人参皂甙Rb1,Rg1,Re和Rh1对细胞脱氢酶活性的影响

应用显微分光光度术,定量地分析了人参皂甙Rb_1、Rg_1、Re、Rh_1对人胚肺成纤维细胞(2BS)和HeLa细胞脱氢酶活性的影响。结果表明,4种单体皂甙增加了高代龄2BS细胞内乳酸脱氢酶(LDH),琥珀酸脱氢酶(SDH),葡萄糖-6-磷酸脱氢酶(G-6-PDH)和丙酮酸脱氢酶(PVO)的活性,降低了HeLa细胞内这几种酶的活性。     

大肠杆菌细胞抽提物制备方案的简化与优化

无细胞蛋白表达系统是一种将目的蛋白在体外进行表达的新技术和新方法,已广泛应用到蛋白质组学、蛋白质结构和功能等领域的研究中。在无细胞蛋白表达系统中,细胞抽提物的制备是关键因素之一。通过对大肠杆菌细胞抽提物制备过程中离心速度、预孵化和透析等参数的考察,利用绿色荧光蛋白作为报告蛋白,可以得到一个细胞抽提物制备的简化方案。采用相对低的转速(12 000×g,10 min),简易空孵化即可制备出活性高的细胞抽提物,用于无细胞体系蛋白表达,其表达的绿色荧光蛋白产量为209μg/mL。与传统的大肠杆菌细胞抽提物S30相比较,新方案将使时间与成本节省62%,产量是传统方法的2.6倍,使无细胞蛋白表达技术的操作快速、高通量的优势更加明显。     

砷对兰州鲇组织中代谢酶活性及RNA和蛋白质含量影响

采用毒性试验方法,研究了安全浓度（1.288 mg/L）条件下亚砷酸钠（NaAsO2）对兰州鲇（Silurus lanzhouensis）脑、鳃、肝脏、肌肉4种组织中6-磷酸葡萄糖脱氢酶（G-6-PDH）和乳酸脱氢酶（LDH）活性,以及RNA和蛋白质含量的影响。结果表明,染毒21d时,As（Ⅲ）可显著降低4种组织中G-6-PDH和LDH活性、RNA和蛋白质含量（P0.05）。撤毒后21d,除脑和肝组织中蛋白质含量未恢复到对照组水平（P0.05）,肝脏中G-6-PDH活性超过了对照组水平（P0.05）外,其余各组织中G-6-PDH和LDH活性、RNA和蛋白质含量均可恢复到对照组水平（P0.05）。以上结果表明,As（Ⅲ）对兰州鲇组织中代谢酶活性具有明显的抑制作用,可致组织细胞RNA损伤和可溶性蛋白质减少,但这种影响是可逆的,撤毒后一定时间内可恢复到正常水平。       

细胞焦亡法破碎微生物细胞在合成生物学与代谢工程的应用

【背景】细胞焦亡是一种细胞程序性死亡。在古菌和细菌中,gasdermin同源蛋白（GSDM）能够被特定的活化caspase （protease）酶切,从而激活类似于细胞焦亡的效应,产生细胞破碎效果。【目的】合成生物学、代谢工程和生物制造等应用过程中,细胞破碎是不可或缺的一步。利用细胞焦亡法破碎细胞取代传统的破碎方法,可以简化操作、提高生产效益。【方法】在大肠杆菌（Escherichia coli） BW25113中共表达protease和不同来源的GSDM,选择有明显细胞焦亡效应即来源Runella sp.的GSDM进行蛋白截短改造,使其在诱导表达蛋白截短体GSDMJD后能直接激活细胞焦亡效应。对GSDMJD进行过表达优化,获得可控大肠杆菌细胞焦亡菌株。进一步以重组表达蔗糖磷酸化酶为研究模型,验证本系统应用于细胞破碎释放蛋白的效果。【结果】实现了大肠杆菌中细胞焦亡的人为可控。焦亡菌株在诱导表达焦亡相关蛋白2 h后大肠杆菌细胞破碎死亡,内容物释放。将上述系统和超声法应用于制备蔗糖磷酸化酶粗酶液,细胞焦亡法制备的粗酶液的相对酶活显著高于超声法制备的粗酶液。在制备粗酶液的菌液OD₆₀₀值为2.0时,细胞焦亡法制备的粗酶液相对酶活最高并且相较于超声法制备粗酶液,提高了60%的相对酶活。【结论】细胞焦亡提供了一种更加简单快捷、绿色环保的微生物细胞破碎方式,为合成生物学与代谢工程的发展奠定了基础。     

Providencia rettgeri strain HNR菌株羟胺氧化酶超声波法提取的研究

为了更有效和便捷的提取菌株脱氮过程中的关键酶,本文利用超声波破碎法对异养脱氮菌Providencia rettgeri strain HNR进行破碎,通过单因素实验及正交试验,确定出最佳破碎条件,便于后续试验。实验结果表明:最佳超声破碎条件为菌液OD_(600)为1.996,工作次数为70次,工作/间歇为5/5s功率为400W的组合为最佳工作条件。在此工作条件下破碎后得到的酶粗提液对羟胺有降解,得到羟胺氧化酶。     

无细胞蛋白表达系统新进展及在生物制药工程中的应用

无细胞蛋白表达系统作为体外合成蛋白表达技术的一种,其基础是细胞抽提物。具备反应操控简便和遗传背景简单等诸多优点。经过大量研究人员的努力,无细胞蛋白表达系统取得新进展,具有较大潜力,可应用于生物制药工程。现就无细胞蛋白表达系统发展进程进行分析,总结该系统在生物制药工程中的应用。     

Tn细胞凋亡抑制蛋白(TnIAP)在粉纹夜蛾细胞中的表达和活性研究

在粉纹夜蛾(Trichoplusia ni)细胞Tn-5B1-4中,高效表达了来自粉纹夜蛾细胞Tn-5B1-4能够抑制细胞凋亡的TnIAP蛋白.聚丙烯酰胺凝胶电泳和免疫印迹分析表明,表达的重组TnIAP只有少部分是可溶性蛋白,大部分以不溶的形式存在.这一结果与以往在昆虫细胞中往往表达出可溶性蛋白不同.活性实验表明,可溶的重组TnIAP能够直接抑制caspase-9酶解Ac-LEHD-AFC的活性,也能抑制caspase-9激活HEK293细胞抽提物酶解Ac-DEVD-AFC的活性.结果进一步证明,昆虫和哺乳动物的细胞凋亡分子机制在进化上是极为保守的.     

重组N-乙酰鸟氨酸脱乙酰基酶的高效表达与纯化

利用质粒pET-22b( )为表达载体,成功构建了高效表达N-乙酰鸟氨酸脱乙酰基酶的基因工程菌BL21-pET-22b( )-argE。研究了该菌的最适超声破壁条件及硫酸铵分级沉淀的最适范围,并以金属螯合亲和层析法纯化含有6-His-Tag的目的蛋白。结果表明:26℃诱导表达的菌体,最佳超声破碎条件为功率200 W,超声时间为15min;目的蛋白主要存在于40%~50%硫酸铵沉淀中。通过Ni-NTA亲和层析柱纯化目的蛋白,可以达到SDS-PAGE电泳纯,并显示单亚基相对分子质量为43 000。纯化倍数为139倍,回收率为11.1%。     

GA3处理对低温胁迫条件下玉米种子呼吸代谢的影响

以低温敏感型的"丰禾1号"和耐低温型的"郑单958"两个玉米品种为实验材料,采用GA3浸种的处理方式("丰禾1号"为20 mg·L-1、"郑单958"为5 mg·L-1),探究了GA3对低温胁迫条件下玉米种子萌发过程中种胚中可溶性糖和可溶性蛋白含量及淀粉酶活性和呼吸途径关键酶活性的影响。结果表明:低温胁迫条件下,GA3浸种处理显著提升了玉米种胚中可溶性糖含量及可溶性蛋白的积累,增强了低温胁迫下细胞的渗透势;α-淀粉酶、β-淀粉酶和总淀粉酶活性显著提高;提高了苹果酸脱氢酶(MDH)、丙酮酸激酶(PK)、联合酶(G-6-PDH和6-PGDH)的活性,提高了糖酵解(EMP)、三羧酸循环(TCA)、磷酸戊糖途径(PPP)途径的运转效率,保证了细胞的物质代谢和能量供应;GA3浸种处理可以显著提高种子对低温的抵抗能力,从而在低温胁迫条件下促进其萌发。     

克雷伯杆菌产1,3-丙二醇关键酶甘油脱水酶的酶活分析方法研究

有氧条件下,建立了克雷伯杆菌破碎方法及其生产1,3-丙二醇代谢途径中关键酶甘油脱水酶的酶活测定方法。甘油脱水酶酶活测定时在超声时间25min,功率为300w的条件下最适破碎频率为破碎时间1s,停息时间4s;甘油脱水酶酶活测定所用磷酸盐缓冲液的最适浓度为0.045mol/L,最适pH值7.2。甘油脱水酶酶活测定反应的最佳温度为37℃,甘油脱水酶酶活测定反应液的最佳吸收波长为290nm。     

Glucose-6-phosphate Dehydrogenase Activity under Conditions of Water Limitation: a Possible Model System for Enzyme Reactions in Unimbibed Resting Seeds and its Relevance to Seed Viability

A model system has been used to measure glucose-6-phosphatedehydrogenase (G-6-PDH) activity when the water contents ofthe reactants are comparable to the water contents of dry restingseeds. The activity of G-6-PDH is reduced by 10²–10³ whenthe water content is limited to between 1.5 and 25 per cent.G-6-PDH activity is affected by temperature and by the proteincontent of the model system. The glucose 6-phosphate (7.03 nmolg^–1 embryo) and the NADP⁺ (25.0 nmol g^–1 embryo)contents of barley embryos were measured. Using these measurements,together with the measurements in the model system of G-6-PDHactivity at low water concentrations, an estimate is made ofthe G-6-PDH activity in resting barley embryo. A cor-relationbetween estimated G-6-PDH activity at different water contentsand the periods for which seeds remain viable is indicated.The limitations of the model system are discussed.     

Histochemical observations on the occurrence of glycolytic and pentose phosphate cycle enzymes in the hepatopancreas and their possible relation to eyestalk factor(s) in the crab Scylla serrata (Forskal).

Histochemical studies were carried out on some of the glycolytic enzymes viz. phosphorylase, aldose, alpha-glycerophosphate dehydrogenase (alpha-GPDH) and lactic dehydrogenase (LDH) and a key enzyme of the pentose phosphatase cycle, glucose-6-phosphate dehydrogenase (G-6-PDH), in the hepatopancreas of Scylla serrata (Forskal). 1. Weak activities of phosphorylase and aldolase and strong-activities of alpha-GPDH and LDH were noticed mainly in the brush border of the tubules and R-cell cytoplasm. A trace activity of G-6-PDH was noticed in the brush border. 2. Bilateral eyestalk removal results in inhibition of both phosphorylase and aldolase. However, enhanced activities of alpha-GPDH and LDH were noticeable 4 h after the operation. The G-6-PDH activity remained unaltered till 24 h. 3. Injection of eyestalk extract into both intact and destalked crabs activated all the enzymes.     

Histochemical study on the localization of glucose-6-phosphate dehydrogenase induced by androgen or thyroxine in the convoluted tubules of mouse submandibular gland.

The effects of 5 alpha-dihydrotestosterone (DHT) and thyroxine (T4) on glucose-6-phosphate dehydrogenase (G-6-PDH) activity in mouse submandibular gland were investigated histochemically. A strong positive histochemical reaction for G-6-PDH was observed in the excretory ducts of untreated male and female mice, with a slight reaction in the basal portion of the convoluted tubules (striated ducts) of males. Administration of DHT to female mice increased G-6-PDH activity specifically in the convoluted tubules. T4 increased the enzyme activity in the tubules more than DHT. The induction of G-6-PDH activity by T4 in adrenalectomized mice suggests that T4 has a direct effect on the submandibular gland.     

Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots

The pivotal role of glucose-6-phosphate dehydrogenase (G-6-PDH)-mediated nitric oxide (NO) production in the tolerance to oxidative stress induced by 100 mM NaCl in red kidney bean (Phaseolus vulgaris) roots was investigated. The results show that the G-6-PDH activity was enhanced rapidly in the presence of NaCl and reached a maximum at 100 mM. Western blot analysis indicated that the increase of G-6-PDH activity in the red kidney bean roots under 100 mM NaCl was mainly due to the increased content of the G-6-PDH protein. NO production and nitrate reductase (NR) activity were also induced by 100 mM NaCl. The NO production was reduced by NaN(3) (an NR inhibitor), but not affected by N(omega)-nitro-L-arginine (L-NNA) (an NOS inhibitor). Application of 2.5 mM Na(3)PO(4), an inhibitor of G-6-PDH, blocked the increase of G-6-PDH and NR activity, as well as NO production in red kidney bean roots under 100 mM NaCl. The activities of antioxidant enzymes in red kidney bean roots increased in the presence of 100 mM NaCl or sodium nitroprusside (SNP), an NO donor. The increased activities of all antioxidant enzymes tested at 100 mM NaCl were completely inhibited by 2.5 mM Na(3)PO(4). Based on these results, we conclude that G-6-PDH plays a pivotal role in NR-dependent NO production, and in establishing tolerance of red kidney bean roots to salt stress.     

The biochemical and morphological changes in the myocardium of patients with an acute surgical vascular pathology (based on data from early autopsies)]

Biochemical and morphological disorders of glucose 6-phosphate dehydrogenase (G-6-PDH) were studied in the myocardium of 9 patients who had died from different vascular surgical diseases. The inhibition of G-6-PDH activity most prominent in lung artery thromboembolism was shown biochemically. Histological and histoenzymological findings demonstrate low G-6-PDH activity in different myocardial regions and solitary defects of cardiomyocytes. The data obtained evidence significant sensibility of the myocardium in surgical patients to different influences.     

Histochemical study on the localization of glucose-6-phosphate dehydrogenase induced by androgen or thyroxine in the convoluted tubules of mouse submandibular gland

Summary The effects of 5-dihydrotestosterone (DHT) and thyroxine (T₄) on glucose-6-phosphate dehydrogenase (G-6-PDH) activity in mouse submandibular gland were investigated histochemically. A strong positive histochemical reaction for G-6-PDH was observed in the excretory ducts of untreated male and female mice, with a slight reaction in the basal portion of the convoluted tubules (striated ducts) of males. Administraition of DHT to female mice increased G-6-PDH activity specifically in the convoluted tubules. T₄ increased the enzyme activity in the tubules more than DHT. The induction of G-6-PDH activity by T₄ in adrenalectomized mice suggests that T₄ has a direct effect on the submandibular gland.     

Effect of tuftsin on enzyme activity in the energy metabolism of lymphocytes

The cytochemical technique was used to measure the activity of succinate dehydrogenase (SDH), lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) of peripheral blood lymphocytes of mice and rats given intraperitoneal injections of an endogenous immunostimulant tuftcin (Tre-Lys-Pro-Arg) in a dose of 0.3 mg/kg. A significant decrease of SDH activity was observed both in mice and rats 4 and 6 hours following injection, respectively. In mice, that activity returned to normal in 12, while in rats in 24 hours. An opposite action was produced by tuftcin on G-6-PDH, causing the maximum elevation of the enzyme activity in rat lymphocytes 6 hours after peptide administration. The decrease to the initial level was observed in 24 hours. Tuftcin did not affect the activity of LDH. The data obtained indicate that the immunological effect of tuftcin is coupled with the changes in the activity of Krebs cycle enzymes (SDH) and pentose phosphate cycle enzymes (G-6-PDH).     

Adaptogenic effect of the vitamin D3 containing supplement "videchol" on glucose-6-phosphate dehydrogenase activity in erythrone of irradiated rats

Two fast migrating, major, multiple molecular forms (MMF) of glucose-6-phosphate dehydrogenase [EC:1.1.1.49]: G-6-PDH-1 and G-6-PDH-2, and two minor forms: G-6-PDH-3 and G-6-PDH-4 were revealed in the electrophoregrams of both erythrocytes haemolisates as well in the homogenates of bone marrow cellular lines of rats at control conditions. Daily 1 cGy irradiation of rats up to a cumulative dose of 20 cGy led to a drop of G-6-PDH total activity and it caused a redistribution of the MMF of the enzyme in bone marrow cellular populations. However, G-6-PDH activity in erythrocytes exceeded the control means in all the experimental terms. The calculation of the local redistribution coefficient (l(G-6-FDH-i)) showed that these changes are mainly determined by the increase of the activity of the isoform G-6-PDH-3. Vitamin D3 administration to rats generated a correction of G-6-PDH activity in all studied cellular populations. Meanwhile, the MMF profiles were characterized by multidirectional rearrangements in the bone marrow erythroid and granulocyte-monocyte cells and in erythrocytes. The specificity of changes in the distribution of the MMF of G-6-PDH in the three studied cellular populations depends on the particularities of their energetic metabolism at irradiation conditions and on the modifying action of the natural adaptogen 1,25-dihydroxicholecalciferol.     

Activity of energy metabolism related enzymes in the myocardium and liver following administration of large doses of noradrenaline]

The activity of the glycolytic enzymes and of the glucose-6-phosphate dehydrogenase (G-6-PDH) were compared with the content of noradrenaline in rat myocardium and the liver after the intraperitoneal injection of high doses of noradrenaline. It was shown that 24 hours after int noradrenaline injection which caused exhaustion of endogenous catecholamine supply, the lactate content and the activities of lactic dehydrogenase were increased in the myocardium; the activity of hexokinase and G-6-PDH in rat myocardium and the liver were also increased, whereas the glucokinase activity was decreased. In these experiments alterations of the enzyme activities were shown to be similar to the alterations in the dystrophic tissues in which the catecholamine content was sharply decreased. The role of the sympathetic nervous system and its mediators in the mechanism of the enzyme regulation of the energy metabolism in the myocardium and the liver is discussed.