Analysis of the potential biological value of pyruvate dehydrogenase E1 subunitβin human cancer

BACKGROUND The pyruvate dehydrogenase E1 subunitβ(PDHB)gene which regulates energy metabolism is located in mitochondria.However,few studies have elucidated the role and mechanism of PDHB in different cancers.AIM To comprehensive pan-cancer analysis of PDHB was performed based on bioinformatics approaches to explore its tumor diagnostic and prognostic value and tumor immune relevance in cancer.In vitro experiments were performed to examine the biological regulation of PDHB in liver cancer.METHODS Pan-cancer data related to PDHB were obtained from the Cancer Genome Atlas(TCGA)database.Analysis of the gene expression profiles of PDHB was based on TCGA and Genotype Tissue Expression Dataset databases.Cox regression analysis and Kaplan-Meier methods were used to assess the correlation between PDHB expression and survival prognosis in cancer patients.The correlation between PDHB and receiver operating characteristic diagnostic curve,clinicopathological staging,somatic mutation,tumor mutation burden(TMB),microsatellite instability(MSI),DNA methylation,and drug susceptibility in pan-cancer was also analyzed.Various algorithms were used to analyze the correlation between PDHB and immune cell infiltration and tumor chemotaxis environment,as well as the co-expression analysis of PDHB and immune checkpoint(ICP)genes.The expression and functional phenotype of PDHB in single tumor cells were studied by single-cell sequencing,and the functional enrichment analysis of PDHB-related genes was performed.The study also validated the level of mRNA or protein expression of PDHB in several cancers.Finally,in vitro experiments verified the regulatory effect of PDHB on the proliferation,migration,and invasion of liver cancer.RESULTS PDHB was significantly and differently expressed in most cancers.PDHB was significantly associated with prognosis in patients with a wide range of cancers,including kidney renal clear cell carcinoma,kidney renal papillary cell carcinoma,breast invasive carcinoma,and brain lower grade glioma.In some cancers,PDHB expression was clearly associated with gene mutations,clinicopathological stages,and expression of TMB,MSI,and ICP genes.The expression of PDHB was closely related to the infiltration of multiple immune cells in the immune microenvironment and the regulation of tumor chemotaxis environment.In addition,single-cell sequencing results showed that PDHB correlated with different biological phenotypes of multiple cancer single cells.This study further demonstrated that down-regulation of PDHB expression inhibited the proliferation,migration,and invasion functions of hepatoma cells.CONCLUSION As a member of pan-cancer,PDHB may be a novel cancer marker with potential value in diagnosing cancer,predicting prognosis,and in targeted therapy.

Glycolytic potential enhanced by blockade of pyruvate influx into mitochondria sensitizes prostate cancer to detection and radiotherapy

Objective:This study aimed to evaluate the effects of mitochondrial pyruvate carrier(MPC)blockade on the sensitivity of detection and radiotherapy of prostate cancer(PCa).Methods:We investigated glycolysis reprogramming and MPC changes in patients with PCa by using metabolic profiling,RNASeq,and tissue microarrays.Transient blockade of pyruvate influx into mitochondria was observed in cellular studies to detect its different effects on prostate carcinoma cells and benign prostate cells.Xenograft mouse models were injected with an MPC inhibitor to evaluate the sensitivity of 18F-fluorodeoxyglucose positron emission tomography with computed tomography and radiotherapy of PCa.Furthermore,the molecular mechanism of this different effect of transient blockage towards benign prostate cells and prostate cancer cells was studied in vitro.Results:MPC was elevated in PCa tissue compared with benign prostate tissue,but decreased during cancer progression.The transient blockade increased PCa cell proliferation while decreasing benign prostate cell proliferation,thus increasing the sensitivity of PCa cells to 18F-PET/CT(SUVavg,P=0.016;SUVmax,P=0.03)and radiotherapy(P<0.01).This differential effect of MPC on PCa and benign prostate cells was dependent on regulation by a VDAC1-MPC-mitochondrial homeostasis-glycolysis pathway.Conclusions:Blockade of pyruvate influx into mitochondria increased glycolysis levels in PCa but not in non-carcinoma prostate tissue.This transient blockage sensitized PCa to both detection and radiotherapy,thus indicating that glycolytic potential is a novel mechanism underlying PCa progression.The change in the mitochondrial pyruvate influx caused by transient MPC blockade provides a critical target for PCa diagnosis and treatment.

Effects of Ethyl Pyruvate on Myocardial Apoptosis and Expression of Bcl-2 and Bax Proteins after Ischemia-reperfusion in Rats

In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion （I/R） in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups （n=8 in each group）： control group was perfused for 120 min. In the I/R group, after 30 min stabilization the injury was induced by 30 min global ischemia followed by 60 min reperfusion. Ethyl pyruvate （EP） group was set up with the same protocol as I/R group except that it was supplied with 2 mmol/L EP 15 rain before ischemia and throughout reperfusion. Myocardial malonaldehyde （MDA） content was measured. Myocardial apoptotic index （AI） was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling （TUNEL） method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group （P〈0.05）. These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.

Delayed ethyl pyruvate therapy attenuates experimental severe acute pancreatitis via reduced serum high mobility group box 1 levels in rats

AIM： To investigate the effect of delayed ethyl pyruvate （EP） delivery on distant organ injury, survival time and serum high mobility group box 1 （HMGB1） levels in rats with experimental severe acute pancreatitis （SAP）. METHODS： A SAP model was induced by retrograde injection of artificial bile into the pancreatic ducts of rats. Animals were divided randomly into three groups （n = 32 in each group）： sham group, SAP group and delayed EP treatment group. The rats in the delayed EP treatment group received EP （30 mg/kg） at 12 h, 18 h and 30 h after induction of SAP. Animals were sacrificed, and samples were obtained at 24 h and 48 h after induction of SAP. Serum HMGB1, aspartate arninotransferase （AST）, alanine arninotransferase （ALT）, blood urea nitrogen （BUN）, and creatinine （Cr） levels were measured. Lung wet-to-dry-weight （W/D） ratios and histological scores were calculated to evaluate lung injury. Additional experiments were performed between SAP and delayed EP treatment groups to study the influence of EP on survival times of SAP rats. RESULTS： Delayed EP treatment significantly reduced serum HMGB1 levels, and protected against liver, renal and lung injury with reduced lung W/D ratios （8.22 ±0.42 vs 9.76 ± 0.45, P 〈 0.01）, pulmonary histological scores （7.1 ± 0.7 vs 8.4 ± 1.1, P 〈 0.01）, serum AST （667 ± 103 vs 1 368 ± 271, P 〈 0.01）, ALT （446 ± 91 vs 653 ± 98, P 〈 0.01） and Cr （1.2 ± 0.3 vs 1.8 ± 0.3, P 〈 0.01） levels. SAP rats had a median survival time of 44 h. Delayed EP treatment significantly prolonged median survival time to 72 h （P 〈 0.01）. CONCLUSION： Delayed EP therapy protects against distant organ injury and prolongs survival time via reduced serum HMGBllevels in rats with experimental SAP. EP may potentially serve as an effective new therapeutic option against the inflammatory response and multiple organ dysfunction syndrome （MODS） in SAP patients.

Signal transducer and activator of transcription 3 promotes the Warburg effect possibly by inducing pyruvate kinase M2 phosphorylation in liver precancerous lesions

BACKGROUND Study shows that signal transducer and activator of transcription 3(STAT3) can increase the Warburg effect by stimulating hexokinase 2 in breast cancer and upregulate lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 in myeloma. STAT3 and pyruvate kinase M2(PKM2) can also be activated and enhance the Warburg effect in hepatocellular carcinoma. Precancerous lesions are critical to human and rodent hepatocarcinogenesis. However, the underlying molecular mechanism for the development of liver precancerous lesions remains unknown. We hypothesized that STAT3 promotes the Warburg effect possibly by upregulating p-PKM2 in liver precancerous lesions in rats.AIM To investigate the mechanism of the Warburg effect in liver precancerous lesions in rats.METHODS A model of liver precancerous lesions was established by a modified Solt-Farber method. The liver pathological changes were observed by HE staining and immunohistochemistry. The transformation of WB-F344 cells induced with Nmethyl-N'-nitro-N-nitrosoguanidine and hydrogen peroxide was evaluated by the soft agar assay and aneuploidy. The levels of glucose and lactate in the tissue and culture medium were detected with a spectrophotometer. The protein levels of glutathione S-transferase-π, proliferating cell nuclear antigen(PCNA), STAT3,and PKM2 were examined by Western blot and immunofluorescence.RESULTS We found that the Warburg effect was increased in liver precancerous lesions in rats. PKM2 and p-STAT3 were upregulated in activated oval cells in liverprecancerous lesions in rats. The Warburg effect, p-PKM2, and p-STAT3 expression were also increased in transformed WB-F344 cells. STAT3 activation promoted the clonal formation rate, aneuploidy, alpha-fetoprotein expression,PCNA expression, G1/S phase transition, the Warburg effect, PKM2 phosphorylation, and nuclear translocation in transformed WB-F344 cells.Moreover, the Warburg effect was inhibited by stattic, a specific inhibitor of STAT3, and further reduced in transformed WB-F344 cells after the intervention for PKM2.CONCLUSION The Warburg effect is initiated in liver precancerous lesions in rats. STAT3 activation promotes the Warburg effect by enhancing the phosphorylation of PKM2 in transformed WB-F344 cells.

Ethyl pyruvate prevents inflammatory factors release and decreases intestinal permeability in rats with D-galactosamine-induced acute liver failure

BACKGROUND: The pathogenesis and progression of acute liver failure (ALF) are closely associated with intestinal endotoxemia because of the high permeability of the intestinal wall. Treatment with ethyl pyruvate (EP) has been shown to protect liver failure effectively. The current study aimed to explore the relationship between proinflammatory cytokines and intestinal permeability, and to investigate whether EP administration might prevent the release of multiple proinflammatory cytokines and decrease intestinal permeability and therefore, protect the liver from injury. METHODS: The ALF model was induced by D-galactosamine in rats. The rats were randomly divided into control (saline i.p.), model (D-galactosamine, 1.2 g/kg, i.p.), prevention [EP injection (40 mg/kg) 2 hours ahead of D-galactosamine] and treatment groups (EP injection 2 hours after D-galactosamine) Samples were obtained at 12 and 24 hours after ALF induction respectively. The histology of liver and intestinal tissue was accessed. Serum alanine aminotransferase, endotoxin, D(-) lactate, diamine oxidase (DAO), tumor necrosis factor-alpha (TNF-α), interferon-γ (IFN-γ) and high mobility group box-1 (HMGB1) were evaluated. The survival of rats was also recorded. RESULTS: The rats in model group showed severe damage to liver tissue and intestinal mucosa 12 and 24 hours after ALF induction. EP significantly improved liver or intestinal injury In addition, serum endotoxin, D(-)-lactate, DAO, TNF-α IFN-γ and HMGB1 levels were significantly increased in the model group compared with the control group. There was a positive correlation between intestinal permeability andproinflammatory cytokines. EP significantly reduced serum endotoxin, D(-)-lactate, DAO, TNF-α, IFN-γ and HMGB1 levels. The median survival time was significantly prolonged in both prevention and treatment groups (126 and 120 hours compared with 54 hours in the model group). CONCLUSIONS: EP has protective and therapeutic effects on intestinal mucosa. EP decreases intestinal permeability, and inhibits the release of multiple proinflammatory cytokines in rats with ALF.

Pyruvate is a prospective alkalizer to correct hypoxic lactic acidosis

Type A lactic acidosis resulted from hypoxic mitochondrial dysfunction is an independent predictor of mortality for critically ill patients. However, current therapeutic agents are still in shortage and can even be harmful. This paper reviewed data regarding lactic acidosis treatment and recommended that pyruvate might be a potential alkalizer to correct type A lactic acidosis in future clinical practice. Pyruvate is a key energy metabolic substrate and a pyruvate dehydrogenase(PDH) activator with several unique beneficial biological properties, including anti-oxidant and antiinflammatory effects and the ability to activate the hypoxia-inducible factor-1(HIF-1α)-erythropoietin(EPO) signal pathway. Pyruvate preserves glucose metabolism and cellular energetics better than bicarbonate, lactate, acetate and malate in the efficient correction of hypoxic lactic acidosis and shows few side effects. Therefore, application of pyruvate may be promising and safe as a novel therapeutic strategy in hypoxic lactic acidosis correction accompanied with multi-organ protection in critical care patients.

Expression of pyruvate dehydrogenase is an independent prognostic marker in gastric cancer

AIM:To investigate the expression and prognostic role of pyruvate dehydrogenase(PDH) in gastric cancer(GC).METHODS:This study included 265 patients(194 male,71 female,mean age 59 years(range,29-81 years) with GC who underwent curative surgery at the First Affiliated Hospital of China Medical University from January 2006 to May 2007.All patients were followed up for more than 5 years.Patient-derived paraffin embedded GC specimens were collected for tissue microarrays(TMAs).We examined PDH expression by immunohistochemistry in TMAs containing tumor tissue and matched nonneoplastic mucosa.Immunoreactivity was evaluated independently by two researchers.Overall survival(OS) rates were determined using the Kaplan-Meier estimator.Correlations with other clinicopathologic factors were evaluated by two-tailed χ2 tests or a two-tailed t-test.The Cox proportional-hazard model was used in univariate analysis and multivariate analysis to identify factors significantly correlated with prognosis.RESULTS:Immunohistochemistry showed that 35.47% of total cancer tissue specimens had cytoplasmic PDH staining.PDH expression was much higher in normal mucosa specimens(75.09%;P = 0.001).PDH expression was correlated with Lauren grade(70.77% in intestinal type vs 40.0% in diffuse type;P = 0.001),lymph node metastasis(65.43% with no metastasis vs 51.09% with metastasis;P = 0.033),lymphatic invasion(61.62% with no invasion vs 38.81% with invasion;P = 0.002),histologic subtypes(70.77% in intestinal type vs 40.0% in diffuse type;P = 0.001) and tumor-node-metastasis(TNM) stage(39% in poorly differentiated vs 65.91% in well differentiated and 67.11% in moderately differentiated;P = 0.001) in GC.PDH expression in cancer tissue was significantly associated with higher OS(P < 0.001).The multivariate analysis adjusted for age,Lauren classification,TNM stage,lymph node metastasis,histological type,tumor size,depth of invasion and lymphatic invasion showed that the PDH expression in GC was an independent prognostic factor for higher OS(HR = 0.608,95%CI:0.504-0.734,P < 0.001).CONCLUSION:Our study indicated that PDH expression is an independent prognostic factor in GC patients and that positive expression of PDH may be predictive of favorable outcomes.

Oxidative esterification of acetol with methanol to methyl pyruvate over hydroxyapatite supported gold catalyst: Essential roles of acid-base properties

Acetol is a major light oxygenate and readily produced from staged or fast pyrolysis of lignocellulose biomass. Herein we report that acetol can be selectively converted to methyl pyruvate, an important fine chemical, through oxidative esterification over Au-based catalysts. Detailed experimental studies showed that Au on amphoteric supports with appropriate strength and balanced ratio of acid and base sites can facilitate the desired oxidative-esterification pathway without accelerating undesired aldol-condensation or Cannizzaro reactions. In particular, hydroxyapatite (with a Ca/P ratio of 1.62) supported Au achieved 87% selectivity to methyl pyruvate at an acetol conversion of 62%.

Creatine Pyruvate Enhances Lipolysis and Protein Synthesis in Broiler Chicken

To assess the effects ofcreatine pyruvate （Cr-Pyr） on lipid and protein metabolism in broiler chickens, a total of 400 1-day-old male birds （Aconred） were randomly allocated to four groups, with each group replicating four times and each replicate involving 25 birds. The broilers were provided with a commercial diet supplemented with Cr-Pyr at 0, 1, 5, or 10% of the diet, respectively, for a period of 3 wk ad libitum （from 22 to 42 d）. In the present study, body weight （BW） and average daily gain （ADG） of broilers decreased in 10% Cr-Pyr group （P〈0.01）, whereas the relative leg and pectoral muscle weights were significantly higher than they were in the control group （P〈0.05）. 5 or 10% Cr-Pyr of diets decreased the abdominal fat rate （AFR, abdominal fat/live weight） of the broilers. The serum or hepatic triglyceride （TG） concentrations were significantly lower in the 5 and 10% groups （P〈0.01）. In contrast, Cr-Pyr caused a marked increase in the serum nonesterified fatty acid （NEFA）, high-density lipoprotein cholesterol （HDL-C） and creatine kinase （CK） concentrations （P〈0.01）. Supplementation with Cr-Pyr （5 and 10%） in the diet also increased glucagons （GLU）, insulin （INS） or leptin （LEP） contents （P〈0.01）. The expression of hepatic peroxisomal proliferators-activated receptor α （PPAR-α） and carnitine palmitoyl transferase-I （CPT-I）, muscle insulin-like growth factor I （IGF-I） were significantly elevated and myostatin mRNA level was reduced in the 5 and 10% groups （P〈0.05）. It was found that supplementation with 5% Cr-Pyr improves both lipid and protein metabolism by regulating various metabolic parameters of broilers, while not adversely affects growth performance in broiler chickens.

Overexpression of the M2 isoform of pyruvate kinase is an adverse prognostic factor for signet ring cell gastric cancer

AIM:To investigate M2 isoform of pyruvate kinase(PKM2) expression in gastric cancers and evaluate its potential as a prognostic biomarker and an anticancer target.METHODS:All tissue samples were derived from gastric cancer patients underwent curative gastrectomy as a primary treatment.Clinical and pathological information were obtained from the medical records.Gene expression microarray data from 60 cancer and 19 noncancer gastric tissues were analyzed to evaluate the expression level of PKM2 mRNA.Tissue microarrays were constructed from 368 gastric cancer patients.Immunohistochemistry was used to measure PKM2 expression and PKM2 positivity of cancer was determined by proportion of PKM2-positive tumor cells and staining intensity.Association between PKM2 expression and the clinicopathological factors was evaluated and the correlation between PKM2 and cancer prognosis was evaluated.RESULTS:PKM2 mRNA levels were increased more than 2-fold in primary gastric cancers compared to adjacent normal tissues from the same patients(log transformed expression level:7.6 ± 0.65 vs 6.3 ± 0.51,P < 0.001).Moreover,differentiated type cancers had significantly higher PKM2 mRNA compared to undifferentiated type cancers(log transformed expression level:7.8 ± 0.70 vs 6.7 ± 0.71,P < 0.001).PKM2 protein was mainly localized in the cytoplasm of primary cancer cells and detected in 144 of 368(39.1%) human gastric cancer cases.PKM2 expression was not related with stage(P = 0.811),but strongly correlated with gastric cancer differentiation(P < 0.001).Differentiated type cancers expressed more PKM2 protein than did the undifferentiated ones.Well differentiated adenocarcinoma showed 63.6% PKM2-positive cells;in contrast,signet-ring cell cancers showed only 17.7% PKM2-positive cells.Importantly,PKM2 expression was correlated with shorter overall survival(P < 0.05) independent of stage only in signet-ring cell cancers.CONCLUSION:PKM2 expression might be an adverse prognostic factor for signet-ring cell carcinomas.Its function and potential as a prognostic marker should be further verified in gastric cancer.

Faecal pyruvate kinase isoenzyme type M2 for colorectal cancer screening:A meta-analysis

AIM:To present a critical discussion of the efficacy of the faecal pyruvate kinase isoenzyme type M2(faecal M2-PK) test for colorectal cancer(CRC) screening based on the currently available studies.METHODS:A literature search in PubMed and Embase was conducted using the following search terms:fecal Tumor M2-PK,faecal Tumour M2-PK,fecal M2-PK,faecal M2-PK,fecal pyruvate kinase,faecal pyruvate kinase,pyruvate kinase stool and M2-PK stool.RESULTS:Stool samples from 704 patients with CRC and from 11 412 healthy subjects have been investigated for faecal M2-PK concentrations in seventeen independent studies.The mean faecal M2-PK sensitivity was 80.3%;the specificity was 95.2%.Four studies compared faecal M2-PK head-to-head with guaiacbased faecal occult blood test(gFOBT).Faecal M2PK demonstrated a sensitivity of 81.1%,whereas the gFOBT detected only 36.9% of the CRCs.Eight independent studies investigated the sensitivity of faecal M2-PK for adenoma(n = 554),with the following sensitivities:adenoma < 1 cm in diameter:25%;adenoma > 1 cm:44%;adenoma of unspecified diameter:51%.In a direct comparison with gFOBT of adenoma > 1 cm in diameter,47% tested positive with the faecal M2-PK test,whereas the gFOBT detected only 27%.CONCLUSION:We recommend faecal M2-PK as a routine test for CRC screening.Faecal M2-PK closes a gap in clinical practice because it detects bleeding and nonbleeding tumors and adenoma with high sensitivity and specificity.

Tumor pyruvate kinase M2:A promising molecular target of gastrointestinal cancer

Gastrointestinal(GI) cancer is one of the most common causes of cancer-related deaths worldwide.Tumor markers are valuable in detecting post-surgical recurrence or in monitoring response to chemotherapy.Pyruvate kinase isoform M2(PKM2),a glycolytic enzyme catalyzing conversion of phosphoenolpyruvate(PEP) to pyruvate,confers a growth advantage to the tumor cells and enables them to adapt to the tumor microenvironment.In this review,we have summarized current research on the expression and regulation of PKM2 in tumor cells,and its potential role in GI carcinogenesis and progression.Furthermore,we have also discussed the potential of PKM2 as a diagnostic and screening marker,and a therapeutic target in GI cancer.

Oxygen dependent pyruvate oxidase expression and production in Streptococcus sanguinis

The objective of this study was to characterize the oxygen dependent regulation of pyruvate oxidase （SpxB） gene expression and protein production in Streptococcus sanguinis （S. sanguinis）. SpxB is responsible for the generation of growth-inhibiting amounts of hydrogen peroxide （H202） able to antagonize cariogenic Strepto- coccus mutans （S. mutans）. Furthermore, the ecological consequence of H202 production was investigated in its self-inhibiting ability towards the producing strain. Expression of spxB was determined with quantitative Real-Time RT-PCR and a fluorescent expression reporter strain. Protein abundance was investigated with FLAG epitope engineered in frame on the C-terminal end of SpxB. Self inhibition was tested with an antagonism plate assay. The expression and protein abundance decreased in cells grown under anaerobic conditions. S. sanguinis was resistant against its own produced H202, while cariogenic S. mutans was inhibited in its growth. The results suggest that S. sanguinis produces H202 as antimicrobial substance to inhibit susceptible niche competing species like S. mutans during initial biofilm formation, when oxygen availability allows for spxB expression and Spx production.

Deletions in the pyruvate pathway of Salmonella Typhimurium alter SPI1-mediated gene expression and infectivity

Background： Salmonella enter/ca serovar Typhimurium is a major foodborne pathogen worldwide. S. Typhimurium encodes type III secretion systems via Salmonella pathogenicity islands （SPI）, producing the major effector proteins of virulence. Previously, we identified two genes of Salmonella pyruvate metabolism that were up-regulated during chicken cell infection： pyruvate formate lyase I （pf/B） and b/functional acetaldehyde-CoA/alcohol dehydrogenase （adhE）. We were therefore interested in examining the role these genes may play in the transmission of Salmonella to humans. Methods： Mutant strains of Salmonella with single gene deletions for pflB and adhE were created. Invasion and growth in human HCT-8 intestinal epithelial cells and THP-1 macrophages was examined. Quantitative PCR was performed on 19 SPI-1 genes. Results： In HCT-8 cells, both mutant strains had significantly higher intracellular counts than the wild-type from 4 to 48 h post-infection. Various SPI-1 genes in the mutants were up-regulated over the wild-type as early as 1 h and lasting until 24 h post-infection. In THP-1 cells, no significant difference in internal Salmonella counts was observed; however, SPI-1 genes were largely down-regulated in the mutants during the time-course of infection. We also found five SPI-1 genes - hilA, hiIC hill）, sicP and rtsA - which were up-regulated in at least one of the mutant strains in log-phase broth cultures alone. We have therefore identified a set of SPI-1 virulence genes whose regulation is effected by the central metabolism of Salmonella.

MODIFICATION OF TRANSITION METAL CATIONS TO POLYMER-STABILIZED PLATINUM COLLOIDAL CLUSTERS IN ENANTIOSELECTIVE HYDROGENATION OF METHYL PYRUVATE

Modification of transition metal cations to polymer-stabilized Pt colloidal clusters modified with cinchonidine was studied in enantioselective hydrogenation of methyl pyruvate.Compared to the enantiomeric excess(e.e.)value(71.4%) obtained without the presence of metal cations,obvious e.e.enhancement(up to 82.5%)was resulted from the addition of Zn^(2+) but with a certain decrease in activity.The reaction parameters in the presence of Zn^(2+) were also studied.It was found that the Pt colloidal catalysts in the presence of metal cations performed very differently from that in the absence of metal cations.

Identification of the amino acid involved in the regulation of bacterial pyruvate, orthophosphate dikinase and phosphoenolpyruvate synthetase

Pyruvate, orthophosphate dikinase (PPDK) and phosphoenolpyruvate synthetase (PEPS) catalyze the conversion of pyruvate to phosphoenolpyruvate (PEP). Both are regulated by a phosphorylation-dephosphorylation mechanism involving a bifunctional serine/ threonine kinase and a pyrophosphorylase (PPDK regulatory protein, PDRP, and PEPS regulatory protein, PSRP, respectively). In plants the regulatory mechanism involves phosphorylation of a threonine residue that is separated by a single amino acid from the histidine residue that forms a phosphorylated intermediate during catalysis. Using antibodies, we demonstrated that the regulation of both Listeria monocytogenes PPDK and Escherichia coli PEP synthetase involves the phosphorylation of a threonine residue located close to the catalytic histidine residue. The amino acid located between the regulatory threonine and the catalytic histidine is highly conserved being serine in PPDK and cysteine in PEPS. Using site-directed mutagenesis we have shown that both PPDK and PEPS in which the serine and cysteine residues, respectively, were substituted with an alanine the enzymes could be regulated indicating that the serine and cysteine residues, respectively, are not essential for regulation. We also demonstrated that altering the intermediate amino acid did not alter the specificity of the regulatory proteins for their protein substrates.

The Novel Pyruvated Glucogalactan Sulfate Isolated from the Red Seaweed, Hypnea pannosa

The polysaccharide was isolated from Hypnea pannosa which was grown in Okinawa, Japan. The yield of the polysaccharide was 17.2%, and the total carbohydrates, pyruvic acid, sulfuric acid and ash contents were 55.2%, 3.8%, 35.2% and 24.3%, respectively. 3,6-Anhydro-α-D-galactose, β-D-galactose, α-D-galactose and D-glucose were identified by liquid and thin-layer chromatography. Fourier transform infrared (FTIR) spectra of the polysaccharide resembled that of ι-carrageenan. From the 1H- and 13C-NMR spectra, 1,3-linked β-D-galactose, 1,4-linked anhydro-α-D-galactose, 1,4-linked α-D-galactose, 1,4-linked β-D-glucose and pyruvic acid (carboxyl acetal, methyl proton and methyl carbon) were assigned. Methylation analysis revealed terminal D-galactose 0.1 mol), 1,4-linked D-glucose (1.0 mol) and 1,2,3,4,6-linked D-galactose (3.7 mol) for native polysaccharide, and terminal D-galactose, 1,4-linked D-galactose (1.9 mol), 1,4-linked D-glucose (1.0 mol), 1,3- linked D-galactose (1.7 mol), and 1,3,4,6-linked D-galactose (0.3 mol) which substituted with pyruvate group at 4 and 6 positions for desulfated polysaccharide. The polysaccharide was the novel pyruvated glucogalactan sulfate, the structure of which was proposed.

Biocatalytic Synthesis of Pyruvate from DL-lactate with Enzymes in Pseudomonas sp.

A novel method of preparing pyruvate from DL-lactate catalyzed by enzymes from a bacterial strain of Pseudomonas sp. SM-6 was proposed. Catalytic processes of cell-free extract enzymes and immobilized enzymes were evaluated. The kinetic data were studied, too.

Effect of Catalase on Biocatalytic Synthesis of Pyruvate by Enzymes from Pseudomonas sp.

Pyruvate was produced from DL-lactate by a kind of green-chemical biocatalyst cell-free extract from bacterial strain Pseudomonas sp. SM-6. Catalase in cell-free extract, which could stabilize the pyruvate formed by lactate oxidase, played an important role in pyruvate preparation. The effect of catalase in conversion process was evaluated.