鸡溶菌酶和重组大肠杆菌融合蛋白(NADP-GDH)体外分子交联的初步研究

用PCR方法克隆的大肠杆菌NADP特异性谷氨酸脱氢酶 (NADP specificglutamatedehydrogenase ,NADP GDH)基因和其突变基因 ,插入表达载体pTrcHisC构建重组蛋白质表达体系 .经过IPTG诱导表达 ,用Talon固定化金属亲和层析树脂纯化出重组的天然和突变NADP GDH ,将它们和溶菌酶 (Lysozyme)通过热诱导去折叠方法进行体外蛋白质分子交联实验 ,在去折叠反应液中加入还原剂二硫苏糖醇 (DTT)后 ,不但没有分子间二硫键交联形成 ,同时也没有其他分子间共价键 (异肽键 )交联的形成 .另外 ,半胱氨酸残基定点突变后的NADP GDH重组蛋白质 ,无法参与形成分子间二硫键 ,实验证实经过热诱导去折叠后也没有分子间共价键 (异肽键 )交联 .这些结果进一步证明蛋白质分子间二硫键的形成能够促进蛋白质其他分子间共价键 (异肽键 )的形成 .     

屎肠球菌JT1701对人体肠系细菌的调控作用

用屎肠球菌JT1701与人体肠系有害菌（大肠杆菌、产气荚膜梭菌、脆弱拟杆菌）和人体肠系益生菌（婴儿双歧杆菌、嗜酸乳杆菌）分别及共同培养发现,肠球球菌对这些菌均有抑制作用。它能抑制这些菌氨的产生,降低培养基的pH。其原因是,屎肠球菌与氨产生相关的尿酶、氨基酸脱氨酶的种类少,酶活性亦低,而与NH4^ 同化相关的谷氨酸脱氢酶、谷氨酸合酶、谷酰氨合成酶的活性要高得多。表明屎肠球菌对人体肠系菌群具有调控作用。     

利用番茄U3snRNA基因上游启动区构建植物表达载体及对烟草的转化

利用番茄U3snRNA基因上游启动区和ACC合成酶反义RNA-核酶嵌合基因DNA片段,构建含U3snRNA基因上游启动区-ACC合成酶的反义RNA-核酶嵌合序列的表达载体,重组于植物双元表达载体pGA643中,得到pGU3R.用三亲融合法导入农杆菌LBA4404中,采用叶盘法转化烟草,诱导再生小植株,获得了卡那霉素的抗性植株.提取抗性植株总DNA,通过PCR、PCRSouthern杂交检测并分别用启动区序列和ACC合成酶的反义RNA-核酶嵌合序列作探针,通过Southern杂交检测,已筛选出整合有外源基因的转化植株.为进一步研究U3snRNA上游启动区增强反义RNA-核酶基因的表达奠定了基础.     

不同生长期上海四膜虫（Tetrahymena shanghaiensis S1）的同工酶的比较研究

用高分辨力的聚丙烯酰胺凝胶电泳分析和比较了不同生长期的上海四膜虫的五种同工酶。发现对数期细胞和衰老期细胞的酸性磷酸酶同工酶，酯酶同工酶和苹果酸脱氢酶同工酶及其它的同工酶的蛋白条带有明显差异。对数生长期细胞的同工酶活力和数量明显高于衰老期细胞的同工酶的活力和数量。说明同工酶不但是细胞代谢的调节者，亦能作为细胞衰老的一种指标。     

Electrogenic glutamate uptake in glial cells is activated by intracellular potassium

Uptake of glutamate into glial cells in the CNS maintains the extracellular glutamate concentration below neurotoxic levels and helps terminate its action as a neurotransmitter. The co-transport of two sodium ions on the glutamate carrier is thought to provide the energy needed to transport glutamate into cells. We have shown recently that glutamate uptake can be detected electrically because the excess of Na+ ions transported with each glutamate anion results in a net current flow into the cell. We took advantage of the control of the environment, both inside and outside the cell, provided by whole-cell patch-clamping and now report that glutamate uptake is activated by intracellular potassium and inhibited by extracellular potassium. Our results indicate that one K+ ion is transported out of the cell each time a glutamate anion and three Na+ ions are transported in. A carrier with this stoichiometry can accumulate glutamate against a much greater concentration gradient than a carrier co-transporting one glutamate anion and two Na+ ions. Pathological rises in extracellular potassium concentration will inhibit glutamate uptake by depolarizing glial cells and by preventing the loss of K+ from the glutamate carrier. This will facilitate a rise in the extracellular glutamate concentration to neurotoxic levels and contribute to the neuronal death occurring in brain anoxia and ischaemia.     

壳聚糖对活性污泥处理味精废水的作用

探讨了厌氧活性污泥经壳聚糖处理后，在不同pH值条件下，处理味精废水的活力情况及脱氢酶活力与沉降性能变化。结果表明，厌氧菌群经壳聚糖处理后，在最适pH7时，其沉降性能、脱氢酶活力、去除味精废水CODcr的活力均有明显提高。考察了不同污染负荷的味精废水（味精废水经预处理后）通过UASB（上流式厌氧污泥床）生化反应器后的CODcr值变化情况，表明提高废水污染负荷及延长其在反应器中停留时间有助于提高CODcr去除率。     

黄色短杆菌中谷氨酸脱氢酶的分离纯化及特性

经硫酸铵盐析、DEAE-纤维素层析、苯-琼脂糖CL-4B(Phenyl-SepharoseOL-4B)层析和交联葡聚糖G-200(Sephadex G-200)层析等步骤从谷氨酸产生菌——黄色短杆菌中分离纯化的谷氨酸脱氢酶,酶活提高了230倍。该酶的分子量约为30万,由六个分子量约为5万的亚基组成,最适pH为8.9,并对热较稳定。在pH8.2时,以谷氨酸和NADP+为底物时的米氏常数值分别为0.294mol/L和0.1mmol/L。     

Effect of exogenous ammonium on glutamine synthetase, 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. Foundation item: Supported by the National Natural Science Foundation of China (No. 3987052), Natural Science Foundation of Hubei Province and International Rice Research Institute, P.O. Box. 3217, 1271 Makati City, Philippines. Biography: LI Ze-song(1968-), male, Graduate student.     

Localization of nitric oxide synthase indicating a neural role for nitric oxide.

Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed by cytotoxic macrophages, in adrenal gland and in brain tissue, where it mediates the stimulation by glutamate of cyclic GMP formation in the cerebellum. Stimulation of intestinal or anococcygeal nerves liberates NO, and the resultant muscle relaxation is blocked by arginine derivatives that inhibit NO synthesis. It is, however, unclear whether in brain or intestine, NO released following nerve stimulation is formed in neurons, glia, fibroblasts, muscle or blood cells, all of which occur in proximity to neurons and so could account for effects of nerve stimulation on cGMP and muscle tone. We have now localized NO synthase protein immunohistochemically in the rat using antisera to the purified enzyme. We demonstrate NO synthase in the brain to be exclusively associated with discrete neuronal populations. NO synthase is also concentrated in the neural innervation of the posterior pituitary, in autonomic nerve fibres in the retina, in cell bodies and nerve fibres in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells. These prominent neural localizations provide the first conclusive evidence for a strong association of NO with neurons.     

转betA/als基因棉花生存竞争力和基因漂流的调查

对导入乙酰乳酸合成酶突变基因als及来自大肠杆菌的胆碱脱氢酶基因betA的棉花纯合株系T3、T4代植株的田间农艺学性状和基因漂流进行了研究。 结果表明,与野生型对照鲁棉研19相比,转betA/als基因棉花涉及生存竞争力的一些农艺学性状,如种子繁殖能力、贮存后活力以及盐碱地条件下的经济产量等显著提高,转基因棉花的棉花纤维马克隆值下降,其它农艺学性状无明显差别;获得的转基因性状之一除草剂抗性遗传稳定,并在田间表达良好;在转基因棉花释放区面积为6?m×6?m的条件下,als基因通过花粉介导进入野生型棉花的频率随着非转基因棉花种植区与释放区之间的距离增大而迅速降低,采取适当的隔离距离（大于200?m）,可以避免外源基因逃逸事件的发生。但设置隔离区时应考虑昆虫对花粉传播等不确定因素的影响,适当加大隔离距离。     

氯化钴对人eNOS基因启动子转录活性的影响

目的:建立氯化钴诱导的人脐静脉血管内皮细胞-12(HUVEC-12)缺氧模型,研究人血管内皮细胞一氧化氮合酶(eNOS)基因的启动子活性变化.方法:用含不同浓度氯化钴的培养基培养细胞,检测细胞培养上清中的乳酸脱氢酶(LDH)含量;将已构建的pGL2-eNOS-p质粒转染HUVEC-12细胞,利用双荧光素酶报告基因技术检测在不同浓度氯化钴和不同作用时间下的eNOS启动子转录活性.结果:氯化钴刺激下HUVEC-12细胞培养上清的LDH含量随氯化钴作用浓度增加而提高;氯化钴刺激后转染细胞的eNOS启动子活性呈现随氯化钴剂量增加而升高的趋势,随作用时间的延长而上升的趋势.结论:成功建立氯化钴诱导HUVEC-12细胞缺氧的体外模型.     

Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake

Glutamate uptake into nerve and glial cells usually functions to keep the extracellular glutamate concentration low in the central nervous system. But one component of glutamate release from neurons is calcium-independent, suggesting a non-vesicular release that may be due to a reversal of glutamate uptake. The activity of the electrogenic glutamate uptake carrier can be monitored by measuring the membrane current it produces, and uptake is activated by intracellular potassium ions. Here we report that raising the potassium concentration around glial cells evokes an outward current component produced by reversed glutamate uptake. This current is activated by intracellular glutamate and sodium, inhibited by extracellular glutamate and sodium, and increased by membrane depolarization. These results demonstrate a non-vesicular mechanism for the release of glutamate from glial cells and neurons. This mechanism may contribute to the neurotoxic rise in extracellular glutamate concentration during brain anoxia.     

谷氨酰胺在小麦花药培养中的作用之探讨

以Ｃ１７为基本培养基,研究分析了在诱导培养基中添加不同浓度的谷氨酰胺对小麦花药愈伤诱导的影响效应。三年来多个基因型的试验结果表明,诱导培养基中添加谷氨酰胺对小麦花药愈伤诱导没有促进效应,这可能是和添加的谷氨酰胺与培养基中的亮氨酸在进入三羧酸循环时产生竞争抑制有关。     

脯氨酸的代谢

本文简要地介绍了脯氨酸的代谢情况。脯氨酸是由谷氨酸或乌氨酸合成的,分解为谷氨酸、有机酸和CO_2。P5C在脯氨酸代谢中起中心作用。脯氨酸还向蛋白质掺入,并被转化成羟脯氨酸。     

Evolution and metabolic significance of the urea cycle in photosynthetic diatoms

Diatoms dominate the biomass of phytoplankton in nutrient-rich conditions and form the basis of some of the world's most productive marine food webs. The diatom nuclear genome contains genes with bacterial and plastid origins as well as genes of the secondary endosymbiotic host (the exosymbiont), yet little is known about the relative contribution of each gene group to diatom metabolism. Here we show that the exosymbiont-derived ornithine-urea cycle, which is similar to that of metazoans but is absent in green algae and plants, facilitates rapid recovery from prolonged nitrogen limitation. RNA-interference-mediated knockdown of a mitochondrial carbamoyl phosphate synthase impairs the response of nitrogen-limited diatoms to nitrogen addition. Metabolomic analyses indicate that intermediates in the ornithine-urea cycle are particularly depleted and that both the tricarboxylic acid cycle and the glutamine synthetase/glutamate synthase cycles are linked directly with the ornithine-urea cycle. Several other depleted metabolites are generated from ornithine-urea cycle intermediates by the products of genes laterally acquired from bacteria. This metabolic coupling of bacterial- and exosymbiont-derived proteins seems to be fundamental to diatom physiology because the compounds affected include the major diatom osmolyte proline and the precursors for long-chain polyamines required for silica precipitation during cell wall formation. So far, the ornithine-urea cycle is only known for its essential role in the removal of fixed nitrogen in metazoans. In diatoms, this cycle serves as a distribution and repackaging hub for inorganic carbon and nitrogen and contributes significantly to the metabolic response of diatoms to episodic nitrogen availability. The diatom ornithine-urea cycle therefore represents a key pathway for anaplerotic carbon fixation into nitrogenous compounds that are essential for diatom growth and for the contribution of diatoms to marine productivity.     

Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

Glutamate is taken up avidly by glial cells in the central nervous system. Glutamate uptake may terminate the transmitter action of glutamate released from neurons, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.     

Microbial starch-binding domains are superior to granule-bound starch synthase I for anchoring luciferase to potato starch granules

Microbial starch-binding domains (SBD) and granule-bound starch synthase I (GBSSI) are proteins which are accumulated in potato starch granules. The efficiency of SBD and GBSSI for targeting active luciferase reporter proteins to granules during starch biosynthesis was compared. GBSSI or SBD sequences were fused to the N- or C-terminus of the luciferase (LUC) gene, via an artificial Pro-Thr encoding linker sequence. The genes were introduced into an amylose-free (amf) potato mutant. It appeared that SBD was superior to GBSSI as a targeting sequence, mainly because the luciferase retained higher activity in the SBD-containing fusion proteins than in the GBSSI-containing ones.     

海带多糖对四氯化碳致小鼠肝损伤的保护作用

目的：主要研究海带多糖对四氯化碳致小鼠急性肝损伤的保护作用．方法：小鼠随机分为6组,给药4d后,腹腔注射0．3％四氯化碳（CCl4）,16h后取血、肝组织匀浆测定．结果：不同剂量的海带多糖均能阻止血清中丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、乳酸脱氢酶（LDH）的升高;能增加血清中一氧化氮（NO）含量、一氧化氮合酶（NOS）活力;增加组织中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-PX）活力．结论：海带多糖对四氯化碳致小鼠急性肝损伤有保护作用．其机理可能是海带多糖能清除氧自由基,提高机体的抗氧化能力．     

胀果甘草查尔酮合成酶基因cDNA的克隆及序列分析

为进一步利用基因工程手段调控甘草黄酮的合成,通过反转录-聚合酶链式反应(RT-PCR)方法,从胀果甘草愈伤组织中克隆查尔酮合成酶(chalcone synthase,CHS)基因的cDNA,运用DNAMAN软件对序列进行分析,运用PHYLIP 3.67软件绘制系统进化树.克隆得到的基因片段全长为1 170 bp,包含1个完整的开放阅读框架(ORF),编码1个由389个氨基酸残基组成的多肽.该基因片段与紫花苜蓿、大豆、豌豆等几种豆科植物的CHS核苷酸同源率高达80%以上,氨基酸同源率高达90%以上.表明克隆得到了胀果甘草chs基因cDNA,序列提交GenBank注册,序列号为EU706287.