黔北麻羊毛色差异皮肤组织的蛋白组学研究

【目的】开展黔北麻羊黑白毛色差异的蛋白组学分析,明确影响其毛色差异的特异性蛋白及其通路,为揭示黔北麻羊特征毛色的形成机制提供参考依据。【方法】分别采集3只黔北麻羊公羊背部黑色羊毛皮肤组织块和腹部白色羊毛皮肤组织块,通过SDT裂解法提取黔北麻羊皮肤组织蛋白并进行蛋白定量分析,然后对筛选出的显著差异表达蛋白分别进行GO功能注释分析、KEGG通路富集分析及亚细胞定位分析。【结果】通过组间比较共筛选出420个显著差异表达蛋白,与白色羊毛对照组（White）相比,黑色羊毛试验组（Black）有247个显著差异表达蛋白呈上调表达、173个显著差异表达蛋白呈下调表达,其中与形成黑色羊毛的黑色素相关蛋白共有15个,包括表皮视黄醇脱氢酶2(SDR16C5)、视黄醇脱氢酶16(RDH16)、视黄醇饱和酶（RETSAT）和角蛋白79(KRT79)等9个上调蛋白,以及蛋白激酶B(AKT)、β抑制蛋白1(ARRB1)和分泌型卷曲相关蛋白1(SFRP1)等6个下调蛋白。GO功能注释分析结果表明,显著差异表达蛋白注释到生物学过程、分子功能和细胞组分三大功能的51条GO功能条目上;亚细胞定位分析显示有159个蛋白定位...     

三氯化锑比色法测定牛肝视黄醇

依据视黄醇可与三氯化锑溶液发生呈色反应生成兰色可溶性物质的原理，用比色法进行视黄醇含量的测定，该方法与常规的异丙醇溶解法测定结果对照，具有相对误差小，准确度高的特点，使用三氯化锑比色法对牛肝中视黄醇含量进行了平行测定，测定结果平均值为０．０２５３ｍｇ／ｇ，相对标准偏差为１．１９％，实验结果显示精密度良好，操作方法简便可靠，具有普遍性使用价值。     

Effects of DHRS3 in C2C12 Myoblast Differentiation and Mouse Skeletal Muscle Injury

Myoblast differentiation is an essential process during skeletal muscle development. C2 C12 myoblast is a commonly used experimental model to study muscle cell differentiation in vitro. Dehydrogenase/reductase(SDR family) member 3(DHRS3) is a highly conserved member in short-chain alcohol dehydrogenase/reductase superfamily and has been shown to be involved in the metabolism of retinol. Previous experimental results showed that the expression of DHRS3 increased significantly during the differentiation of myoblasts differentiation. However, the effect of DHRS3 on mouse muscle cell differentiation was unclear. The objective of current study was to determine if DHRS3 affected muscle cell differentiation, and if DHRS3 was involved in muscle regeneration. Protein expression was determined by western blot and immunofluorescence analysis. The activation and inhibition of DHRS3 increased and decreased C2 C12 myoblast differentiation respectively, which indicated that DHRS3 could affect C2 C12 myoblast differentiation. DHRS3 expression was significantly changed during muscle regeneration, with the regeneration of muscle injury, the expression of DHRS3 tended to increase first and then decrease. It suggested that DHRS3 might be involved in muscle regeneration. In summary, this study confirmed the involvement of DHRS3 in C2 C12 myoblast differentiation and mouse skeletal muscle regeneration and provided a theoretical basis for further elucidating the molecular mechanism of muscle development.     

视黄醇对仔猪睾丸支持细胞体外生物学特性的影响

支持细胞是构成曲细精管上皮的重要组成细胞,视黄醇及其衍生物是雄性动物睾丸发育和精子发生中所必须的物质,支持细胞是睾丸内视黄醇及其衍生物作用的靶细胞。本研究以3~5周龄仔猪睾丸支持细胞为材料,通过分析视黄醇对体外支持细胞活力、增殖和凋亡相关基因表达、相关细胞因子转录及合成的影响,评价视黄醇对支持细胞体外生物学特性的影响。结果显示,与对照组相比,视黄醇添加浓度达到5.000、0.125 μmol·L^-1时,显著抑制培养24 h和72 h的细胞活性(P<0.05);添加1.250 μmol·L^-1视黄醇组和对照组相比,增殖相关基因PCNA、BCL-2和C-MYC显著下调(P<0.05),凋亡相关基因BAX显著上调(P<0.05),且支持细胞内GDNF、CSF-1和EGF在基因表达和蛋白质水平上均显著降低(P<0.05)。体外培养条件下,添加视黄醇抑制支持细胞活性,促进细胞凋亡。     

脂代谢通路对鸡雄性生殖细胞分化的调控机制

【目的】探索家鸡雄性生殖细胞分化过程中脂代谢相关基因以及信号通路的调控机制,以期为完善体外诱导体系提供依据。【方法】采用流式细胞分选法获得纯度较高的胚胎干细胞(embryonic stem cell,ESC)、原始生殖细胞(primitive germ cells,PGC)和精原干细胞 (spermatogonial stem cell,SSC),提取细胞总RNA。利用RNA-seq高通量分析方法对这三种细胞进行转录组水平测序,进行GO（Gene ontology）分析和KEGG通路富集,寻找脂代谢相关基因以及信号通路;并利用脂代谢--视黄醇代谢通路终产物视黄酸（retinoic acid,RA）以及抑制剂苯磺酰异羟肟酸 (piloty’s acid)在体外诱导ESC向雄性生殖细胞分化和体外鸡胚注射后孵化,qRT-PCR（quantitative real time PCR）检测视黄醇代谢通路上差异基因的表达变化,与RNA-seq结果进行比较分析同时检测雄性生殖细胞标记基因的变化情况。【结果】GO功能显著性富集和 Pathway 显著性富集分析结果发现：在ESC向SSC分化过程中共存在328个基因持续参与脂代谢调控,富集到27条脂代谢通路中,包括视黄醇代谢、初级胆汁酸的合成、甾类激素的生物合成、脂肪酸代谢、甘油酯代谢以及类固醇的生物合成等通路。在视黄醇代谢通路中ADH5在PGC中特异表达,ALDH1A1在整个发育过程中持续上调,ADH5和ALDH1A1在细胞中参与视黄酸的合成;CYP26b1在整个发育过程中持续上调,参与视黄酸的降解过程;RA体外诱导ESC向SSC方向诱导试验结果表明ADH5、ALDH1A1和CYP26b1家族基因在体外RA诱导过程中变化与体内分化过程一致,RA诱导组产生的SSC样细胞显著高于控制组和Piloty’s Acid+RA组。鸡胚注射抑制剂试验结果表明视黄醇通路被抑制后孵化至18d后SSC细胞表面特异标记基因integrinα6和integrinβ1的表达水平显著的低于正常的孵化组(CON)和阴性对照组（BLANK）【结论】脂代谢--视黄醇代谢通路在家鸡雄性生殖细胞的分化过程中起重要作用,体外利用视黄醇代谢通路终产物RA进行诱导时第2天出现类胚体,第4、6天类胚体逐渐增大,第8天开始裂解,在第10天出现SSC样细胞,而在诱导过程中抑制视黄醇通路信号后则会降低SSC样细胞的产生,而在体外孵化过程中视黄醇通路抑制后也能影响SSC细胞的产生。     

Dehydrogenase Activities in Dystrophic Mice

The levels of activity of glucose-6-phosphate dehydrogenase, isocitric dehydrogenase, glutathione reductase, lactic dehydrogenase, and a-glycerophosphate dehydrogenase have been studied in the gastrocnemius muscle of mice with "dystrophia muscularis." The activity of enzymes requiring triphosphopyridine nucleotide as a cofactor is increased relative to the control littermates, whereas the activity of those enzymes requiring diphosphopyridine nucleotide is decreased.     

Esterase, malate, and lactate dehydrogenases activity in murine neuroblastoma

Mouse neuroblastoma tumors have only the fifth isozyme band (A(4)) of lactate dehydrogenase, whereas this band is missing in the brain which contained four other bands of lactate dehydrogenase. The alpha-esterase isozyme patterns of tumors, kidney, and brain are similar except that there is an additional slowest-moving form of esterase in all tumor tissues. The malate dehydrogenase pattern is not altered in any of the tissues.     

捻转血矛线虫Hc-FAR-4蛋白的表达特性及其与配体结合能力分析

【目的】捻转血矛线虫(Haemonchus contortus)寄生在牛羊等反刍动物的皱胃中,引起的捻转血矛线虫病在我国呈全国性流行。文章通过研究脂肪酸与视黄醇结合相关蛋白Hc-FAR-4的表达特性及配体结合能力,以了解其在捻转血矛线虫生长、发育、繁殖过程中的作用。【方法】对Hc-far-4进行克隆、并且构建原核表达载体pET-30a-Hc-far-4,pET-30a-Hc-far-4重组质粒经过PCR与双酶切鉴定准确无误之后转化到E. coli BL21感受态细胞中,用0.1 mmol·L ^-1IPTG(isopropyl-β-d-thiogalactoside)进行重组蛋白的诱导表达。收集重组蛋白rHc-FAR-4,利用荧光分析法,研究rHc-FAR-4蛋白与DAUDA、视黄醇、油酸的结合能力。配体结合试验是依据荧光物质retinol与脂肪酸类似物DAUDA在极性和非极性溶液中,在某一波长的激发光激发下,所发出的荧光光谱随之变化的特性,判断rHc-FAR-4蛋白是否具有与DAUDA、retinol结合的能力。再在体系中加入非荧光长链脂肪酸油酸,根据荧光光谱的变化情况判定油酸能否分别与DAUDA、retinol竞争目的蛋白的脂肪酸结合位点,间接说明目的蛋白能否与非荧光脂肪酸油酸结合。同时制备鼠源多克隆抗体,利用ELISA技术检验免疫后小鼠的抗体效价,抗体效价合适即可收集小鼠血清。收集的血清用于免疫组织荧光（IHF）试验,探究Hc-FAR-4的表达部位,从而推测其功能。该试验过程为：对捻转血矛线虫进行石蜡包埋,石蜡切片,抗原修复,3%的BSA 于4℃封闭过夜,自制鼠源多克隆抗体作为一抗孵育1 h; Alexa Fluor? 488 nm羊抗鼠IgG作为二抗避光孵育1 h,DAPI染色30 min,激光共聚焦显微镜检查染色情况。利用荧光定量PCR技术分析Hc-far-4在捻转血矛线虫各个主要阶段的表达特性。【结果】成功克隆目的基因Hc-far-4,测序结果与Sanger数据库中捻转血矛线虫far-4基因序列（>HCISE00908800.t1）相似度为 99.9%;重组质粒 pET-30a-Hc-far-4在E. coli BL21中成功表达,在诱导8 h后达到峰值。经过ELISA检测,结果显示制备的鼠源多克隆抗体效价达到1﹕1 024 000—1﹕2 048 000,可用于后续试验。Western Blot鉴定结果显示rHc-FAR-4重组蛋白带有His标签,并且条带大小为25 kD,结果符合预期。制备的鼠源多克隆抗体经过Western Blot鉴定,能够与天然Hc-FAR-4 蛋白结合,说明该抗体可用于IHF试验。配体结合试验结果表明 rHc-FAR-4蛋白具有结合脂肪酸与视黄醇的能力。荧光定量PCR分析表明,Hc-far-4在四期幼虫中的转录水平最高;IHF试验表明,Hc-FAR-4主要表达在捻转血矛线虫的肠壁、性腺中。综上推测Hc-FAR-4蛋白主要在寄生生活阶段参与了转运脂肪酸与视黄醇,为捻转血矛线虫的生长发育与生殖提供营养物质。【结论】捻转血矛线虫 FAR-4 蛋白能够结合脂肪酸类似物DAUDA和视黄醇,但是与油酸的结合能力较弱,主要表达在肠壁,在性腺、角皮中也有少量表达,其基因在进行营寄生生活阶段时表达量达到峰值。     

Cytochemical localization of lactate dehydrogenase in muscular dystrophy of the mouse

By use of phenazine methosulfate and the "ncubation mixture film method," lactate dehydrogenase activity has been demonstrated in the dystrophic muscle fibers of strain 129 mice. The results indicate that for demonstration of lactate dehydrogenase activity in dystrophic muscle fibers phenazine methosulfate is necessary. This finding is typical for the "white" muscle fibers in the normal muscle and suggests that the dystrophy affects primarily the "white" muscle fibers.     

Genetic control of lactate dehydrogenase expression in mammalian tissues

The amount of lactate dehydrogenase isozyme 4 in erythrocytes of mice is controlled by alleles at the Ldr-1 locus. The A subunits of lactate dehydrogenase from erythrocytes deficient in isozyme 4 cannot assemble in vitro with B subunits to form active isozyme. The inability to form hybrid enzyme is not due to a mutation in the structural gene for the A polypeptide. Rather, a factor that is bound to the A subunits of erythrocytes restricts free exchange with B subunits.     

Histochemical and functional correlations in anterior horn neurons of the cat spinal cord

The histochemical reaction for phosphorylase is completely lost from anterior horn neurons rich in phosphorylase within 72 hours after proximal or distal axonal section. Using this new type of axonal reaction as a marking technique in the anterior horn of the seventh lumbar spinal cord segment of the cat, we demonstrated that (i) alpha motor neurons of slow twitch motor units, like those of fast twitch motor units, are rich in phosphorylase and poor in succinate dehydrogenase, and (ii) interneurons and Renshaw neurons are rich in succinate dehydrogenase and poor in phosphorylase. Gamma motor neurons, because of their small size, are considered to be rich in succinate dehydrogenase and poor in phosphorylase. Thus, anterior horn neurons capable of higher firing frequencies (Renshaw neurons, interneurons, and gamma motor neurons) are richer in mitochondrial oxidative enzyme activity as marked by succinate dehydrogenase. Those firing at lower frequencies (both types of alpha motor neurons) are richer in phosphorylase activity and glycogen content and, thus, apparently better equipped for anaerobic glycolysis.     

乌江上游四川裂腹鱼和昆明裂腹鱼5种同工酶的比较

采用聚丙烯酰胺凝胶垂直平板电泳法(PAGE)对四川裂腹鱼和昆明裂腹鱼4种组织(肌肉、肝脏、尾鳍和性腺)的5种同工酶(LDH、MDH、ADH、EST和G-6-PDH)进行比较研究,对5种同工酶在2种鱼的组织分布、位点表达及酶谱表型作了对比分析。结果表明:LDH、MDH、ADH、EST和G6PDH 5种同工酶酶谱表型和酶带的活性在种间和种内不同组织中均表现出一定差异。同时依据其同工酶酶谱表达的复杂性和个体间多样性,探讨功能重复基因和哑基因存在的可能。     

Lactate dehydrogenase isozymes in parthenogenetic teiid lizards (Cnemidophorus)

Heterozygosity occurs at the lactate dehydrogenase b-locus in two diploid parthenogenetic species of Cnemidophorus. Each such species produces two different B subunits, one of which is also found in two sexual species and in two triploid parthenogenetic species; the other occurs also in a third sexual species. Interspecific hybridization between sexual species carrying different b-alleles and producing different B subunits may be responsible for the heterozygosity at the lactate dehydrogenase b-locus in diploid parthenogenetic Cnemidophours.     

土壤脱氢酶与蛋白酶对酞酸酯污染的动态响应

为评价农田土壤酞酸酯(Phthalate acid esters,PAEs)污染的生态风险,采用室内模拟法,在土壤受邻苯二甲酸二(乙基-己基)酯[Dis(2-ehylhexyl)phthalate ester,DEHP]和邻苯二甲酸二丁酯(Di-n-butyl phthalate ester,DBP)2种酞酸酯类化合物单一和复合不同污染水平下,测定土壤脱氢酶与蛋白酶活性在培养期内对这2种化合物单一污染与复合污染的动态变化。结果表明:土壤中浓度小于10 mg/kg的DBP和浓度小于20 mg/kg的DEHP对土壤脱氢酶与蛋白酶活性没有明显影响,浓度为20 mg/kg时DBP对土壤脱氢酶表现出抑制-激活-恢复效应;在DBP浓度为20 mg/kg时土壤蛋白酶短期内有明显的激活效应;当土壤中DBP或DEHP污染浓度大于或等于50mg/kg时,土壤蛋白酶与土壤脱氢酶均被显著抑制;DBP与DEHP复合污染对土壤脱氢酶表现出协同抑制效应,而对蛋白酶没有类似的效应特征,主成分分析(PAC)也佐证了以上结果。因此,土壤脱氢酶与蛋白酶可以作为土壤酞酸酯污染生态风险评价的生物学指标,但对酞酸酯不同污染水平,这两种土壤酶活性的响应特征有所差异。     

Lactate Dehydrogenases in Human Testes

A unique form of lactate dehydrogenase was observed in the starch-gel electrophoretic patterns of adult human testes. It was present in sperm, but absent in prepubertal testes. Its electrophoretic mobility, heat stability, kinetic behavior with pyridine nucleotide analogs, and chromatographic characteristics on diethylaminoethyl cellulose were intermediate to those observed for lactate dehydrogenase isozymes 3 and 4.     

Erythrocyte acid phosphomonesterase and glucose-6-phosphate dehydrogenase deficiency in Caucasians

Caucasian patients with erythrocyte glucose-6-phosphate dehydrogenase deficiency also have a deficiency in erythrocyte acid phosphomonoesterase. This acid phosphomonoesterase deficiency is not present in Negroes with the glucose-6-phosphate dehydrogenase deficiency.     

尖孢镰刀菌异核体及其不同核型分离子的孢子萌发时期同工酶分析

在获得尖孢镰刀菌棉花萎蔫专化型异核体菌株及其3个稳定的不同核型分离子的基础上，选择孢子萌发时期的幼嫩菌丝制备粗酶提取液，采用聚丙烯酰胺凝胶电泳方法进行了7种同工酶分析。结果表明，酯酶和乳酸脱氢酶电泳图谱在4个样品中完全一致；没有致病能力的菌株在葡萄糖－6磷酸脱氢酶在苹果酸脱氢酶的表达量高于致病力强的菌株；致病力强的菌株没有乙醇脱氢酶的表达。在4个样品中都没有检测到苹果酸酶和过氧化氢酶。     

The tricarboxylic acid cycle in cyanobacteria

It is generally accepted that cyanobacteria have an incomplete tricarboxylic acid (TCA) cycle because they lack 2-oxoglutarate dehydrogenase and thus cannot convert 2-oxoglutarate to succinyl-coenzyme A (CoA). Genes encoding a novel 2-oxoglutarate decarboxylase and succinic semialdehyde dehydrogenase were identified in the cyanobacterium Synechococcus sp. PCC 7002. Together, these two enzymes convert 2-oxoglutarate to succinate and thus functionally replace 2-oxoglutarate dehydrogenase and succinyl-CoA synthetase. These genes are present in all cyanobacterial genomes except those of Prochlorococcus and marine Synechococcus species. Closely related genes occur in the genomes of some methanogens and other anaerobic bacteria, which are also thought to have incomplete TCA cycles.     

Glucose-6-phosphate dehydrogenase: homologous molecules in deer mouse and man

Two forms of glucose-6-phosphate dehydrogenase, A and B, have been reported in deer mouse tis sues. The B enzyme, which showed autosomally controlled polymorphism, is now found to be equally active to ward glucose-6-phosphate and galac tQse-6- phosphate; the A enzyme is specific for the former. Human and horse livers also have two forms of glucose-6-phosphate dehydrogenase which exhibit the same substrate spe cifities as those in the deer mouse. A wide variety of electrophoretic patterns was seen in the human galactose-active enzyme.