Prokaryotic Expression of Gene Encoding Glutamate Dehydrogenase of Streptococcus suis Serotype 2 and Preparation of Polyclonal Antibodies against Its Expressed Products

[ Objective] To obtain detection antigen for diagnosis of Streptococcus suis infection. [ Method] The complete ORF of glutamate dehy- drogenase （GDH） gene was amplified from the genomic DNA of Streptococcus suis serotype 2 strain SC22 isolated in Sichuan Province by poly- merase chain reaction （PCR）. The resulting product was cloned into the prokaryotic expression vector pET-30a, which was then transformed into E. coil BL21 （DE3）. The identified positive transformants were screened for expression induced by IPTG. The expression products were subjected to SDS-PAGE and the recombinant protein was purified by nickel ion-agarose affinity chromatography. New Zealand rabbits were immunized with the purified recombinant GDH protein to prepare polyclonal antibodies. Titers of the anti-serum were determined by indirect ELISA and Western blot assay. [ Result] The recombinant GDH protein was effectively expressed in the host bacteria, and highly pure recombinant protein was obtained by nickel ion-agarose affinity chromatography. High-titer anti-serum against the recombinant protein was obtained. As evidenced by western blot as- say, the sera could react specifically with the lysates of all detected Streptococcus suis strains. In addition, the recombinant GDH protein could re- act specifically with serum samples collected from five pigs experimentally infected by strain SC22. [ Conclusion] The expressed GDH fusion protein has some common epitopes of natural GDH and can be used as detection antigen to develop ELISA and other diagnostic methods.

Effect of Exogenous Ammonium on GlutamineSynthetase, Glutamate Synthase, and Glutamate Dehydrogenase in the Root of Rice Seedling

Root biomass of rice seedlings was increased at lower concentration of exogenous NH 4 + , but it was decreased at higher concentration of exogenous NH 4 + . The level of free NH 4 + in the roots was accumulated gradually with the increase of NH 4 + concentration in the nutrient solution. The content of the soluble proteins was essentially constant at higher NH 4 + . The activities of glutamine synthetase (GS), NADH-dependent glutamate synthase (NADH-GOGAT), and NADH-dependent glutamate dehydrogenase (NADH-GDH) were risen with exogenous NH 4 + concentration at the lower NH 4 + concentration range. But the activities of GS and NADH-GOGAT were declined, and the level of NADH-GDH activity was kept constant under higher NH 4 + concentration. The GS/GDH ratio suggested that NH 4 + was assimilated by GS-GOGAT cycle under lower NH 4 + concentration, but NADH-GDH was more important for NH 4 + assimilation and detoxifying NH 4 + to the tissue cells at the higher NH 4 + level. According to the growth and the activity changes of these ammonium-assimilating enzymes of rice seedling roots, 10. 0 μg/mL NH 4 + -N in nutrient solution was more suitable to the rice growth.

Optimization of Cowpea Dry Grain Yield through the Stimulation of the RNA Synthetic Activity of NADH-Glutamate Dehydrogenase

Several potentially practical biochemical processes in plant systems still remain hidden, especially the NADH-glutamate dehydrogenase (GDH) synthesis of nongenetic code-based RNA that optimizes crop nutritious yield by degrading superfluous genetic code-based RNA. In continued characterization of the biochemistry of cowpea grain yield, GDH was purified by electrophoresis from seeds of cowpea treated with solutions of stoichiometric mixes of mineral salts. The GDH was made to synthesize RNAs in the amination (α-KG/NADH/) and then in the deamination (L-Glu/NAD+) direction. The initial product RNAs were captured and sequenced. The grand challenge was to discover the specific molecular roles of the redox enzyme in the optimization of cowpea grain yields. In the amination direction, the GDH hexamers synthesized plus-RNA, but in the deamination direction, they synthesized minus-RNA. The plus-RNAs and minus-RNAs were homologous to about the same numbers of different mRNAs encoding the key enzymes that regulate photosynthesis;saccharide biochemistry and glycolysis;phenylpropanoid biosynthesis;nodulation nitrogen fixing processes;dehydrin drought and glutathione environmental stress resistance processes;purine, pyrimidine, DNA, RNA and essential amino acid biosynthesis;storage protein vicilin accumulation;isoflavone earliness of cowpea maturity;peroxidase synthesis of lignin and sequestration of CO2 to enrich soil organic carbon contents;triglyceride physiology in the biosynthesis of bioactive compounds that render cowpea resistant to insects and fungi;etc., all of which constitute the GDH chemical pathways for discrimination of biochemical, physiological, metabolic, genetic reactions;and optimization of cowpea dry grain yields. Each stoichiometric mix of mineral salts produced optimally yielding biochemical variant of purple hull cowpea;the K + K + K mix was spectacular because it increased the grain yield to 7598 kg from the 3644 kg·ha-1 in the control cowpea. Optimized nutritious staple crop yield buttresses food security. The synthesis of plus-RNA in amination and minus-RNA in deamination is an economic tactical plan in biochemistry for the selection of superfluous mRNAs that would be degraded to assure the survival of cowpea growing under unfavorable environmental conditions.

Glutamate dehydrogenase and Na^+-K^+ ATPase expression and growth response of Litopenaeus vannamei to different salinities and dietary protein levels

Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture.The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp(L.vannamei) were investigated.This involved an examination of growth performance,glutamate dehydrogenase(GDH) and Na+-K+ ATPase mRNA expression,,and GDH activity in muscles and gills.Three experimental diets were formulated,containing 25%,40%,and 50% dietary protein,and fed to the shrimp at a salinity of 25.After 20 days,no significant difference was observed in weight gain,though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels.Subsequently,shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5,respectively,and sampled at weeks 1 and 2.Shrimp fed with 40% protein at 25 in salinity(optimal conditions) were used as a control.Regardless of the salinities,shrimp fed with 50% dietary protein had significantly higher growth performance than other diets;no significant differences were found in comparison with the control.Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks.Ambient salinity change also stimulated the hepatosomatic index,which increased in the first week and then recovered to a relatively normal level,as in the control,after 2 weeks.These findings indicate that in white shrimp,the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism.Increased dietary protein level could improve the osmoregulation capacity of L.vannamei with more energy resources allocated to GDH activity and expression.

Purification and characterization of a thermostable glutamate dehydrogenase from a thermophilic microorganism from Deception Island, Antarctica

Glutamate dehydrogenase （GDH） catalyzes the oxidative deamination of glutamate to a-ketoglutarate and ammonium ions. Currently the determination of ammonium and glutamate is carried out using a bovine GDH enzyme, which lacks optimal thermostability for long term storage at room temperature. From samples of Deception Island, Antarctica, we obtained the thermophilic bacteria PID 15 belonging to the Bacillus genera with high GDH specific activity. This new enzyme exhibited NAD＋ dependent activity and no activity was observed when NADP＋ was used. This enzyme shows a specific activity of 4.7 U.mg-1 for the oxidative deamination reaction and 15.4 U·mg-1 for the reduction of a-ketoglutarate. This enzyme has an optimum temperature of 65℃ and pH of 8.5 for the oxidative deamination. For the reduction of a-ketoglutarate, the optimum temperature is 60℃, with a pH of 8.0. One of the most important characteristics of this enzyme is its ability to retain more than 60% of its activity when it is incubated for 8 h at 65℃. The enzyme is also able to retain full activity when it is incubated for 48 d at 4℃ and over 80% of its activity when it is incubated at 25℃. Characterization of its kinetics suggests that it primarily catalyzes the formation of α-ketoglutarate. This enzyme has an important biological role in the catabolism of glutamate and may have some interesting biotechnological applications based on its thermostable properties.

Involvement of the ammonium assimilation mediated by glutamate dehydrogenase in response to heat stress in the scleractinian coral Pocillopora damicornis

Glutamate dehydrogenase(GDH)plays an important role in the ammonium assimilation and nitrogen metabolism by catalyzing the reversible oxidative deamination of L-glutamate toα-ketoglutarate.In the present study,the potential functions of GDH in response to heat stre ss were explored in the scleractinian coral Pocillopora damicornis(designated as PdGDH).The cDNA of PdGDH contained an open reading frame of 1611 bp encoding a polypeptide of 536 amino acids,which exhibited the highest sequence identity to GDH of Stylophora pistillata(96%identity),and the deduced PdGDH protein was predicted to contain one GdhA domain(from Val95 to Tyr525).The recombinant protein of PdGDH(rPdGDH)was expressed in Escherichia coli BL21(DE3)-Transetta,and its catalytic activity was measured under different temperatures,pH conditions and epigallocatechin-3-gallate(EGCG,a GDH inhibitor)concentrations.The purified rPdGDH only used reduced coenzyme nicotinamide adenine dinucleotide(NADH)as coenzyme,and its highe st activity was observed at 35℃and pH 7.5,re spectively.The rPdGDH activity was negatively correlated with the concentration of EGCG,and was inhibited by more than half(65%,P<0.05)at 10mol/L EGCG.No significant alteration of PdGDH mRNA expression was detected at 12 h after exposure to heat and ammonium(P>0.05).Furthermore,the activities of NADH-GDH in the scleractinian coral P.damicornis increased significantly at 12 h after the heat and ammonium stress,and the NADH-GDH activity in the heat stress group(32.66 U/mg,P<0.05)was significantly higher than that in the heat and ammonium stress group(11.26 U/mg).These results collectively suggested that PdGDH,as a homologue of glutamate dehydrogenase in the scleractinian coral P.damicornis,could respond to heat stress at the protein level,which would have ability to further promote ammonium assimilation to increase the heat acclimatization of the coral-Symbiodiniaceae symbiotic association.

The Role of Glutamate Dehydrogenase Activity in Development of Neurodegenerative Disorders

The specific role of Glutamate dehydrogenase (GLDH) in the brain is not yet clear, but it is an important enzyme in protein degradation as well as a metabolism regulator of glutamate as a neurotransmitter. The enzyme probably provides crucial protection for postsynaptic membranes against the neurotoxic effects of glutamate neurotransmitters. In men, GLDH activity declines almost evenly through the ages;in women, it declines faster in the first five decades. In the years of menopause, GLDH activity declines slower. The diminished GLDH activities in leukocytes and in the brain vary considerably, but they are parallel with the progress of neurodegenerative diseases. The GLDH activity is partly deficient in the brain, particularly in the leukocytes of patients with heterogeneous neurological disorders and degeneration of multiple neuronal systems. We found a statistically significant difference of GLDH activity in the cerebrospinal fluid in patients with neurological diseases and unexpected in patients with degenerative and inflammatory disorders. The decrease in GLDH activity in the cerebrospinal fluid of patients with neurodegenerative disorders may be one of the reasons for the neuro-excito-toxic glutamate effect. Defining the GLDH activity in leukocytes is at the moment the sole experimental method. The second one could be the measurement in cerebrospinal fluid. The results suggest a possibility to regulate glutamate level in human brain through activation of GLDH.

Research on Glutamate Dehydrogenase Activity in Sugar Beet (Beta vulgaris L) under Different Nitrogen Levels

The experiment of Glutamate Dehydrogenase (GDH) activity in various plant parts under different nitrogen levels in frame culture during the whole period of growth was carried out on campus of Northeast Agricltural University in 1993. The result showed that GDH activity in leaf blades under four nitrogen applied levels rose rapidly to the acme from the seedling to foliage rapid growth stage, then diminished rapidly to the lower level at the latter stage of foliage rapid growth. This level was kept to harvest. GDH activity in roots at each growth stage under all nitrogen levels exhibited little disparity and did not show ostensible regularity of changes. GDH activity in leaf blades was stimulated with nitrogen, however, it reduced with nitrogen fertilizer applying further. GDH activity in leaf blades was the biggest compared with crowns, petioles and roots, which suggested that it could represent the highest enzyme activityof the whole plant.

ldhA基因敲除对Corynebacterium glutamicum TCCC11822发酵生产L-谷氨酸的影响

针对L-谷氨酸发酵过程中乳酸产量偏高的问题,以L-谷氨酸生产菌谷氨酸棒杆菌(Corynebacterium glutamicum)TCCC11822为出发菌株,通过构建敲除质粒pK18mobsacB△ldh并采用同源重组技术敲除其乳酸脱氢酶编码基因ldhA,以期达到减少副产物、提高L-谷氨酸产量和转化率的目的。结果表明,与出发菌株相比ldhA基因敲除株的乳酸合成量降低了85.6%,L-谷氨酸的产量和转化率分别提高7.6%和5.5%,但生物量略有下降。本研究可为L-谷氨酸及其他氨基酸生产菌株的理性改造提供参考。

GLUD1 在恶性黑色素瘤及非黑色素瘤皮肤癌中的表达及意义

目的比较谷氨酸脱氢酶1(GLUD1)在正常皮肤、光线性角化病(AK)、皮肤鳞状细胞癌(cSCC)、基底细胞癌(BCC)、皮内痣及恶性黑色素瘤(MM)组织中的表达情况。方法通过免疫组化链霉亲和素-生物素-过氧化物酶复合物技术(SABC法)检测30例AK、30例BCC、30例cSCC与30例正常皮肤组织中GLUD1的表达情况;采用相同方法比较30例皮内痣与30例MM组织标本中GLUD1的表达差异。结果GLUD1在cSCC组、BCC组和AK组中阳性细胞率分别为(40.73±3.50)%、(33.11±2.90)%和(29.68±4.08)%,均显著高于正常皮肤组(16.37±2.14)%,其中cSCC组中阳性细胞率显著高于AK组及BCC组。GLUD1在MM组中阳性细胞率显著高于皮内痣组[(48.43±4.66)%比(19.64±2.45)%]。GLUD1在正常皮肤、AK、BCC和cSCC组织中染色强阳性率分别为0.00%(0/30)、13.33%(4/30)、3.33%(1/30)和26.67%(8/30),cSCC组显著高于正常皮肤组,差异有统计学意义(P<0.05)。GLUD1在皮内痣组和MM组的染色强阳性率分别为0.00%(0/30)和50.00%(15/30),差异有统计学意义(P<0.05)。结论GLUD1的高表达可能与AK、BCC、cSCC和MM的发病相关。

Cloning and characterization of the glutamate dehydrogenase gene in Bacillus licheniformis

The gdhA genes of IRC-3 GDH strain and IRC-8 GDH+ strain were cloned, and they both successfully complemented the nutritional lesion of an E. coli glutamate auxotroph, Q100 GDH". However, the gdhA gene from the mutant IRC-8 GDH+ strain failed to complement the glutamate deficiency of the wild type strain IRC-3. The gdhA genes of the wild type and mutant origin were sequenced separately. No nucleotide difference was detected between them. Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant. Additionally, no GDH inhibitor was found in the wild type strain IRC-3. It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression. Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the family I -type hexameric protein, while the GDH of Bacillus subtilis belongs to family II.

L-leucine stimulates glutamate dehydrogenase activity and glutamate synthesis by regulating mTORC1/SIRT4 pathway in pig liver

The liver is the most essential organ for the metabolism of ammonia, in where most of ammonia is removed by urea and glutamine synthesis. Regulated by leucine, glutamate dehydrogenase(GDH) catalyzes the reversible inter-conversion of glutamate to ammonia. To determine the mechanism of leucine regulating GDH, pigs weighing 20 ± 1 kg were infused for 80 min with ammonium chloride or alanine in the presence or absence of leucine. Primary pig hepatocytes were incubated with or without leucine. In the in vivo experiments with either ammonium or alanine as the nitrogen source, addition of leucine significantly inhibited ureagenesis and promoted the production of glutamate and glutamine in the perfused pig liver(P < 0.05). Similarly, leucine stimulated GDH activity and inhibited sirtuin4(SIRT4)gene expression(P < 0.01). Leucine could also activate mammalian target of rapamycin complex 1(m TORC1) signaling(P < 0.05), as evidenced by the increased phosphorylation levels of ribosomal protein S6 kinase 1(S6 K1) and ribosomal protein S6(S6). Interestingly, the leucine-induced m TORC1 pathway activation suitably correlated with increased GDH activity and decreased expression of SIRT4.Similar results were observed in primary cultured hepatocytes. Notably, leucine exerted no significant change in GDH activity in SIRT4-deficient hepatocytes(P > 0.05), while m TORC1 signaling was activated.Leucine exerted no significant changes in both GDH activity and SIRT4 gene expression in rapamycin treated hepatocytes(P > 0.05). In conclusion, L-leucine increases GDH activity and stimulates glutamate synthesis from different nitrogen sources by regulating m TORC1/SIRT4 pathway in the liver of pigs.

A chronoamperometry method for study on effect of lanthanide ions on glutamate dehydrogenase

Glutamate dehydrogenase occupies a pivotal position in the metabolism of most organisms. The reductive amination of α-ketoglutarate catalyzed by the enzyme is one of the most important reactions in the nitrogen assimilation with NADH as coenzyme.

过表达AngdhA对水稻生长与氮素利用效率的影响

【目的】研究过表达黑曲霉AngdhA基因对水稻生长、氮代谢关键酶基因表达、碳氮含量、氮素利用效率、产量及耐盐性的影响,为水稻节本增效、高产和高抗多目标育种提供科学参考。【方法】以水稻转基因株系TG3、TG13与野生型(wild-type,WT)为材料进行水培试验。常规生长试验在水稻幼苗3叶期取样,分析谷氨酰胺合成酶、谷氨酸合酶编码基因的表达及碳、氮含量;成熟期取样测定水稻产量、碳和氮含量及氮素利用效率。抗盐胁迫试验以3叶期幼苗为试材,设置营养液盐胁迫浓度分别为0、50、100 mmol/L NaCl,在胁迫开始后0、2、4天,取样分析水稻幼苗相对生长速率和脯氨酸含量,调查叶片萎蔫率和各部位生物量。【结果】PCR扩增和电泳检测证实黑曲霉AngdhA基因成功导入水稻TG3和TG13株系,qRT-PCR荧光定量分析发现,TG3和TG13株系中黑曲霉AngdhA基因表达量分别为内参UBI基因的248和41倍。但TG3和TG13株系中氮代谢关键酶基因OsGS1;1、OsGS1;2和OsGOGAT表达量与WT差异不显著,仅TG13的OsGS2表达量极显著低于WT。同时,导入黑曲霉AngdhA基因并未明显影响转基因株系TG3和TG13苗期叶片、根和成熟期籽粒、茎叶、根中氮含量和碳/氮值,但TG3和TG13植株体内碳和氮的积累量增加,其中籽粒中碳和氮的积累量均显著高于WT。另外,与WT相比,转基因株系TG3和TG13的氮素利用率分别提高了14.67%和44.12%,氮素籽粒生产效率分别提高了26.96%和39.83%,氮素偏生产力分别提高了24.53%和39.67%。转基因株系TG3和TG13的穗粒数、每株穗数和单株产量显著或极显著提高,其中单株产量较WT分别提高29.18%和43.95%。不同盐浓度胁迫下转基因株系TG3和TG13的相对生长速率极显著高于WT,尤其是在50 mmol/L NaCl条件下,TG3和TG13相对生长速率甚至高于对照条件(0 mmol/L)。盐胁迫下水稻脯氨酸含量明显提高,其中TG3和TG13脯氨酸含量在50 mmol/L NaCl条件下显著高于WT,但在100 mmol/L条件下极显著低于WT。【结论】水稻TG3和TG13株系中的外源AngdhA基因不扰乱转基因水稻体内的碳氮平衡,能促进水稻对氮素的吸收、利用和再活化,提高水稻氮素利用效率、产量和耐盐能力,但TG3中AngdhA基因的超量表达可能增加谷氨酸合成与分解途径的底物循环,使转基因水稻对氮素的吸收、利用和产量增幅下降。盐胁迫下脯氨酸的差异积累可能与水稻对胁迫程度的响应与耐受度存在差异有关。

Effect of Purified Paper Wasp Ropalidia marginata Venom Toxin Enzyme Activity in Blood Serum, Liver, and Gastrocnemius Muscle of Albino Mice

In the present, investigation effects of sub-lethal dose of purified paper wasp Ropalidia marginata venom toxins were evaluated on important metabolic enzymes i.e. ALP ACP, GPT, GOT, LDH, and AchE enzyme activity in serum, liver, and gastrocnemius muscles of albino mice. Alkaline phosphatase was found to be increased up to 119.9% at the 6th hr of the toxin injection in comparison to control. This elevation may be due to cytolysis. Maximum increase i.e., 153.33% level of glutamate pyruvate transaminase (GPT) was found at 6 hrs of 40% of 24-h LD50 treatment while it was found to be 151.1% at 6 hrs of 24 hr 80% of LD50, venom injection. A significant elevation was observed in LDH activity in serum, liver, and muscles, while the activity of AchE was decreased in serum, liver, and gastrocnemius muscles of albino mice after injecting the sub-lethal dose of Ropalidia marginata venom. This increase in the activity of LDH produces liver damage, massive disintegration and necrosis of hepatic cells. This elevation in LDH level led to a significant increase in the glucose catabolism and elevated oxidative stress in muscle and liver cells. It also displays insufficient oxygen supply and consequently leads to cell death. In experimental animals, venom toxin treatment decreased AchE level, and animals showed muscular paralysis. When mice were treated with 40% and 80% of 24-h LD50 of purified venom caused a significant (p < 0.05) elevation in the level of ACP, GOT, GPT, and LDH while the reduction in ALP and AChE level. Present study will be useful in the development of prototypes for study of pharmacological and therapeutic effects of various venom toxins. For this purpose structure activity relationship of enzyme and venom toxin, its due interaction to various metabolic enzymes and receptors must be explored.

Effects of Purified Indian Cattle Tick Rhipicephalus microplus Saliva Toxins on Various Enzymes in Blood Serum, Liver and Muscles of Albino Mice

In the present investigation, in vivo effects of purified ticks’ saliva toxin were evaluated on the level of certain important cellular metabolic enzymes i.e. acid phosphatase (ACP), alkaline phosphatase (ALP), glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and lactic dehydrogenase. For this purpose, sub-lethal doses, 40% and 80% of 24 h LD50 purified saliva toxins of Rhipicephalus microplus (Canestrini, 1888) were injected subcutaneously in the albino mice. In treated mice saliva toxins targeted membrane-bound enzymes i.e. serum acid phosphatase and alkaline phosphatase, its level was increased from 118.30% to 163.63% at the 6th hr in comparison to the control. Besides this, the levels of serum glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) and lactic dehydrogenase (LDH) also increased up to 161.11% (at 6th hr), 148.27 (at 8th hr) and 125.45% (at 6th hr) respectively in comparison to control. An increase in the level of LDH showed insufficient oxygen supply, massive disintegration of cells and leakage of the enzyme into the circulation. It clearly indicated the toxic effects of saliva toxins on the membrane of blood cells, hepatocytes and myocardial muscle cell functions in albino mice. On the other hand activity of acetyl cholinesterase was reduced by 65.51% at the 6th hr of the saliva toxin injection in comparison to the control. This inhibition of acetyl cholinesterase activity caused the accumulation of acetylcholine molecules at the synaptic junctions and led to prolonged activation of acetylcholine receptors. It caused permanent stimulation of nerves and muscle cells that may result in muscular paralysis and finally death of the animal.

十二指肠贾第虫谷氨酸脱氢酶原核表达及动力学分析

为探索十二指肠贾第虫(Giardia duodenalis)谷氨酸脱氢酶(glutamate dehydrogenase,GDH)在滋养体中的分布和酶动力学等生物学特性,对GDH进行了克隆并构建原核表达质粒pET-28a-GDH,然后转化至BL21(DE3)plysS感受态细胞中经IPTG诱导表达,对表达产物进行SDS-PAGE和Western blot分析,以Ni 2+亲和层析纯化。对纯化的重组GDH进行体外酶动力学分析并免疫小鼠制备多克隆抗体,最后用间接免疫荧光技术对滋养体中的GDH进行定位。结果显示,贾第虫GDH的编码序列长1350 bp,试验成功构建pET-28a-GDH重组表达质粒并诱导表达,重组蛋白约为49.7 ku,且大部分以可溶性形式存在。ELISA和Western blot结果表明,贾第虫GDH具有良好免疫原性,获得的多克隆抗体具有较强特异性。间接免疫荧光定位显示GDH主要位于滋养体的胞质中。酶动力学研究结果显示,GDH最适反应pH约为9.3、最适温度约为42℃,对L-谷氨酸和NADP+的Km值分别为4.485 mmol/L±0.5062 mmol/L和119.5μmol/L±10.1μmol/L。研究结果为深入研究贾第虫GDH的生物学功能奠定了基础。

OGDHL与BRCA2蛋白在胃癌组织中的表达及其临床意义

目的:探讨类草酸谷氨酸脱氢酶(OGDHL)与乳腺癌2号基因(BRCA2)蛋白在胃癌组织中的表达及其临床意义。方法:选取本院2016年6月至2018年12月收治的136例胃癌患者癌组织(病例组)及癌旁组织(癌旁组)为研究对象,采用实时荧光逆转录(RT-PCR)及免疫组织化学法检测胃癌组织和癌旁组织中OGDHL、BRCA2 mRNA及蛋白水平,采用χ2检验及Cox回归分析胃癌患者临床病理特征、预后与OGDHL、BRCA2表达的相关性。结果:病例组OGDHL、BRCA2 mRNA表达低于癌旁组(P<0.05)。病例组OGDHL、BRCA2蛋白水平和阳性率低于癌旁组(P<0.05)。OGDHL、BRCA2蛋白水平与胃癌患者年龄无关(P>0.05),与肿瘤最大径、TNM分期、浸润深度、淋巴血管间隙浸润、淋巴结转移、复发相关,且肿瘤最大径≥5 cm、TNM分期越高、浸润深度越深、有淋巴血管间隙浸润、淋巴结转移、复发的患者OGDHL、BRCA2蛋白阳性表达率越低(P<0.05)。OGDHL、BRCA2阳性组生存率明显高于OGDHL、BRCA2阴性组(P<0.05)。OGDHL、BRCA2阴性表达是影响胃癌患者预后的独立危险因素(HR=2.99、4.30,95%CI:1.92~4.56、2.03~9.13,P<0.05)。结论:胃癌组织中OGDHL、BRCA2表达减少,其表达水平与胃癌临床病理特征相关,OGDHL、BRCA2阴性表达是胃癌预后的危险因素。

稀土元素对谷氨酸发酵产酸及其谷氨酸脱氢酶的影响

以目前我国谷氨酸发酵广泛使用的谷氨酸棒杆菌S9114 为实验菌株 ,研究了稀土元素对谷氨酸发酵产酸及其谷氨酸脱氢酶的影响。结果表明LaCl3、CeCl3和NdCl3浓度分别为 0 72 0、0 0 71和 0 0 0 7mmol/L时 ,促使谷氨酸发酵产酸水平提高 6%～ 8% ,对菌体的GDH NADPH的酶活性有显著的激活作用。实验还表明 ,稀土元素对纯化GDH

不同氮源对水稻幼苗根氨同化酶的影响

将氮素含量相同、但来源不同的氮源分别加入到水稻培养液中 ,分析了不同氮源对水稻幼苗根氨同化酶活性的影响 .与无氮培养的水稻根相比 ,( NH4) 2 SO4,NH4NO3,KNO3,谷氨酸 ( Glu)和谷氨酰胺 ( Gln)都能分别促进水稻根谷氨酰胺合成酶 ( GS)、依赖于 NADH的谷氨酸合酶 ( NADH- GOGAT)和依赖于 NADH的谷氨酸脱氢酶 ( NADH- GDH)活性 .( NH4) 2 SO4对 GS和 NADH- GOGAT活性促进最为显著 ,而 Gln对 GS和 Glu对 NADH-GOGAT的活性分别影响最小 .Glu对 NADH- GDH活性的诱导稍强于其它氮源 .这些结果提示 。