The gene encoding thermostable lactate dehydrogenase (Tm-LDH) was cloned into the plasmid pHsh from Thermotoga maritima, and expressed in Escherichia coli JM 109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 33 kDa. The optimal temperature and pH of Tm-LDH were observed 95 degrees C and 7.0. The purified enzyme had a half-life of 2 h at 90 degrees C, and exhibited better stability over a pH range from 5.5 to 8.0. The K(m) and V(max) values were 1.7 mmol/L, 3.8 x 10(4) U/mg of protein for pyruvate, and 7.2 mmol/L and 1.1 x 10(5) U/mg for NADH, respectively. The expression of Tm-LDH in T7 system could not obtain high efficiency, but it has been soluble over-expression in pHsh system and reached 340 mg/L. The superior stability and productivity of Tm-LDH will lay the foundation of its industrial-scale fermentation and application in the NAD regeneration.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; metabolism ; L-Lactate Dehydrogenase ; biosynthesis ; Molecular Weight ; Recombinant Proteins ; biosynthesis ; Temperature ; Thermotoga maritima ; enzymology

OBJECTIVETo explore the effect of different culture media on viability and expression of tau protein in organotypic hippocampal slice.

METHODSBrain slices (400 microm) from 1, 2, 4, and 8 week-old Wistar rats were prepared and cultured in minimum essential medium (MEM) or Dulbecco's modified eagle medium: nutrient mixture (DMEM/F12) medium respectively for 21 days. Viability of the slices was measured by lactate dehydrogenase (LDH) assay and expression of tau protein was detected by Western blot.

RESULTSThe viability of the slices was not influenced significantly by the two different culture media, while the expression level of tau protein was significantly higher in DMEM/F12 than in MEM (P < 0.05), especially in the slices from 2 and 4 week-old rats.

CONCLUSIONThe slices from 2 or 4 week-old rat hippocampi and DMEM/F12 medium may be the preferred choice for tau associated researches. An ideal Alzheimer's disease model may be established based on the results of these researches.

Animals ; Culture Media ; Hippocampus ; growth & development ; metabolism ; L-Lactate Dehydrogenase ; biosynthesis ; Organ Culture Techniques ; methods ; Rats ; Rats, Wistar ; tau Proteins ; biosynthesis ; genetics

In order to obtain a yeast strain able to produce L-lactic acid under the condition of low pH and high lactate content, one wild acid-resistant yeast strain isolated from natural samples, was found to be able to grow well in YEPD medium (20 g/L glucose, 20 g/L tryptone, 10 g/L yeast extract, adjusted pH 2.5 with lactic acid) without consuming lactic acid. Based on further molecular biological tests, the strain was identified as Candida magnolia. Then, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into C. magnolia by electroporation method. Subsequently, a recombinant L-lactic acid producing yeast C. magnolia-2 was obtained. The optimum pH of the recombinant yeast is 3.5 for lactic acid production. Moreover, the recombinant strain could grow well and produce lactic acid at pH 2.5. This recombinant yeast strain could be useful for producing L-lactic acid.
Candida ; genetics ; isolation & purification ; metabolism ; Genetic Vectors ; genetics ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Lactic Acid ; biosynthesis ; Metabolic Engineering ; Recombination, Genetic ; Rhizopus ; enzymology ; genetics ; Transformation, Bacterial

OBJECTIVESTo construct a prokaryotic recombinant vector for mouse lactate dehydrogenase-C and to detect its expression in BL21.

METHODSThe coding sequence of mouse lactate dehydrogenase subunit C was amplified from mouse testis RNA with specific primers, and cloned into pGEX-2T after the restriction digestion with BamH I and EcoR I. GST fusion protein was expressed after induction with IPTG.

RESULTSSequencing and restriction digestion of the recombinant plasmid revealed the existence of coding sequence for mouse lactate dehydrogenase subunit C. A protein band of about 60,000 could be induced by IPTG in the recombinant plasmid.

CONCLUSIONSThe coding sequence of mouse lactate dehydrogenase subunit C was introduced into the pGEX-2T plasmid and a GST-fused protein could be induced at a high level.

Animals ; Escherichia coli ; genetics ; Glutathione Transferase ; genetics ; Isoenzymes ; genetics ; L-Lactate Dehydrogenase ; genetics ; Male ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; Spermatozoa ; enzymology

OBJECTIVETo establish stress adaptation model of mouse fibroblast cell line NIH-3T3, to provide a group of parallel object for stress adaptation research, and to explore the function and mechanism of HSP90 in stress adaptation.

METHODSA stress-adapted cell model was established by thermal preconditioning (42 degrees C, 20 minutes), and the adaptation result was evaluated by observing the change of the membrane injury and the damage of DNA induced by the heat stress for the second time (44 degrees C, 20 minutes). The HSP90 content was detected by Western blot.

RESULTSAccording to the membrane injury and HSP90 synthesis induced by the heat stress for the second time, it was primarily confirmed that 6 hours after thermal preconditioning were the optimum stress protection time. When cells underwent heat stress for the second time 6 hours after thermal preconditioning, the membrane injury (15.4% +/- 2.6% vs 41.2% +/- 5.1%), damage of DNA (15.1% vs 26.3%) were decreased compared with the control group in which there was no preconditioning. The OD(HSP90)/OD(control) value indicated that the cellular HSP90 contents was decreased immediately after heat stress (44 degrees C, 40 min). The content of HSP90 was 0.82 +/- 0.18 in the heat stress group, 1.70 +/- 0.52 in the preconditioning group and 1.41 +/- 0.16 in the heat stress after preconditioning group.

CONCLUSIONWith the preconditioning for the NIH-3T3, the time point for the stress protection is confirmed, the model for the cellular stress adaptation is established and the protective effect of HSP90 is primarily confirmed in this model.

Adaptation, Physiological ; physiology ; Animals ; DNA Damage ; HSP90 Heat-Shock Proteins ; biosynthesis ; Heat Stress Disorders ; metabolism ; physiopathology ; L-Lactate Dehydrogenase ; metabolism ; Mice ; NIH 3T3 Cells

Corynebacterium glutamicum SA001 is a mutant with lactate dehydrogenase (ldhA) deletion. In order to increase metabolic flux from isocitrate to succinate, and to improve the production of succinate under anaerobic conditions,we transducted the gene aceA coding isocitrate lyase (ICL) from Escherichia coli K12 into Corynebacterium glutamicum SA001 (SA001/pXMJ19-aceA). After 12 h aerobic induction by adding 0.8 mmol/L of IPTG, the recombinant strain was transferred to anaerobic fermentation for 16 h. Succinate reached 14.84 g/L, with a productivity of 0.83 g/(L x h). Compared to C. glutamicum SA001, the activity of ICL of the recombinant strain was increased 5.8-fold, and the succinate productivity was increased 48%. Overexpression of isocitrate lyase will increase the metabolic flux of glyoxylate bypass flowing to succinate.
Corynebacterium glutamicum ; genetics ; metabolism ; Escherichia coli ; enzymology ; genetics ; Gene Deletion ; Industrial Microbiology ; Isocitrate Lyase ; biosynthesis ; genetics ; L-Lactate Dehydrogenase ; genetics ; Succinic Acid ; metabolism ; Transduction, Genetic

Glycerol from oil hydrolysis industry is being considered as one of the abundent raw materials for fermentation industry. In present study, the aerobic and anaerobic metabolism and growth properties on glycerol by Esherichia coli CICIM B0013-070, a D-lactate over-producing strain constructed previously, at different temperatures were investigated, followed by a novel fermentation process, named temperature-switched process, was established for D-lactate production from glycerol. Under the optimal condition, lactate yield was increased from 64.0% to 82.6%. Subsequently, the yield of D-lactate from glycerol was reached up to 88.9% while a thermo-inducible promoter was used to regulate D-lactate dehydrogenase transcription.
Aerobiosis ; Anaerobiosis ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Glycerol ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lactic Acid ; biosynthesis ; Promoter Regions, Genetic ; genetics ; Temperature

OBJECTIVEA simple liver cold preservation model was established to study the synthesis of heat shock protein 70 (HSP70) induced by zinc (ZnSO(4), i.p.) and its protection during liver cold preservation in rat.

METHODSMale Wistar rats were divided into 5 groups (n = 6). In control group rat received no pretreatment; in Zn-1 group, Zn-2 group, and Zn-3 group rats were pretreated with zinc sulfate at a dose of 5 mg/kg, 10 mg/kg, 15 mg/kg respectively; and in H group rat received heat shock preconditioning (42.5 degrees C x 15 min). Livers were preserved in UW solution for 6, 12 and 24 h, respectively. HSP70 was analyzed by Western blot. Aspartate transaminase (AST) and lactate dehydrogenase (LDH) values of the perfusion solution and the histology of the liver were evaluated.

RESULTSHSP70 expression was markedly elevated after pretreatment with zinc and heat shock. AST and LDH values in the Zn-1, Zn-2 and H groups were significantly lower than those in the control group, respectively (P < 0.05). There was no significant difference among the three groups (P > 0.05), whereas the AST and LDH values in the Zn-3 group were much higher than those in the control group. Histology results showed that liver injury in the Zn-1, Zn-2 and H groups were minimal, while it was severe in the Zn-3 group.

CONCLUSIONSZn(2+) is a potent and feasible inducer of HSP expression and is able to protect liver from cold preservation injury. The proper inducing dosage of Zn(2+) ranged from 5 mg/kg to 10 mg/kg. The dosage of 15 mg/kg for Zn(2+) as a HSP inducer is not indicated for its severe toxicity to the liver.

Animals ; Aspartate Aminotransferases ; blood ; Dose-Response Relationship, Drug ; HSP70 Heat-Shock Proteins ; biosynthesis ; L-Lactate Dehydrogenase ; blood ; Liver ; pathology ; Male ; Organ Preservation ; Rats ; Rats, Wistar ; Zinc ; pharmacology

To investigate the effects of metallothionein (MT) on isolated rat heart, 16 Wistar rats were randomly divided into 2 groups. In control group (group C), distilled water was injected intraperitoneally and 24 h later isolated hearts were perfused with Langendorff and stored at 4 degrees C for 3 h with histidine-tryptophan-ketoglutarate (HTK) solutions, and then isolated hearts were perfused for 2 h by Langendorff. In experimental group (group E), 3.6% ZnSO(4) was injected intraperitoneally, 24 h later isolated hearts were perfused by Langendorff and stored at 4 degrees C for 3 h with HTK solutions, and then the isolated hearts were perfused for 2 h with Langendorff. MT content, the recovery of hemodynamics, myocardial water content (MWC), lactate dehydrogenase (LDH) and creatine kinase (CK) leakage, adenosine triphosphate (ATP) and malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, myocardial cell Ca(2+) content, Ca(2+)-ATPase activity of mitochondria ([Ca(2+)-ATPase](m)) and its Ca(2+) content ([Ca(2+)](m)), synthesizing ATP activity of mitochondria ([ATP](m)), and the ultrastructure of cells were examined. There were a significant increase in group E in hemodynamic recovery, ATP content, SOD activity, [Ca(2+)-ATPase](m) activity, [ATP](m) activity, and substantial reduction in MWC, LDH and CK leakage, MDA content, myocardial cell Ca(2+) content, [Ca(2+)](m) content, and the ultrastructural injury were obviously milder than that of group C. This study demonstrated that MT has protective effects on isolated rat heart.
Cardiotonic Agents/*pharmacology ; Creatine Kinase/*metabolism ; L-Lactate Dehydrogenase/metabolism ; Metallothionein/biosynthesis ; Metallothionein/*pharmacology ; Myocardium/*metabolism ; Myocardium/ultrastructure ; Random Allocation ; Rats, Wistar ; Superoxide Dismutase/metabolism ; Zinc Sulfate/pharmacology

Escherichia coli NZN111 is a double mutant with lactate dehydrogenase (ldhA) and pyruvate formate-lyase (pflB) inactivated. Under anaerobic conditions, disequilibrium of coenzyme NADH and NAD+ causes Escherichia coli NZN111 losing the glucose utilizing capability. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-mdh and overexpressed the mdh gene with 0.3 mmol/L of IPTG under anaerobic fermentation condition in sealed bottles. The specific malate dehydrogenase (MDH) activity in the recombinant strain was 14.8-fold higher than that in E. coli NZN111. The NADH/ NAD+ ratio decreased from 0.64 to 0.26 and the concentration of NAD+ and NADH increased 1.5-fold and 0.2-fold respectively. Under anaerobic conditions, the recombinant strain possessed the capability of growth and glucose absorption. We took dual-phase fermentation for succinate production. After the dry cell weight (DCW) reached 6.4 g/L under aerobic conditions, the cell culture was changed to anaerobic conditions. After 15 h, 14.75 g/L glucose was consumed and succinic acid reached 15.18 g/L. The yield of succinic acid was 1.03 g/g Glu and the productivity of succinic acid was 1.012 g/(L x h).
Acetyltransferases ; genetics ; Anaerobiosis ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Gene Knockout Techniques ; Glucose ; metabolism ; L-Lactate Dehydrogenase ; genetics ; Malate Dehydrogenase ; genetics ; metabolism ; Mutation ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Succinic Acid ; metabolism