Laboratory proficiency evaluation of the detection ability of malic enzyme 1 and isocitrate dehydrogenase 1 in mouse kidneys / 中国实验动物学报

Objective To investigate the detection capacity of malic enzyme 1 and isocitrate dehydrogenase 1 ( Mod1&Idh1) in the laboratory animal monitoring laboratories in China in order to understand the detection capacity of la-boratories and to improve the detection level of laboratory animals’ quality.Methods Based on the program approved by CNAS, samples preparing, homogeneity test and stability test of malic enzyme 1 and isocitrate dehydrogenase 1 in the mouse kidneys were carried out.Standard operation procedure and samples with random numbers were distributed to the la-boratories.The laboratories should submit the result reports before the time limit expires.If the laboratory reports were the same with the standard results, the laboratories will receive satisfactory remark.If laboratory reports were not the same with the standard results, the laboratories will receive unsatisfactory remark.If a laboratory did not submit report, the laboratory will also receive unsatisfactory result.Results Eight laboratories out of 10 (80%) enrolled laboratories reported satisfac-tory experiment results, and two laboratories (20%) presented unsatisfactory results.Conclusions The whole detection level of laboratories in Mod1 &Idh1 is relatively high in the laboratory animals monitoring laboratories in China.It can re-flect the detection level of laboratories to conduct the laboratory capacity evaluation.

Effects of overexpression of carboxylation pathway genes and inactivation of malic enzymes on malic acid production in Escherichia coli / 生物工程学报

Malic acid is a dicarboxylic acid that is widely used in food, pharmaceutical and chemical industries. We studied the effects of overexpression of carboxylation pathway genes and inactivation of malic enzymes on the aerobic production of malic acid. Over expression of phosphoenolpyruvate (PEP) carboxylase (ppc) generated strain E21, which increased malic acid production from 0.57 g/L to 3.83 g/L. Then pyc gene from Coryenbacterium glutamicus and pck gene from Actinobacillus succinogenes were overexpressed in E21 separately. The resulting strains E21 (pTrcpyc) and E21 (pTrc-A-pck) produced 6.04 and 5.01 g/L malate with a yield of 0.79 and 0.65 mol/mol glucose, respectively. Deleting two malic enzymes (encoded by maeA and maeB) also led to an increase of 36% in malic acid production with a production of 5.21 g/L. However, the combination of malic enzymes deletion and pyc overexpression could not further increase the yield of malic acid. After optimization of fermentation conditions, strain E21 (pTrcpyc) produced 12.45 g/L malic acid with a yield of 0.84 mol/mol which is 63.2% of the theoretical yield.

A Pilot Study for Discovering Candidate Genes of Chromosome 18q21 in Methamphetamine Abusers: Case-control Association Study

OBJECTIVE: It was previously suggested that the malic enzyme 2 (ME2) as the candidate gene for psychosis in fine mapping of chromosome 18q21. Chromosome 18q21 is also one of the possible regions that can contribute to addiction. METHODS: We performed a pilot study for discovering candidate gene of chromosome 18q21 in the methamphetamine abusers for elucidating the candidate gene for methamphetamine addiction leading to psychosis. We have selected 30 unrelated controls (16 males, 14 females; age=59.8+/-10.4) and 37 male methamphetamine abusers (age=43.3+/-7.8). We analyzed 20 single nucleotide polymorphisms (SNPs) of 7 neuronal genes in chromosome 18q21 for DNA samples that was checked for the data quality and genotype error. The association between the case-control status and each individual SNP was measured using multiple logistic regression models (adjusting for age and sex as covariates). And we controlled false discovery rate (FDR) to deal with multiple testing problem. RESULTS: We found 3 significant SNPs of 2 genes in chromosome 18q21 (p-value<0.05; adjusting for age as covariate) in methamphetamine abusers compared to controls. We also found 2 significant SNPs of 1 gene (p-value<0.05; adjusting for age and sex as covariates) (rs3794899, rs3794901:MAPK4). Two SNPs in MAPK4 gene were significant in both statistical groups. CONCLUSION: MAPK4, the gene for mitogen-activated protein kinase 4, is one of the final 6 candidate genes including ME2 in 18q12-21 in our previous finemapping for psychosis. Our results suggest that MAPK4 can be a candidate gene that contribute to the methamphetamine addiction leading to psychosis.

Effects of the purified extracts from Lycii Cortex Radicis and ginger on lipid statusand serum cytokine levels in rats fed high fat diet

The present study was to investigate the effects of Lycii Cortex Radicis (LCR), the root bark of lycium (Lycium chenese Miller) and ginger (Gin) on body lipid status and serum levels of cytokines. Sprague-Dawley (SD) male rats weighing 193.6 +/- 16.8 g were divided into five groups, including one low fat (LF) and four high fat groups, i.e. HF-Control, HF-LCR, HF-Gin and HF-LCR + Gin groups. Diets for HF-LCR, HF-Gin and HF-LCR + Gin groups contained purified extracts having 0.2 g LCR tyramine, ginerol and 0.1 g tyramine plus 0.02 g gingerol per kg, respectively. Compared with those of the HF-Control total serum cholesterol level decreased, and HDL-cholesterol level increased in the HF-LCR group and serum triglyceride levels decreased in the three experimental groups fed the purified extracts. Liver cholesterol level was lower in the HF-LCR group than the HF-Control group, but triglyceride levels, which were increased by high fat diets were not changed by significantly by LCR or ginger extracts. Fecal lipid excretion was higher in the HF-LCR and HF-Gin groups, but cholesterol excretion was lower in the HF-Gin group than in the HF-Control group. The activities of liver cytosolic glucose-6-phosphate dehydrogenase and malic enzyme were lower in the HF-LCR + Gin group than in the HF-Control group. Serum adiponectin levels did not differ among the five groups, while leptin level was lower in the HF-Gin group and C-reactive protein levels were lower in the HF-Gin and the HF-LCR + Gin groups than in the HF-Control group. It is concluded that LCR can be utilized as an ingredient for lipid-lowering functional foods in the form of purified extract and addition of small amount of ginger extract would be useful for reducing one of the inflammatory cytokines to help prevent atherosclerosis.

Hydrogenosomal activity of Trichomonas vaginalis cultivated under different iron conditions

To evaluate whether iron concentration in TYM medium influence on hydrogenosomal enzyme gene expression and hydrogenosomal membrane potential of Trichomonas vaginalis, trophozoites were cultivated in irondepleted, normal and iron-supplemented TYM media. The mRNA of hydrogenosomal enzymes, such as pyruvate ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin and malic enzyme, was increased with iron concentrations in T. vaginalis culture media, measured by RT-PCR. Hydrogenosomal membrane potentials measured with DiOC6 also showed similar tendency, e.g. T. vaginalis cultivated in iron-depleted and iron-supplemented media for 3 days showed a significantly reduced and enhanced hydrogenosomal membrane potential compared with that of normal TYM media, respectively. Therefore, it is suggested that iron may regulate hydrogenosomal activity through hydrogenosomal enzyme expression and hydrogenosomal membrane potential.

Effect of Overexpression of NAD+-dependent Malic Enzyme on Anaerobic Mixed Acid Fermentation of E.coli FMJ39 / 中国生物工程杂志

In order to investigate the effect of malic enzyme on anaerobic mixed acid fermentation,the gene encoding NAD+-dependent malic enzyme was amplified from E.coli DH5? genome by using PCR and cloned into vector pTrc99a to give an expression vector pTrc99a-sfcA.Malic enzyme was overproduced by E.coli FMJ39(ldh,pfl)harboring pTrc99a-sfcA under the condition of IPTG induction.The results of anaerobic fermentation indicated overexpression of malic enzyme would have influence on the pathway of formate,acetate and succinate of FMJ39.The concentration of formate and acetate was 17.58% and 15.27% higher than FMJ39,the succinate concentration was reduced by 26.87%.There was no obvious citrate concentration change.The experimental results also indicated that high concentration of L-Thr and L-Ser would induce the Tdc operon to convert pyruvate into formate and acetate even when pfl gene encoding the pyruvate formate lyase was inactivated.