世界首创用昆虫细胞表达重组猪卵泡刺激激素

群马大学生理活性物质中心助教授加藤幸雄小组用昆虫细胞表达重组猪卵泡刺激激素(FSH),确认重组FSH具有与天然FSH相同的生物活性。据说目前有用人、牛生产重组FSH的,但是用猪生产重组FSH是世界首创。得到了纯度高的FSH。它除对卵泡刺激激素的机能和作用机制的分析有用外,还能应用于猪体外受精系统的研究。于10日2～8日在新泻召开的日本畜产学会上发表了这项成果。 群马大学与日生研、大阪府立大学、东北农业试验场、冈山大学合作,在一个杆状病毒载体上插入了FSH的α链和β链两个基因,使昆虫细胞Tn5感染。以前用个别载体感染α链和β链时FSH滞留在昆虫细     

长效山羊促卵泡素类似物真核表达载体的构建及其在CHO细胞内的稳定表达

通过PCR重叠延伸法将人绒毛膜促性腺激素（hCG）的羧基末端延长肽（CTP）基因连接至山羊促卵泡素（FSH）15亚基的羧基末端,克隆入双表达载体pVITRO,测序证实后将表达载体转染CHO细胞,用潮霉素B筛选稳定表达的CHO细胞株。结果表明获得了长效山羊FSH基因,并得到稳定表达的CHO细胞株,表达量为0．105mU／mL,为进一步研究CTP结构与功能的关系,以及长效激素的理化特性奠定了基础。     

新型长效重组促卵泡素的纯化和性质及药代动力学研究

促卵泡素(Follicle-stimulating hormone,FSH)用于治疗男女不孕不育症,由于FSH的半衰期较短,需要频繁注射给药,因此制备长效FSH制剂一直是该类药物的研发方向。首次将来自绒促性素(human chorionic gonadotropin,HCG)中的羧基端肽(carboxy-terminal peptide,CTP)和人免疫球蛋白G2(immunoglobulin G2,IgG2)Fc片段变体(vFc)与单链FSH有序连接,通过基因工程和哺乳动物细胞培养技术实现了其在中国仓鼠卵巢细胞中的稳定表达,然后利用Protein A柱层析手段进行纯化,制备了新型重组FSH-CTP-vFc融合蛋白,同时分析了不同细胞培养时间表达的重组融合蛋白的理化性质和体内外活性。动物试验结果表明,纯化的FSH-CTP-vFc融合蛋白具有显著的体内生物活性,药代动力学分析显示该重组融合蛋白的半衰期是重组FSH的近10倍。这种新型长效重组FSH-CTP-vFc融合蛋白在临床上可大大减少注射次数和患者痛苦,提高患者用药依从性。     

山羊重组促卵泡素长效类似物基因在毕赤酵母中表达

为了获得长效FSH制剂, 利用重叠PCR技术将山羊FSH的a, b亚单位, 通过hCGb亚单位羧基末端延长肽(CTP)基因序列的连接, 构建成单链长效类似物基因FSHb-CTP-a。将其克隆至表达载体pPIC9K, 重组载体线性化处理后电转化至毕赤酵母GS115中, 经判型和G418筛选后获得高拷贝菌株His+Mut+。经甲醇诱导表达后, 进行SDS-PAGE和Western blot分析表明: 重组FSHb-CTP-a的转化子可以正确有效地表达目的蛋白, 分子量约为29 kD。放射免疫分析法(RIA)测定表达上清, 高拷贝转化子的平均表达量为91.849 mIU/mL, 显著高于低拷贝转化子的平均37.419 mIU/mL的表达量, 为FSH的结构研究和长效FSH制剂的生产奠定了基础。     

山羊重组促卵泡素长效类似物基因在毕赤酵母中表达

为了获得长效FSH制剂, 利用重叠PCR技术将山羊FSH的a, b亚单位, 通过hCGb亚单位羧基末端延长肽(CTP)基因序列的连接, 构建成单链长效类似物基因FSHb-CTP-a。将其克隆至表达载体pPIC9K, 重组载体线性化处理后电转化至毕赤酵母GS115中, 经判型和G418筛选后获得高拷贝菌株His+Mut+。经甲醇诱导表达后, 进行SDS-PAGE和Western blot分析表明: 重组FSHb-CTP-a的转化子可以正确有效地表达目的蛋白, 分子量约为29 kD。放射免疫分析法(RIA)测定表达上清, 高拷贝转化子的平均表达量为91.849 mIU/mL, 显著高于低拷贝转化子的平均37.419 mIU/mL的表达量, 为FSH的结构研究和长效FSH制剂的生产奠定了基础。     

扬子鳄卵巢性类固醇激素受体的免疫细胞化学研究

为探讨扬子鳄卵巢内不同性类固醇激素受体在卵泡发育中的调控作用,研究采用组织学和免疫细胞化学方法,运用激光共聚焦显微镜,对扬子鳄不同发育时期卵泡中的雌激素受体、雄激素受体和孕激素受体进行了检测。结果发现,3种类固醇激素受体在卵巢各期滤泡细胞中均有表达,在4月Ⅱ-Ⅳ期卵泡的滤泡细胞中阳性反应最强;9月卵巢的滤泡细胞中阳性反应最弱;ER和AR不仅在各期滤泡细胞中存在阳性位点,在6月卵泡的卵母细胞胞质中也有表达。结果说明,在扬子鳄卵母细胞生长发育和成熟过程中,3种激素受体通过与其对应的激素结合对滤泡细胞的发育、卵黄的合成与积累以及排卵起着重要的调控作用。       

IGF-Ⅰ及其受体、IGF结合蛋白-2和LH受体mRNA在卵泡中的表达

利用原位杂交和原位DNA－3’末端标记的方法研究了胰岛素样生长因子河（IG－I）、IGF－I受体、IGF结合蛋白－2、和促性腺激素受体的信使核糖核酸（mRNA）在不同生长与闭锁阶段的大鼠卵巢卵泡中表达的变化。结果表明：IGF－I主要在正常生长的初级卵泡、窦前卵泡和小窦状卵泡中表达。在各生长与成熟阶段的卵泡中都检测到IGF－I受体mRNA,闭锁卵泡的IGF－I受体表达降低。窦前与窦状的生长和闭锁卵泡均表达IGFBP－2。促卵泡激素（FSH）受体在窦前和小窦状卵泡的表达水平比其在大卵泡中的高。窦前与小窦状卵泡仅在膜细胞中表达黄体生成素（LH）受体mRNA,大卵泡的膜细胞与颗粒细胞均表达LH受体,在闭锁卵泡中仅在膜细胞中观察到LH受体的信号。综上结果,提示IGF－I,IGF－I受体和FSH受体在窦前和小窦状卵泡中的协同表达对卵泡的早期发育有重要作用。LH受体mRNA特异地在大卵泡的颗粒细胞中表达可能与优势卵泡选择相关。     

牛促卵泡激素在毕赤酵母中的表达

目的:实现重组牛促卵泡激素在毕赤酵母中的表达。方法:依据毕赤酵母的密码子偏爱性设计并利用PCR方法合成了牛促卵泡激素的α亚基和带有6×his-tag的β亚基相应的DNA序列,构建表达载体pHIL-S-bFSH,转化毕赤酵母,表达蛋白进行ELISA定量和Western blot鉴定。结果:重组牛促卵泡激素的表达量为0.26mg/L,Western blot鉴定分子量为35kDa。结论:在毕赤酵母中实现了具有免疫源活性的重组牛促卵泡激素的表达。     

新型重组糖蛋白表达载体--盘基网柄菌

近年来,盘基网柄菌作为异源重组糖蛋白表达载体的研究受到了学术界的重视,已经有多种具有生物活性的复杂糖蛋白成功地得到了表达。通过对表达产物的研究发现,盘基网柄菌具有各种翻译后加工机制,例如磷酸化、酰基化及形成GPI(糖基磷脂酰基醇)锚点等,具有类似于高等动物的糖基化修饰能力。与哺乳动物细胞表达载体相比较,盘基网柄菌具有培养成本低廉、细胞生长迅速及易于大规模培养的优势。盘基网柄菌有可能发展成为一种有重要应用前景的糖蛋白表达载体系统。     

奶牛卵泡超数同步发育及其卵母细胞的发育潜能

目的建立促奶牛卵泡超数同步发育的方法,并利用体细胞核移植和分子技术评价来自这些卵泡的卵母细胞发育潜能。方法用E2-P4对淘汰的高产奶牛进行联合处理后,分别对其实施不同组合的外源促性腺激素处理[FSH12h组,FSH48h组,FSH48h-LH6h组（简称LH6h组）,FSH48h-LH26h组（简称LH26h组）]以促进其卵泡超数同步发育;然后从卵泡中回收COCs进行体外成熟,排放第一极体的卵供作优质种牛的体细胞核转移（nucleartransfer,NT）受体,以评价卵的发育潜能。结果E2-P4能成功地诱导奶牛卵巢中产生一个新的卵泡起始波,并能保证卵泡在外源促性腺激素作用下实现超数同步发育;上述四组不同组合外源促性腺激素处理的卵母细胞衍生的NT重构胚经体内培养7d后,后3组的囊胚发育率显著高于FSH12h组和对照组（P〈0.01）;将这些克隆囊胚移植同步发情的受体牛子宫后,仅LH26h组的卵所获得的克隆胚能实现全程发育（直至克隆小牛出生）。经RT-PCR分析颗粒细胞中FSHr、LHr和连接蛋白43（Connexin43,Cx43）等mRNA含量变化,以及Western印迹分析其Bcl-2和Bax蛋白表达水平的变化,证实该组卵巢提供的同步发育卵泡为早期闭锁卵泡。结论E2-P4-FSH48h-LH26h组合处理可人为调控牛卵泡的同步发育;处于早期闭锁状态卵泡的卵母细胞具备较高的发育潜能。     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

2017年中国植物科学若干领域重要研究进展

2017年中国植物科学继续保持高速发展态势, 重大成果频出, 具体表现在中国植物学家在国际顶级学术期刊发表的文章数量平稳上升。中国植物科学领域的研究工作者成果精彩纷呈, 如新型广谱抗病机制的发现、水稻广谱抗病遗传基础及机制和疫霉菌诱发病害成灾机制研究等。2017年中国生命科学领域十大进展评选中, 有两项植物科学领域的研究成果入选。水稻生物学、进化与基因组学和激素生物学等领域学科发展突出。另外, 值得一提的是, 长期从事高等植物与代谢途径调控分子网络研究和水稻品种设计育种的李家洋院士的研究成果“水稻高产优质性状形成的分子机理及品种设计”荣获2017年国家自然科学一等奖。这一具有重大国际影响的开创性贡献标志着中国植物科学在该领域的国际科学前沿居于引领和卓越地位。该文对2017年中国本土科学家在植物科学若干领域取得的重要研究成果进行了系统梳理, 旨在全面追踪和报道当前中国植物科学领域发展的最新前沿动态, 与广大读者共同分享我国科学家所取得的辉煌成就。     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义。综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望。     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义.综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望.     

Changes in aquatic vegetation in Rhine floodplain streams in Alsace in relation to disturbance

Abstract. Changes are described in aquatic vegetation in oligotrophic, groundwater-fed Rhine floodplain streams in Alsace (eastern France), resulting from disturbance. Disturbance factors include changes in nutrients, either permanent ones - effluent from a waste water treatment plant or trout hatcheries - or periodic ones: flooding. Regular inputs of high levels of phosphate and ammonia modified the macrophyte vegetation in these streams. The floristic composition, which was characteristic of oligotrophic waters upstream of the eutrophicated sector, changed to that of a eutrophic situation as originally found downstream. Periodic disturbance by floods which normally occur once a year, irregularly eutrophicates the small streams, causing the development of a mixture of eutrophic and oligotrophic species. Six macrophyte communities are distinguished, indicating different trophic levels. The aquatic vegetation is adapted to the variations of phosphate and ammonia levels. Hence, aquatic macrophytes can be used as bio-indicators of fluctuations in water nutrient levels in relation to the type of disturbance.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.     

Apoptosis in experimental myocardial infarction in situ and in the perfused heart in vitro

We report the appearance of apoptotic cells in experimental myocardial infarction (rabbit heart) in in situ and in vitro preparations. Apoptosis was recognized by intravital staining with Hoechst 33342 (Ho342), by nick-end labeling (TUNEL) and by DNA laddering. A steady rise in the relative number of apoptotic cardiomyocytes (apoptotic index) was noted in in situ preparations. Apoptosis was first noted 6 h after the onset of ischemia with its highest value occurring after 72 h. Apoptotic nuclei were absent in remote areas of the left and right ventricles. Apoptotic nuclei within the infarcted area showed diminished intensity of Ho342 fluorescence. Three days after ischemia, a border zone adjacent to the infarcted area consisting of apoptotic macrophages was recognized. A novel finding was the appearance of apoptotic cardiomyocytes in the isolated perfused ischemic heart. Occurring as early as 50 min after the onset of ischemia, a high apoptotic index was present adjacent to the ligature placed around the coronary artery. This observation provides the opportunity to selectively examine factors leading to apoptosis in the ischemic heart under controlled experimental conditions.