Hypomethylation of glycine dehydrogenase promoter in peripheral blood mononuclear cells is a new diagnostic marker of hepatitis B virus-associated hepatocellular carcinoma

Background: Glycine dehydrogenase(GLDC) plays an important role in the initiation and proliferation of several human cancers. In this study, we aimed to detect the methylation status of GLDC promoter and its diagnostic value for hepatitis B virus-associated hepatocellular carcinoma(HBV-HCC). Methods: We enrolled 197 patients, 111 with HBV-HCC, 51 with chronic hepatitis B(CHB), and 35 healthy controls(HCs). The methylation status of GLDC promoter in peripheral mononuclear cells(PBMCs) was identified by methylation specific polymerase chain reaction(MSP). The mRNA expression was examined using real-time quantitative polymerase chain reaction(q PCR). Results: The methylation frequency of the GLDC promoter was significantly lower in HBV-HCC patients(27.0%) compared to that in CHB patients(68.6%) and HCs(74.3%)( P < 0.001). The methylated group had lower alanine aminotransferase level( P = 0.035) and lower rates of tumor node metastasis(TNM) Ⅲ/Ⅳ( P = 0.043) and T3/T4( P = 0.026). TNM stage was identified to be an independent factor for GLDC promoter methylation. GLDC mRNA levels in CHB patients and HCs were significantly lower than those in HBV-HCC patients( P = 0.022 and P < 0.001, respectively). GLDC mRNA levels were significantly higher in HBV-HCC patients with unmethylated GLDC promoters than those with methylated GLDC promoters( P = 0.003). The diagnostic accuracy of alpha-fetoprotein(AFP) combined with GLDC promoter methylation for HBV-HCC was improved compared with that of AFP alone(AUC: 0.782 vs. 0.630, P < 0.001). In addition, GLDC promoter methylation was an independent predictor for overall survival of HBV-HCC patients( P = 0.038). Conclusions: The methylation frequency of GLDC promoter was lower in PBMCs from HBV-HCC patients than that from patients with CHB and HCs. The combination of AFP and GLDC promoter hypomethylation significantly improved the diagnostic accuracy of HBV-HCC.

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.

Direct detection of a single[4Fe-4S]cluster in a tungsten-containing enzyme:Electrochemical conversion of CO_(2) into formate by formate dehydrogenase

The conversion of CO_(2) into fuels and valuable chemicals is one of the central topics to combat climate change and meet the growing demand for renewable energy.Herein,we show that the formate dehydrogenase from Clostridium ljungdahlii(ClFDH)adsorbed on electrodes displays clear characteristic voltammetric signals that can be assigned to the reduction and oxidation potential of the[4Fe-4S]^(2+/+)cluster under nonturnover conditions.Upon adding substrates,the signals transform into a specific redox center that engages in catalytic electron transport.ClFDH catalyzes rapid and efficient reversible interconversion between CO_(2) and formate in the presence of substrates.The turnover frequency of electrochemical CO_(2) reduction is determined as 1210 s^(-1) at 25℃ and pH 7.0,which can be further enhanced up to 1786 s^(-1) at 50℃.The Faradaic efficiency at−0.6 V(vs.standard hydrogen electrode)is recorded as 99.3%in a 2-h reaction.Inhibition experiments and theoretical modeling disclose interesting pathways for CO_(2) entry,formate exit,and OCN−competition,suggesting an oxidation-state-dependent binding mechanism of catalysis.Our results provide a different perspective for understanding the catalytic mechanism of FDH and original insights into the design of synthetic catalysts.

Combinatorial co-expression of xanthine dehydrogenase and chaperone XdhC from Acinetobacter baumannii and Rhodobacter capsulatus and their applications in decreasing purine content in food

This study investigated the combinatorial expression of xanthine dehydrogenase(XDH)and chaperone XdhC from Acinetobacter baumannii and Rhodobacter capsulatus and their applications in decreasing purine content in the beer,beef and yeast.Naturally occurring xdhABC gene clusters of A.baumannii CICC 10254 and R.capsulatus CGMCC 1.3366 as well as two refactored clusters constructed by exchanging their xdhC genes were overexpressed in Escherichia coli and purified to near homogeneity.RcXDH chaperoned by AbXdhC showed nearly the same catalytic performance as that by RcXdhC,except for the decreased substrate affinity.While the AbXDH co-expressed with RcXdhC displayed enhanced acidic adaptation but weakened catalytic activity.All the XDHs degraded purines in beer,beef and yeast extract effectively,indicating potential applications in low-purine foods to prevent hyperuricemia and gout.The study also presents a method for exploiting the better chaperone XdhC and novel XDHs by functional complement activity using existing XdhCs such as RcXdhC.

F-box only protein 2 exacerbates non-alcoholic fatty liver disease by targeting the hydroxyl CoA dehydrogenase alpha subunit

BACKGROUND Non-alcoholic fatty liver disease(NAFLD)is a major health burden with an increasing global incidence.Unfortunately,the unavailability of knowledge underlying NAFLD pathogenesis inhibits effective preventive and therapeutic measures.AIM To explore the molecular mechanism of NAFLD.METHODS Whole genome sequencing(WGS)analysis was performed on liver tissues from patients with NAFLD(n=6)and patients with normal metabolic conditions(n=6)to identify the target genes.A NAFLD C57BL6/J mouse model induced by 16 wk of high-fat diet feeding and a hepatocyte-specific F-box only protein 2(FBXO2)overexpression mouse model were used for in vivo studies.Plasmid transfection,co-immunoprecipitation-based mass spectrometry assays,and ubiquitination in HepG2 cells and HEK293T cells were used for in vitro studies.RESULTS A total of 30982 genes were detected in WGS analysis,with 649 up-regulated and 178 down-regulated.Expression of FBXO2,an E3 ligase,was upregulated in the liver tissues of patients with NAFLD.Hepatocyte-specific FBXO2 overexpression facilitated NAFLD-associated phenotypes in mice.Overexpression of FBXO2 aggravated odium oleate(OA)-induced lipid accumulation in HepG2 cells,resulting in an abnormal expression of genes related to lipid metabolism,such as fatty acid synthase,peroxisome proliferator-activated receptor alpha,and so on.In contrast,knocking down FBXO2 in HepG2 cells significantly alleviated the OA-induced lipid accumulation and aberrant expression of lipid metabolism genes.The hydroxyl CoA dehydrogenase alpha subunit(HADHA),a protein involved in oxidative stress,was a target of FBXO2-mediated ubiquitination.FBXO2 directly bound to HADHA and facilitated its proteasomal degradation in HepG2 and HEK293T cells.Supplementation with HADHA alleviated lipid accumulation caused by FBXO2 overexpression in HepG2 cells.CONCLUSION FBXO2 exacerbates lipid accumulation by targeting HADHA and is a potential therapeutic target for NAFLD。

Carbon Chain Length Determines Inhibitory Potency of Perfluoroalkyl Sulfonic Acids on Human Placental 3β-Hydroxysteroid Dehydrogenase 1:Screening,Structure-Activity Relationship,and In Silico Analysis

Objective This study aimed to compare 9 perfluoroalkyl sulfonic acids(PFSA)with carbon chain lengths(C4–C12)to inhibit human placental 3β-hydroxysteroid dehydrogenase 1(3β-HSD1),aromatase,and rat 3β-HSD4 activities.Methods Human and rat placental 3β-HSDs activities were determined by converting pregnenolone to progesterone and progesterone secretion in JEG-3 cells was determined using HPLC/MS–MS,and human aromatase activity was determined by radioimmunoassay.Results PFSA inhibited human 3β-HSD1 structure-dependently in the order:perfluorooctanesulfonic acid(PFOS,half-maximum inhibitory concentration,IC50:9.03±4.83μmol/L)>perfluorodecanesulfonic acid(PFDS,42.52±8.99μmol/L)>perfluoroheptanesulfonic acid(PFHpS,112.6±29.39μmol/L)>perfluorobutanesulfonic acid(PFBS)=perfluoropentanesulfonic acid(PFPS)=perfluorohexanesulfonic acid(PFHxS)=perfluorododecanesulfonic acid(PFDoS)(ineffective at 100μmol/L).6:2FTS(1H,1H,2H,2H-perfluorooctanesulfonic acid)and 8:2FTS(1H,1H,2H,2H-perfluorodecanesulfonic acid)did not inhibit human 3β-HSD1.PFOS and PFHpS are mixed inhibitors,whereas PFDS is a competitive inhibitor.Moreover,1–10μmol/L PFOS and PFDS significantly reduced progesterone biosynthesis in JEG-3 cells.Docking analysis revealed that PFSA binds to the steroid-binding site of human 3β-HSD1 in a carbon chain length-dependent manner.All 100μmol/L PFSA solutions did not affect rat 3β-HSD4 and human placental aromatase activity.Conclusion Carbon chain length determines inhibitory potency of PFSA on human placental 3β-HSD1 in a V-shaped transition at PFOS(C8),with inhibitory potency of PFOS>PFDS>PFHpS>PFBS=PFPS=PFHxS=PFDoS=6:2FTS=8:2FTS.

Choline dehydrogenase interacts with SQSTM1 to activate mitophagy and promote coelomocyte survival in Apostichopus japonicus following Vibrio splendidus infection

Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes,leading to the production of excessive reactive oxygen species(ROS)and irreversible apoptotic cell death.Emerging evidence suggests that mitochondrial autophagy(mitophagy)is the most effective method for eliminating damaged mitochondria and ROS,with choline dehydrogenase(CHDH)identified as a novel mitophagy receptor that can recognize non-ubiquitin damage signals and microtubule-associated protein 1 light chain 3(LC3)in vertebrates.However,the functional role of CHDH in invertebrates is largely unknown.In this study,we observed a significant increase in the mRNA and protein expression levels of A.japonicus CHDH(AjCHDH)in response to V.splendidus infection and lipopolysaccharide(LPS)challenge,consistent with changes in mitophagy under the same conditions.Notably,AjCHDH was localized to the mitochondria rather than the cytosol following V.splendidus infection.Moreover,AjCHDH knockdown using si RNA transfection significantly reduced mitophagy levels,as observed through transmission electron microscopy and confocal microscopy.Further investigation into the molecular mechanisms underlying CHDH-regulated mitophagy showed that AjCHDH lacked an LC3-interacting region(LIR)for direct binding to LC3 but possessed a FB1 structural domain that binds to SQSTM1.The interaction between AjCHDH and SQSTM1 was further confirmed by immunoprecipitation analysis.Furthermore,laser confocal microscopy indicated that SQSTM1 and LC3 were recruited by AjCHDH in coelomocytes and HEK293T cells.In contrast,AjCHDH interference hindered SQSTM1 and LC3 recruitment to the mitochondria,a critical step in damaged mitochondrial degradation.Thus,AjCHDH interference led to a significant increase in both mitochondrial and intracellular ROS,followed by increased apoptosis and decreased coelomocyte survival.Collectively,these findings indicate that AjCHDH-mediated mitophagy plays a crucial role in coelomocyte survival in A.japonicus following V.splendidus infection.

Effects of genistein and equol on human and rat testicular 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities

The objective of the present study was to investigate the effects of genistein and equol on 3β-hydroxysteroid de- hydrogenase （3β-HSD） and 17β-hydroxysteroid dehydrogenase 3 （17β-HSD3） in human and rat testis microsomes. These enzymes （3β-HSD and 17β-HSD3）, along with two others （cytochrome P450 side-chain cleavage enzyme and cytochrome P450 17α-hydroxylase/17-20 lyase）, catalyze the reactions that convert the steroid cholesterol into the sex hormone testosterone. Genistein inhibited 3β-HSD activity （0.2 μmol L^-1 pregnenolone） with half-maximal inhibition or a half-maximal inhibitory concentration （IC50） of 87 ± 15 （human） and 636 ± 155 nmol L^-1 （rat）. Genistein＇s mode of action on 3β-HSD activity was competitive for the substrate pregnenolonrge and noncompetitive for the cofactor NAD＋. There was no difference in genistein＇s potency of 3β-HSD inhibition between intact rat Leydig cells and testis microsomes. In contrast to its potent inhibition of 3β-HSD, genistein had lesser effects on human and rat 17β-HSD3 （0.1 μmol L^-1 androstenedione）, with an IC50 〉 100μmol L^-1. On the other hand, equol only inhibited human 3β-HSD by 42%, and had no effect on 3β-HSD and 17β-HSD3 in rat tissues. These observations imply that the ability of soy isoflavones to regulate androgen biosynthesis in Leydig cells is due in part to action on Leydig cell 3β- HSD activity. Given the increasing intake of soy-based food products and their potential effect on blood androgen levels, these findings are greatly relevant to public health.

Influence of selenium induced oxidative stress on spermatogenesis and lactate dehydrogenase-X in mice testis

Aim: To evaluate the effect of oxidative stress on the spermatogenesis and lactate dehydrogenase-X (LDH-X) activity in mouse testis. Methods: For creating different levels of oxidative stress in mice, three selenium (Se) level diets were fed in separate groups for 8 weeks. Group 1 animals were fed yeast-based Se-deficient (0.02 ppm) diet. Group 2 and Group 3 animals were fed with the same diet supplemented with 0.2 ppm and 1 ppm Se as sodium selenite, respectively. After 8 weeks, biochemical and histopathological observations of the testis were carried out. LDH-X levels in the testis were analyzed by western immunoblot and ELISA. Results: A significant decrease in testis Se level was observed in Group 1 animals, whereas it was enhanced in Group 3 as compared to Group 2. The glutathione peroxidase (GSH-Px) activity was significantly reduced in both the liver and testis in Group 1, but not in Group 2 and 3. A significant increase in the testis glutathione-S-transferase (GST) activity was observed in Group 1, whereas no significant change was seen in Groups 2 and 3. Histological analysis of testis revealed a normal structure in Group 2. A significant decrease in the germ cell population in Group 1 was observed as compared to Group 2 with the spermatids and mature sperm affected the most. Decrease in the lumen size was also observed. In the Se-excess group (Group 3), displacement of germ cell population was observed. Further, a decrease in the LDH-X level in testis was observed in Group 1. Conclusion: Excessive oxidative stress in the Se deficient group, as indicated by changes in the GSH-Px/GST activity, affects the spermatogenic process with a reduction in mature sperm and in turn the LDH-X level.

Relationship Between Polymorphism of Methylenetetrahydrofolate Dehydrogenase and Congenital Heart Defect

To investigate the relationship between G1958A gene polymorphism of methylenetetrahydrofolate dehydrogenase (MTHFD) and occurrence of congenital heart disease (CHD) in North China. Methods One hundred and ninety-two CHD patients and their parents were included in this study as case group in Liaoning Province by birth defect registration cards, and 124 healthy subjects (age and gender matched) and their parents were simultaneously selected from the same geographic area as control. Their gene polymorphism of MTHFD G1958A locus was examined with PCR-RFLP, and serum folic acid and homocysteine (Hcy) levels were tested with radio-immunoassay and fluorescence polarization immunoassay (FPIA). Results There existed gene polymorphism at MTHFD G1958A locus in healthy subjects living in North China. The percentages of GG, GA, and AA genotype were 57.98%, 35.57%, and 6.45% respectively, and the A allele frequency was 24.23%, which was significantly different from Western population. No difference was observed when comparing genotype distribution and allele frequency between the case and control groups, so was the result from the comparison between genders. The A allele frequency of arterial septal defect patients’ mothers (10.87%) was significantly lower than that of controls (28.15%) (P=0.014), with OR=0.31 (95% CI: 0.09-0.84), and no difference in the other subgroups. The percentage of at least one parent carrying A allele in arterial septal defect subgroup (43.48%) was significantly lower than that in controls (69.64%) (P=0.017), with OR=0.34 (95% CI: 0.12-0.92). The analysis of genetic transmission indicated that there was no transmission disequillibrium in CHD nuclear families. Their serum folic acid level was significantly higher than that of controls (P=0.000), and Hcy level of the former was higher than that of the latter with no statistical significance (P>0.05). Serum Hcy and folic acid levels of mothers with gene mutation were lower than those of mothers with no mutation. Conclusion No significant difference of genotype distribution and allele frequency existed between CHD patients and healthy population. MTHFD G1958A mutation in parents (particularly in mother) can decrease the risk of arterial septal defect in offspring. The possible mechanism of protection might be mutation, which can increase MTHFD enzyme activity, folic acid metabolism and homocysteine remethylation, and decrease Hcy level.

In Vitro and In Vivo Effects and Safety Assessment of Corn Peptides on Alcohol Dehydrogenase Activities

The in vitro and in vivo effects of corn peptides（CPs） prepared from corn gluten meal by proteolysis with an alkaline protease and fractions of CPs from Sephadex G-15 and G-10 columns on activities of alcohol dehydroge-nase（ADH） were studied. The results show that CPs and fraction 3 of CPs from Sephadex G-10 column enhance in vitro ADH activity. Furthermore, the in vitro accelerating effect of the fraction 3 of CPs on ADH activity was superior to that of glutathione, which was also found even in the presence of ADH inhibitor, such as pyrazole. In the in vivo experiments, the animals were fed with different dosages of CPs and with a dose of Chinese distilled spirit orally, and sacrificed for the measurement of ADH activity. In vivo experimental results indicate that CPS enhanced hepatic ADH activities. To test the safety of CPs as health food, 30 d feeding test was performed. No obvious toxic effects were detected in treated Wistar rats.

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.

Role of aldehyde dehydrogenase 2 in ischemia reperfusion injury:An update

Aldehyde dehydrogenase 2(ALDH2) is best known for its critical detoxifying role in liver alcohol metabolism. However, ALDH2 dysfunction is also involved in a wide range of human pathophysiological situations and is associated with complications such as cardiovascular diseases, diabetes mellitus, neurodegenerative diseases and aging. A growing body of research has shown that ALDH2 provides important protection against oxidative stress and the subsequent loading of toxic aldehydes such as 4-hydroxy-2-nonenal and adducts that occur in human diseases, including ischemia reperfusion injury(IRI). There is increasing evidence of its role in IRI pathophysiology in organs such as heart, brain, small intestine and kidney; however, surprisingly few studies have been carried out in the liver, where ALDH2 is found in abundance. This study reviews the role of ALDH2 in modulating the pathways involved in the pathophysiology of IRI associated with oxidative stress, autophagy and apoptosis. Special emphasis is placed on the role of ALDH2 in different organs, on therapeutic "preconditioning" strategies, and on the use of ALDH2 agonists such as Alda-1, which may become a useful therapeutic tool for preventing the deleterious effects of IRI in organ transplantation.

Identification and expression patterns of alcohol dehydrogenase genes involving in ester volatile biosynthesis in pear fruit

Alcohol dehydrogenase （ADH） catalyzes the interconversion of aldehydes and their corresponding alcohols, and is a key enzyme in volatile ester biosynthesis. However, little is known regarding ADH and ADH encoding genes （ADHs） in pear. We identified 8 ADHs in the pear＇s genome （PbrADHs） by multiple sequences alignment. The PbrADHs were highly ho- mologous in their coding regions, while were diversiform in structure. 9 introns were predicted in PbrADH3-PbrADH8, while 8 introns, generated through exon fusion and intron loss, were predicted in PbrADH1 and PbrADH2. To study the genetic regulation underlying aroma biogenesis in pear fruit, we determined the PbrADH transcripts, ADH activities and volatile contents of fruits during ripening stage for Nanguoli and Dangshansuli, two cultivars having different aroma characteristics. ADH activity was strongly associated with the transcription of ADH~ in the two cultivars during fruit ripening stage. The higher ester content paralleling to a higher ADH activity was detected in Nanguoli than in Dangshansuli, so it is induced that the lower ester content in Dangshansuli fruit may be the result of weak ADH activity. The present study revealed that total ADH activity and volatile ester production correlated with increased PbrADHstranscript levels. PbrADH6 may contribute to ADH activity catalyzing aldehyde reduction and ester formation in pear fruit.

Identification and bioinformatics analysis of lactate dehydrogenase genes from Echinococcus granulosus

Objective:To identify full length cDNA sequence of lactate dehydrogenase(LDH) from adult Echinococcus granulosus(E.granulosus) and to predict the structure and function of its encoding protein using bioinformatics methods.Methods:With the help of NCBI,EMBI, Expasy and other online sites,the open reading frame(ORF),conserved domain,physical and chemical parameters,signal peptide,epitope,topological structures of the protein sequences were predicted and a homology tertiary structure model was created:Vector NT1 software was used for sequence alignment,phylogenetic tree construction and tertiary structure prediction. Results:The target sequence was 1 233 bp length with a 996 bp biggest ORF encoding 331 amino acids protein with typical L-LDH conserved domain.It was confirmed as full length cDNA of LDH from E.granulosus and named as EgLDH(GenBank accession number:HM748917).The predicted molecular weight and isoelectric point of the deduced protein were 3 5516.2Da and 6.32 respectively.Compared with LDHs from Taenia solium,Taenia saginata asiatica,Spirometra erinaceieuropaei.Schistosoma japonicum,Clonorchis sinensis and human,it showed similarity of 86% ,85% ,55% ,58% ,58% and 53% ,respectively.EgLDH contained 3 putative transmembrane regions and 4 major epitopes(54aa-59aa.81aa-87aa,97aa-102aa,307aa-313aa),the latter were significant different from the corresponding regions of human LDH.In addition,some NAD and substrate binding sites located on epitopes 54aa-59aa and 97aa-102aa,respectively.Tertiary structure prediction showed that 3 key catalytic residues 105R,165D and 192H forming a catalytic center near the epitope 97aa-102aa,most NAD and substrate binding sites located around the center.Conclusions:The full length cDNA sequences of EgLDH were identified.It encoded a putative transmembrane protein which might be an ideal target molecule for vaccine and drugs.

Cloning and Sequence Analysis of a Glucose-6-Phosphate Dehydrogenase Gene PsG6PDH from Freezing-tolerant Populus suaveolens

A 1 207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freez- ing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydro- genase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana ta- bacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.

High Level Expression of Glucose-6-phosphate Dehydrogenase Gene PsG6PDH from Populus suaveolens in E. coli

In order to investigate the functions of the gene PsG6PDH and the mechanisms underlying freezing tolerance of Populus suaveolens, the recombinant expression vector pET-G （pET30a-G6PDH）, which contained full encoding region of PsG6PDH gene, was established. The recombinant was identified by lawn-PCR and double enzyme digestion and then transformed into expression host XA90 and induced by isopropyl-a-D-thiogalactoside （IPTG） to express 100 kD polypeptide of G6PDH fusion protein. The results showed that the expressed amount of the fusion protein culminated after 1 mmol·L^-1 IPTG treatment for 4h and that pET-G product was predominately soluble and not extra-cellular secreting.

Relationship between the Expression of Thymidylate Synthase,Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase and Survival in Epithelial Ovarian Cancer

The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression and clinicopathologic features was investigated. The protein location and expression of TS, TP and DPD was examined in the same patients by an avidin-biotin-peroxidase immunohistochemistry. TS and TP mRNA expression levels were significantly higher in tumor group than in normal controls, with the average value of TS and TP mRNA being 6.14±0.62 and 0.59±0.06 in tumor tissue, and 0.71±0.14 and 0.16±0.04 in normal tissue, respectively. DPD mRNA expression levels were significantly lower in tumor group (0.11±0.02) than in normal controls (0.38±0.05). There was statistically significant difference in TS and TP mRNA expression levels among different pathological grades and clinical stages (P<0.05), but histological subtype was not significantly associated with TS and TP mRNA expression. DPD gene expression was not significantly associated with any clinicopathological parameters. Immunohistochemistry revealed that TP protein was mainly distributed in nucleus, and TS and DPD mainly in cytoplasm. The protein expression intensity of TS, TP and DPD was coincided with the mRNA expression levels. It was concluded that TS, TP mRNA and protein expression levels were significantly higher in epithelial ovarian cancer, and DPD mRNA and protein expression levels were significantly lower. The expression levels of TS and DPD were related to the patients’ prognosis and survival. Combined gene expression levels of TS, TP and DPD represent a new variable to predict the clinical outcome in ovarian cancer. The association of TS, TP and DPD expression levels with survival suggests an importance of these genes for tumor occurrence and progression.

Aldehyde dehydrogenase 2 overexpression inhibits neuronal apoptosis after spinal cord ischemia/reperfusion injury

Aldehyde dehydrogenase 2（ALDH2）is an important factor in inhibiting oxidative stress and has been shown to protect against renal ischemia/reperfusion injury.Therefore,we hypothesized that ALDH_2 could reduce spinal cord ischemia/reperfusion injury.Spinal cord ischemia/reperfusion injury was induced in rats using the modified Zivin＇s method of clamping the abdominal aorta.After successful model establishment,the agonist group was administered a daily consumption of 2.5%alcohol.At 7 days post-surgery,the Basso,Beattie,and Bresnahan score significantly increased in the agonist group compared with the spinal cord ischemia/reperfusion injury group.ALDH_2expression also significantly increased and the number of apoptotic cells significantly decreased in the agonist group than in the spinal cord ischemia/reperfusion injury group.Correlation analysis revealed that ALDH_2 expression negatively correlated with the percentage of TUNEL-positive cells（r=-0.485,P〈0.01）.In summary,increased ALDH_2 expression protected the rat spinal cord against ischemia/reperfusion injury by inhibiting apoptosis.

Association of genetic polymorphisms of aldehyde dehydrogenase-2 with esophageal squamous cell dysplasia

AIM:To demonstrate the possible associations between genetic polymorphisms of aldehyde dehydrogenase-2(ALDH2) and esophageal squamous cell dysplasia(ESCD).METHODS:All participants came from an area of high incidence of esophageal cancer and underwent an endoscopic staining examination;biopsies were taken from a non-staining area of the mucosa and diagnosed by histopathology.Based on the examinations,the subjects were divided into the control group with normal esophageal squamous epithelial cells and the ESCD group.ALDH2 genotypes of 396 cases were determined including 184 ESCD cases and 212 controls.The odds ratio(OR) and 95% confidence intervals(95% CI) were calculated by binary logistic regression models.RESULTS:The distribution of ALDH2 genotypes showed significant differences between the two groups.The adjustment factors were gender and age in the logistic regression models.Compared with 2*2/2*2 genotype,2*1/2*1 genotype was found to be a risk factor for ESCD,and the OR(95% CI) was 4.50(2.21-9.19).There were significant correlations between ALDH2 genotypes and alcohol drinking/smoking/history of esophageal cancer.CONCLUSION:The ALDH2 polymorphism is significantly associated with ESCD.