluconeogenic requirement. Citrate synthase and Malate dehydrogenase do not exhibit significant difference, indicating TCA cycle is necessary when utilizing glucose, ethanol or lactic acid as carbon source. Thus, the NADPH loss due to the repressed pentose phosphate pathway could be compensated by TCA cycle in cases of ethanol and lactic acid. The expression of malic enzyme and isocitrate lyase are activated to a large extent when metabolizing ethanol, indicating glyoxylate shunt is essential in transferring ethanol to generate four carbon precursors for the biosynthesis and the NADP-dependent malic enzyme could also serve as compensation mechanism for NADPH loss in this case.
Carbon ; metabolism ; Culture Media ; Culture Techniques ; methods ; Electrophoresis, Gel, Two-Dimensional ; methods ; Ethanol ; metabolism ; Lactic Acid ; metabolism ; Proteome ; analysis ; metabolism ; Proteomics ; Saccharomyces cerevisiae ; genetics ; growth & development ; metabolism ; Saccharomyces cerevisiae Proteins ; analysis ; metabolism

We report a case of 2X-year-old man with G4 chronic kidney disease (CKD G4). He had a decline in estimated glomerular filtration rate (eGFR) after the initiation of BEP (cisplatin, etoposide, bleomycin) therapy followed by VelP (ifosfamide, cisplatin, vinblastine) therapy for mixed germ cell testicular tumor. The patient’s eGFR did not recover during 59 months after chemotherapy, but showed a tendency to improve after treatment with yojinkodakuto. Yojinkodakuto was prepared by the IPCD (immersing powdered crude drugs) method. The IPCD method was adopted to ensure convenience and good quality prescription herbal medicines. Although eGFR fluctuated after yojinkodakuto treatment, at 3 years, serum creatinine (sCr) and eGFR progressed well, with sCr reducing from 3.98 mg/dL at initiation to 2.6 mg/dL and eGFR increasing from 16 mL/min/1.73 m2 to 25.4 mL/min/1.73 m2. In addition, a long-term eGFR plot analysis confirmed the 3-year improvement trend.