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Identification of yak lactate dehydrogenase B gene variants by gene cloning
YuCai Zheng,XingBo Zhao,Jing Zhou,Ying Piao,SuYu Jin,QingHua He,Jian Hong,Ning Li,ChangXin Wu
Science China Life Sciences , 2008, DOI: 10.1007/s11427-008-0036-6
Abstract: Native polyacrylamide gel electrophoresis showed that two types of lactate dehydrogenase (LDH) existed in yaks. Based on the electrophoresis characteristics of LDH isoenzymes, yak LDH variants were speculated to be the gene mutation on H subunit encoded by B gene. According to the mobility in electrophoresis, the fast-band LDH type was named LDH-Hf and the slow-band LDH type LDH-Hs. In order to reveal the gene alteration in yak LDH variants, total RNA was extracted from heart tissues of yaks with different LDH variants, and cDNAs of the two variants were reverse transcripted. Two variants of B genes were cloned by RT-PCR. Sequence analysis revealed that four nucleotides differed between LDH-Bf and LDH-Bs, which resulted in two amino acids alteration. By Deepview software analysis of the conformation of yak LDH1 variants and H subunit, these four nucleotides altered two amino acids that generated new hydrogen bonds to change the hydrogen bonds network, and further caused subtle conformational changes between the two LDH variants.
Cloning and Differential Expression Analysis of Lactate Dehydrogenase Gene in Leiwuqi Yak

- , 2018, DOI: 10.7606/j.issn.1004-1389.2018.10.002
Abstract: 旨在克隆西藏类乌齐牦牛乳酸脱氢酶(LDHB)基因并检测其组织表达情况,为给进一步研究LDHB在高海拔地区的极端低氧适应性及能量代谢中的调控作用提供依据。结果表明,类乌齐牦牛LDHB基因CDS区全长1 004 bp,编码334个氨基酸,存在 2 处碱基突变,导致密码子分别由AAG→GAG(赖氨酸→谷氨酸)、ATC→AGC(异亮氨酸→丝氨酸);编码蛋白分子质量为36.72 ku,为疏水稳定性蛋白,功能预测在糖酵解及氧化还原等过程中发挥主要功能;系统进化树表明,西藏类乌齐牦牛与九龙牦牛、黄牛的氨基酸序列相似性最高,亲缘关系最近,其次为山羊、宽吻海豚、猪、马、羊驼和野生双峰骆驼,与鸡、乌鸦、非洲爪蟾和鲤鱼较远;定量分析显示,LDHB基因在牦牛肺脏中的表达水平显著高于心脏和肝脏,推测该基因与牦牛抗缺氧性状相关。
As a glycolytic enzyme,lactate dehydrogenase (LDHB) shows distinct tissue specificity in vivo,and plays an important role in physiological functions and developmental processes in mammals.The aim of this study is to clone the CDS sequence of LDHB gene and to determine its expression in various tissues in Leiwuqi yak,this will facilitate the function research on how LDHB gene regulates extreme hypoxia adaptability and energy metabolism at high altitudes area.The results showed that the CDS region of LDHB gene was 1 004 bp length,encoding 334 amino acids.A total of 2 nucleotide mutations were found,including a mutation of A362 to G362 and a mutation of T466 to G466,and resulted in the mutation of lysine to glutamate and fromisoleucine to serine respectively.The predicted molecular mass of LDHB protein was 36.72 ku,and predicted to be a hydrophobic stable protein.Functional prediction analysis found that LDHB may played a major role in glycolysis,redox and other processes,mainly involved in the metabolism of organisms.The phylogenetic tree showed that the amino acid sequences of Tibetan leiwuqi yak,Jiulong yak and Bos taurus shared the highest similarity and closest genetic relationship,followed by capra hircus,bottlenose dolphin,sus scrofa,equus caballus,vicugna pacos,and camelus ferus,far from gallus,xenopus laevis,cyprinus carpio.The RT-qPCR analysis showed that the expression of LDHB gene in yak lung was significantly higher than in other tissues,this indicated a potential role of LDHB in anti-hypoxic traits in yak.In conclusion,the structure and function of LDHB gene in yak were relatively conservative.
Identification of yak lactate dehydrogenase B gene variants by gene cloning

ZHENG YuCai,ZHAO XingBo,ZHOU Jing,PIAO Ying,JIN SuYu,HE QingHua,HONG Jian,LI Ning &,WU ChangXin,

中国科学C辑(英文版) , 2008,
Cloning of Lactate dehydrogenase Gene and Effect on the Waterlogging Tolerance of Brassica napus L.
Ben-bo Xu,Ling-li Xie,Yong Cheng,Guang-yuan Lu
Advance Journal of Food Science and Technology , 2012,
Abstract: To investigate the mechanism on waterlogging tolerance in Brassica napus, 12 B. napus cultivars with different waterlogging tolerance were used in the research and Waterlogging Tolerance Index (WTI) was calculated by multiplying relative percentage germination and the relative seedling height. The results indicated that Lactate Dehydrogenase (LDH) enzyme activity rapidly increased at 24 h after waterlogging treatment and reached peak between 48-72 h. WTI was correlated with LDH enzyme activity at 24 h after water logging treatment and the correlation coefficient between them was 0.84. Transcription level of the BnLDH had significant difference in the 12 lines after waterlogging treatment. BnLDH expression level was very low before waterlogging treatment and induced by waterlogging treatment and arrived at peak at 48 h. Correlation analysis indicated correlation coefficient between WTI and BnLDH expression at 24 and 48 h after waterlogging treatment was 0.56 and 0.72, respectively. An LDH gene, denoted BnLDH-1, was cloned from oilseeds by the Rapid Amplification of CDNA Ends (RACE) from 12 materials and the results indicated all of them had same protein sequence.
Cloning, characterization and prokaryotic expression of lactate dehydrogenase C cDNA in Tamias sibiricus

李昊,韩崇选,张冬辉,周智敏,Li Hao,Han Chongxuan,Zhang Donghui,Zhou Zhimin
- , 2015, DOI: 10.13802/j.cnki.zwbhxb.2015.03.027
Abstract: 为研究花鼠乳酸脱氢酶C(lactate dehydrogenase C,LDH-C)对花鼠免疫不育控制的影响,以花鼠睾丸cDNA为模板,通过RT-PCR技术得到花鼠LDH-C基因cDNA编码区,并进行序列分析,构建花鼠LDH-C的原核表达载体,导入到大肠杆菌BL21(DE3)中诱导表达,并采用聚丙烯酰胺凝胶电泳和免疫印迹法对表达产物进行鉴定.结果显示:扩增出的cDNA片段为999 bp,编码332个氨基酸,含有完整的开放阅读框;负电荷残基与正电荷残基均为36个;预测蛋白质分子量为37 kD,理论等电点为7.04,信号肽和跨膜区,推测其是一种非分泌、疏水性蛋白.α螺旋、规则卷曲以及延伸链是sLDH-C蛋白二级结构的主要成分.重组菌在IPTG诱导下获得了约37 kD带有His-Tag的目的蛋白.
In order to investigate the effects of lactate dehydrogenase C (LDH-C) on chipmunks, Tamias sibiricus, immune infertility control, the cDNA of chipmunks LDH-C was cloned from the chipmunks testis by RT-PCR, and its sequence was analyzed. The LDH-C gene was constructed into the prokaryotic expression vector, and this vector was transformed into E. coli BL21 (DE3) and was induced by IPTG. The SDS-PAGE and western-blot were conducted to identify the expression products. The results showed that the cDNA was 999 bp and encoded for a polypeptide of 332 amino acids, which contained complete open reading frame. The amount of negatively charged residues and positively charged residues were both 36 and the predicted molecular mass was around 37 kD. The theoretical isoelectric point (pI) was 7.04, and there was no signal peptide or transmembrane region. The LDH-C protein was predicted as non-secreted and hydrophobicity protein. Alpha helix, random coil and extended strand were the main components of the secondary structure of LDH-C. A 37 kD target protein with His-Tag was obtained from prokaryotic expression induced by IPTG.
NAD-Independent L-Lactate Dehydrogenase Is Required for L-Lactate Utilization in Pseudomonas stutzeri SDM  [PDF]
Chao Gao, Tianyi Jiang, Peipei Dou, Cuiqing Ma, Lixiang Li, Jian Kong, Ping Xu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036519
Abstract: Background Various Pseudomonas strains can use l-lactate as their sole carbon source for growth. However, the l-lactate-utilizing enzymes in Pseudomonas have never been identified and further studied. Methodology/Principal Findings An NAD-independent l-lactate dehydrogenase (l-iLDH) was purified from the membrane fraction of Pseudomonas stutzeri SDM. The enzyme catalyzes the oxidation of l-lactate to pyruvate by using FMN as cofactor. After cloning its encoding gene (lldD), l-iLDH was successfully expressed, purified from a recombinant Escherichia coli strain, and characterized. An lldD mutant of P. stutzeri SDM was constructed by gene knockout technology. This mutant was unable to grow on l-lactate, but retained the ability to grow on pyruvate. Conclusions/Significance It is proposed that l-iLDH plays an indispensable function in Pseudomonas l-lactate utilization by catalyzing the conversion of l-lactate into pyruvate.
Cloning,Soluble Expression and Mutant Activity Analysis of Lactate Dehydrogenase Gene from Plasmodium falciparum

XU Xiao-Ling,YANG Rui-Yi,YANG Xue-Qin,FENG Li-Ling,ZENG Qing-Ping,

生物工程学报 , 2007,
Abstract: To establish a platform for high throughput screening and in vitro evaluating novel metabolic enzyme-targeted inhibitors towards anti-malarial drugs, a lactate dehydrogenase gene of Plasmodium falciparum (PfLDH) was amplified from the Hainan isolate FCC1/HN. The fusion expression vectors, pGEX-2TK and pET-29a( + ), were utilized to introduce the PfLDH gene into strains of Escherichia coli, BL21 and BL21 (DE3), for over-expression. Consequently, the enzymatic activity of PfLDH was successfully detected in the suspension of lytic bacteria. The PfLDH gene cloned in pGEX-2TK was mainly expressed as inclusion bodies, while the same gene cloned in pET-29a( + ) was nearly expressed in a soluble form of PfLDH, demonstrating the latter vehicle might be more suitable for the large-scale preparation of recombinant PfLDH. Furthermore, according to the electrophoregram of SDS-PAGE and the sequencing data, a series of truncated PfLDH sequences generated randomly from gene amplification were screened and cloned, from which four pre-matured genes with a terminator mutation, PfLDH-delta271, -delta236, -delta167 and -delta53 coding for 45, 80, 149 and 263 amino acid residues, were individually recovered. Through the gene expression and enzymatic activity measurement, the effect of pre-matured terminator mutation on the activity of PfLDH was evaluated, which should pave the way for probing the relationship between structure and function of PfLDH.
Lactate dehydrogenase (LDH) isoenzymes patterns in ocular tumours  [cached]
Singh Rajendra,Kaurya OPS,Shukla P,Ramputty Rewanund
Indian Journal of Ophthalmology , 1991,
Abstract: Estimation of lactate dehydrogenase (LDH) isoenzymes in the serum and aqueous humor was carried out in 15 cases of benign ocular tumour, 15 cases of malignant tumor and 15 normal cases. Cases of both sexes aged between 1 year and 75 years were included. LDH, isoenzymes specially LDH4 and LDH5 are higher and LDH1 and LDH2 lower in sera of patients with malignant tumor specially retinoblastoma as compared to benign tumor cases and control cases. LDH isoenzymes in aqueous humor are significantly higher and show a characteristic pattern in retinoblastoma cases, the concentration was presumably too low in the control, malignant tumor other than retinoblastoma and benign tumor cases as its fractionation was not possible.
The Effect of Subclinical Mastitis on Lactate Dehydrogenase in Dairy Cows
Baharak Mohammadian
International Journal of Animal and Veterinary Advances , 2011,
Abstract: Diagnosis of Subclinical Mastitis (SCM) is of increasing importance and appropriate detection methods are needed. The present study aimed at assessing the relationship between lactate Dehydrogenase (LDH) activity and (SCM) occurred naturally on dairy herds. Milk samples were collected from quarters of 25 cows with (SCM) as well as from 35 healthy controls. Blood specimens were also collected from jugular vein of mentioned cows for the assay. According to results the mean activity of (LDH) was higher in milk from udders than in milk from healthy udders. There were no significant differences in blood serum (LDH) of healthy and (SCM) cows (p<0.01). The increment in (LDH) in milk of udders shows the presence of tissue damage provoked by (SCM). Thus, this parameter might be suitable for use in the early diagnosis of (SCM) in cows.
Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans  [PDF]
Stacie A. Brown,Marvin Whiteley
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0007864
Abstract: Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen and the proposed causative agent of localized aggressive periodontitis. A. actinomycetemcomitans is found exclusively in the mammalian oral cavity in the space between the gums and the teeth known as the gingival crevice. Many bacterial species reside in this environment where competition for carbon is high. A. actinomycetemcomitans utilizes a unique carbon resource partitioning system whereby the presence of L-lactate inhibits uptake of glucose, thus allowing preferential catabolism of L-lactate. Although the mechanism for this process is not fully elucidated, we previously demonstrated that high levels of intracellular pyruvate are critical for L-lactate preference. As the first step in L-lactate catabolism is conversion of L-lactate to pyruvate by lactate dehydrogenase, we proposed a model in which the A. actinomycetemcomitans L-lactate dehydrogenase, unlike homologous enzymes, is not feedback inhibited by pyruvate. This lack of feedback inhibition allows intracellular pyruvate to rise to levels sufficient to inhibit glucose uptake in other bacteria. In the present study, the A. actinomycetemcomitans L-lactate dehydrogenase was purified and shown to convert L-lactate, but not D-lactate, to pyruvate with a Km of approximately 150 μM. Inhibition studies reveal that pyruvate is a poor inhibitor of L-lactate dehydrogenase activity, providing mechanistic insight into L-lactate preference in A. actinomycetemcomitans.
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