The multiple molecular forms of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH ) and lactate dehydrogenase (LDH ) were studied in the liver and skeletal muscle of the freshwater catf...The multiple molecular forms of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH ) and lactate dehydrogenase (LDH ) were studied in the liver and skeletal muscle of the freshwater catfish, Clarias batrachus. There were two electrophoretically distinguishable bands (AA andBB) of cMDH and mMDH which suggests that they are apparently encoded at two gene loci (A and B) in both the tissues.However, the presence of a single band (LDH-1 ) of LDH in liver and double bands (LDH-1and LDH-2) in skeletal muscle in which LDH-2 was predominant reflects the differential expression of LDH genes in different metabolic tissues to meet the requirement of energy production. The AA isoform (74 kd) of liver cMDH was smaller than those of the AA form (110 kd) of skeletal muscle. In contrast, the BB isoform of liver (42 kd) and skeletal muscle (54 kd) were more or less similar in size. Unlike the case of cMDH, the molecular weight of AA isoform (115 kd) of liver mMDH was higher than those of the AA form (87kd) of skeletal muscle. Whereas the molecular weight of BB isoform (58 kd) of liver was in proximity to the weight of BB form (44 kd) of skeletal muscle mMDH. The size of AA isoform (74 kd) of liver cMDH was smaller, while the AA isoform (110 kd) of skeletal muscle was larger as compared to AA form of mMDH in the liver (115 kd) and skeletal muscle (87 kd). But the size of BB isoform of both the isozymes was almost equal in these metabolic tissues. The molecular weight of liver LDH-1 (96 kd) was close to the weight of LDH-1 (82 kd) in skeletal muscle. The molecular weight of skeletal muscle LDH-2 was deduced as 37 kd which is much more lower than the weight of LDH-1 in liver and skeletal muscle. The smaller size of LDH-2 in skeletal muscle may be of a physiological significance in this anaerobic tissue展开更多
Objective: To construct a prokaryotic recombinant vector for mouse lactate dehydrogenase-C and to detect its expression in BL21. Methods: The coding sequence of mouse lactate dehydrogenase subunit C was amplified from...Objective: To construct a prokaryotic recombinant vector for mouse lactate dehydrogenase-C and to detect its expression in BL21. Methods: The coding sequence of mouse lactate dehydrogenase subunit C was amplified from mouse testis RNA with specific primers and cloned into pGEX-2T after restriction digestion with BamH I and EcoR I. GST fusion protein was expressed after induction with IPTG. Results: Sequencing and restriction digestion of the recombinant plasmid revealed the coding sequence for mouse lactate dehydrogenase subunit C. A protein band of about 60 000 could be induced by IPTG in the recombinant plasmid. Conclusion: The 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.展开更多
Metabolic engineering of the tumor microenvironment has emerged as a new strategy.Lactate dehydrogenase A(LDHA)is a prominent target for metabolic engineering.Here,we designed a cationic lipid nanoparticle formulation...Metabolic engineering of the tumor microenvironment has emerged as a new strategy.Lactate dehydrogenase A(LDHA)is a prominent target for metabolic engineering.Here,we designed a cationic lipid nanoparticle formulation for LDHA gene editing.The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein(GFP).The delivery efficiency was affected by the ratios of three components:a cationic lipid,cholesterol or its derivative,and a fusogenic lipid.The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA,yielding the lipoplex,pCas9-sgLDHA/F3.The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells.Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium.pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture.In vivo,combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice.This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.展开更多
目的探究肝细胞癌中分泌性富含半胱氨酸的酸性蛋白(secreted protien acidic and rich in cysteine,SPARC)表达及其与糖酵解作用的相关调节机制。方法选取肝癌细胞HepG2、Hep3B、Huh7,分别稳定转染SPARC质粒和SPARC siRNA,应用比色法检...目的探究肝细胞癌中分泌性富含半胱氨酸的酸性蛋白(secreted protien acidic and rich in cysteine,SPARC)表达及其与糖酵解作用的相关调节机制。方法选取肝癌细胞HepG2、Hep3B、Huh7,分别稳定转染SPARC质粒和SPARC siRNA,应用比色法检测2种转染的细胞中糖酵解关键酶己糖激酶(HK)和乳酸脱氢酶-A(LDHA)的活性变化。结果转染SPARC质粒的3种肝癌细胞中HK-Ⅱ、LDHA的活性较对照组明显下降,3种细胞中HK-Ⅱ相对吸光度分别降低至(57.45±5.05)%,(56.1±5.70)%,(49.73±7.06)%;LDHA相对吸光度分别降低至(50.53±8.07)%,(54.38±2.08)%,(42.34±3.44)%;差异均有统计学意义(P<0.05);反之,siRNA抑制SPARC表达的肝癌细胞中,HK-Ⅱ、LDHA活性较对照组明显上调,3种细胞中HK-Ⅱ相对吸光度分别上调至(174.20±8.41)%,(166.69±9.71)%,(152.84±3.39)%;LDHA的相对吸光度分别上调至(176.65±6.85)%;(163.24±6.44)%;(151.25±6.75)%,差异具有统计学意义(P<0.05)。结论肝细胞癌中,SPARC通过负性调控糖酵解作用的关键酶抑制糖酵解。展开更多
用同位32~P标记乳酸脱氢酶-C(LDH-C)cDNA作为探针,与小鼠胸腺、脑、胰、心肌、骨骼肌、睾丸、肾、肺、肝的RNA以及人胰、皋丸、肝、骨骼肌、心肌及脑的RNA分别作点溃杂交(dot blot hybridization)及Northern印迹杂交,证实LDH-C基因只在...用同位32~P标记乳酸脱氢酶-C(LDH-C)cDNA作为探针,与小鼠胸腺、脑、胰、心肌、骨骼肌、睾丸、肾、肺、肝的RNA以及人胰、皋丸、肝、骨骼肌、心肌及脑的RNA分别作点溃杂交(dot blot hybridization)及Northern印迹杂交,证实LDH-C基因只在睾丸中特异性表达。展开更多
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 th...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.展开更多
文摘The multiple molecular forms of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH ) and lactate dehydrogenase (LDH ) were studied in the liver and skeletal muscle of the freshwater catfish, Clarias batrachus. There were two electrophoretically distinguishable bands (AA andBB) of cMDH and mMDH which suggests that they are apparently encoded at two gene loci (A and B) in both the tissues.However, the presence of a single band (LDH-1 ) of LDH in liver and double bands (LDH-1and LDH-2) in skeletal muscle in which LDH-2 was predominant reflects the differential expression of LDH genes in different metabolic tissues to meet the requirement of energy production. The AA isoform (74 kd) of liver cMDH was smaller than those of the AA form (110 kd) of skeletal muscle. In contrast, the BB isoform of liver (42 kd) and skeletal muscle (54 kd) were more or less similar in size. Unlike the case of cMDH, the molecular weight of AA isoform (115 kd) of liver mMDH was higher than those of the AA form (87kd) of skeletal muscle. Whereas the molecular weight of BB isoform (58 kd) of liver was in proximity to the weight of BB form (44 kd) of skeletal muscle mMDH. The size of AA isoform (74 kd) of liver cMDH was smaller, while the AA isoform (110 kd) of skeletal muscle was larger as compared to AA form of mMDH in the liver (115 kd) and skeletal muscle (87 kd). But the size of BB isoform of both the isozymes was almost equal in these metabolic tissues. The molecular weight of liver LDH-1 (96 kd) was close to the weight of LDH-1 (82 kd) in skeletal muscle. The molecular weight of skeletal muscle LDH-2 was deduced as 37 kd which is much more lower than the weight of LDH-1 in liver and skeletal muscle. The smaller size of LDH-2 in skeletal muscle may be of a physiological significance in this anaerobic tissue
文摘Objective: To construct a prokaryotic recombinant vector for mouse lactate dehydrogenase-C and to detect its expression in BL21. Methods: The coding sequence of mouse lactate dehydrogenase subunit C was amplified from mouse testis RNA with specific primers and cloned into pGEX-2T after restriction digestion with BamH I and EcoR I. GST fusion protein was expressed after induction with IPTG. Results: Sequencing and restriction digestion of the recombinant plasmid revealed the coding sequence for mouse lactate dehydrogenase subunit C. A protein band of about 60 000 could be induced by IPTG in the recombinant plasmid. Conclusion: The 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.
基金by grants from the National Research Foundation,Ministry of Science and ICT,Republic of Korea(NRF-2021R1A2B5B03002123,NRF-2018R1A5A2024425,NRF-2021K2A9A2A06044515,2022M3E5F1017919)Ministry of Education,Republic of Korea(NRF-2021R1A6A3A01086428)Korean Health Technology R&D Project(No.HI19C0664),Ministry of Health&Welfare,Republic of Korea.
文摘Metabolic engineering of the tumor microenvironment has emerged as a new strategy.Lactate dehydrogenase A(LDHA)is a prominent target for metabolic engineering.Here,we designed a cationic lipid nanoparticle formulation for LDHA gene editing.The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein(GFP).The delivery efficiency was affected by the ratios of three components:a cationic lipid,cholesterol or its derivative,and a fusogenic lipid.The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA,yielding the lipoplex,pCas9-sgLDHA/F3.The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells.Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium.pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture.In vivo,combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice.This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.
文摘目的探究肝细胞癌中分泌性富含半胱氨酸的酸性蛋白(secreted protien acidic and rich in cysteine,SPARC)表达及其与糖酵解作用的相关调节机制。方法选取肝癌细胞HepG2、Hep3B、Huh7,分别稳定转染SPARC质粒和SPARC siRNA,应用比色法检测2种转染的细胞中糖酵解关键酶己糖激酶(HK)和乳酸脱氢酶-A(LDHA)的活性变化。结果转染SPARC质粒的3种肝癌细胞中HK-Ⅱ、LDHA的活性较对照组明显下降,3种细胞中HK-Ⅱ相对吸光度分别降低至(57.45±5.05)%,(56.1±5.70)%,(49.73±7.06)%;LDHA相对吸光度分别降低至(50.53±8.07)%,(54.38±2.08)%,(42.34±3.44)%;差异均有统计学意义(P<0.05);反之,siRNA抑制SPARC表达的肝癌细胞中,HK-Ⅱ、LDHA活性较对照组明显上调,3种细胞中HK-Ⅱ相对吸光度分别上调至(174.20±8.41)%,(166.69±9.71)%,(152.84±3.39)%;LDHA的相对吸光度分别上调至(176.65±6.85)%;(163.24±6.44)%;(151.25±6.75)%,差异具有统计学意义(P<0.05)。结论肝细胞癌中,SPARC通过负性调控糖酵解作用的关键酶抑制糖酵解。
基金the Science and Technology Fund of Sichuan Province for Young Scholars (Grant No. 05ZQ026-024)Science Foundation of the State Ethnic Affairs Commission of China, the National Basic Research Program of China (Grant No. 2006CB102100)New Century Excellent Talents in Chinese Universities (Grant No. NCET-04-0135)
文摘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.
