稳定表达人源GABAAR-CHO细胞株的建立

目的: 构建α₁亚基诱导表达、β₂和γ_2L亚基稳定表达的人源α₁β₂γ_2L-GABA_AR-CHO(Chinese hamster ovary)细胞株。方法: 从人cDNA文库中扩增α₁、β₂、γ_2L亚基编码基因,分别构建亚基表达载体;将三个亚基表达载体共转染CHO-K1细胞,通过抗性筛选、膜电位检测法进行稳定表达克隆筛选;通过qPCR、Western blot对亚基表达进行鉴定;以激动剂GABA、阳性变构调节剂地西泮(diazepam,Dia)、拮抗剂荷包牡丹碱(bicuculine)为工具药,采用全细胞膜片钳方法及膜电位检测法对稳定表达细胞的药理学功能进行鉴定。结果: 经克隆筛选获得表达量较高的α₁β₂γ_2L-GABA_AR-CHO并对其亚基表达鉴定,结果显示该细胞稳定表达α₁、β₂、γ_2L亚基,构建的α₁β₂γ_2L-GABA_AR-CHO细胞仅在加入四环素(tetracyclin)诱导的情况下表达α₁亚基并与β₂、γ_2L组装成具有功能活性的α₁β₂γ_2L-GABA_AR;对其进行全细胞膜片钳检测研究发现,GABA可对其产生激动效应,引起α₁β₂γ_2L-GABA_AR-CHO细胞产生氯离子通道特征性电流变化,Dia可剂量依赖性地增强GABA对α₁β₂γ_2L-GABA_AR的激动效应;在膜电位检测研究中,获得GABA激动效应EC₅₀为(177.72 ± 15.92)nmol/L,Dia变构效应EC₅₀为(3.63±0.52)μmol/L,拮抗剂Bicuculine拮抗效应IC₅₀为(538.83±29.55)nmol/L。结论: 通过采用诱导表达策略,成功构建了α₁β₂γ_2L-GABA_AR-CHO稳定表达细胞株,该细胞株具有对激动剂、阳性变构剂、拮抗剂特异性检测的药理学功能。     

浙江省主要亚热带森林群落类型物种和谱系水平的α和β多样性比较

了解不同森林群落类型的物种和谱系水平的α和β多样性, 有助于指导森林经营和生物多样性保护。本研究比较了浙江省内不同地点主要森林类型(包括常绿阔叶林、常绿落叶阔叶混交林、落叶阔叶林和针阔叶混交林)的物种α多样性和谱系α多样性, 以及物种β多样性和谱系β多样性。研究表明, 该地区主要森林类型的物种和谱系α多样性均存在较大差异, 但控制了空间和地形因子的作用后, 差异几乎全部消失; 森林类型内部及相互间的物种和谱系β多样性均存在显著差异, 同种森林类型内部的物种和谱系β多样性分别小于不同森林类型之间的物种和谱系β多样性, 且在控制了空间和地形因子的作用后, 以上差异仍然显著。本研究表明影响亚热带主要森林群落类型物种和谱系水平的α和β多样性的因素存在差异: α多样性可能主要受到空间和地形因子等的影响, 而β多样性则可能受到森林类型的重要影响。     

青藏高原北部戈壁植物群落物种、功能与系统发育β多样性分布格局及其影响因素

戈壁荒漠广泛分布于全球干旱和极旱区域, 是我国陆地生态系统的重要组成部分。由于自然环境恶劣和交通条件限制, 目前有关戈壁植物群落物种、功能和系统发育等多维度β多样性形成机制的系统研究还很缺乏, 严重制约着对戈壁植物多样性维持机制的认知。本文以青藏高原北部61个典型戈壁生境植物群落为研究对象, 通过构建系统发育树和测量8个关键功能性状, 获取戈壁生境的物种、功能和系统发育β多样性, 比较3个维度β多样性格局与零模型的差异, 同时量化环境距离和地理距离对其的相对影响, 以探讨戈壁植物多样性的形成机制。结果显示: (1)戈壁植物的物种、功能和系统发育β多样性均表现出显著的距离衰减效应; (2)戈壁植物的物种、功能和系统发育β多样性均表现为非随机的格局; (3)由于功能性状趋同进化, 植物功能和系统发育β多样性变化趋势并不一致; (4)环境差异对植物3个维度β多样性均有着比空间距离更为重要的影响, 且土壤含水量、地表砾石盖度等局域生境因素的影响比气候更为强烈。以上结果表明, 戈壁植物的β多样性可能主要由局域生境过滤作用控制, 且不同维度的β多样性分布格局并不一致。     

细胞色素P450还原酶与CYP17的共表达及其功能分析*

17α-羟基黄体酮(17α-OH-PROG)是甾体激素类药物的关键中间体,其生物合成主要由细胞色素单加氧酶(CYP17)催化生成。在此过程中,细胞色素 P450还原酶(cytochrome P450 reductase,CPR)作为细胞色素P450 酶电子传递链的重要组成部分,直接影响CYP17的催化效率。为研究不同来源CPR与17α-羟化酶的适配性,首先以人源17α-羟化酶作为研究对象,构建了表达质粒pPIC3.5k-hCYP17,获得了重组毕赤酵母菌株。其次筛选获得3种不同来源CPR,构建了表达质粒 pPICZX-CPR,获得17α-羟化酶与CPR共表达菌株,并在毕赤酵母中进行转化实验,对转化产物进行薄层色谱(TLC)和高效液相色谱(HPLC)分析。结果显示,重组菌株具有17α-羟化酶活性,能够催化黄体酮生成目标产物17α-OH-PROG 以及副产物16α-羟基黄体酮(16α-OH-PROG)。不同来源的CPR与17α-羟化酶共表达与仅表达17α-羟化酶的产率相比均有所提高,其中hCPR-CYP17共表达菌株表现出最高的转化水平,17α-OH-PROG产率提高42%。上述结果表明:17α-羟化酶基因与CPR共表达能够提高其黄体酮17α-羟基化水平。为甾体黄体酮17α-羟基化的生物催化研究提供思路,对甾体药物的工业生产具有重要意义。     

毛头鬼伞子实体化学成分及抗糖尿病活性初探

从毛头鬼伞子实体中萃取得到乙醇、乙酸乙酯、石油醚3种有机提取物,采用α-葡萄糖苷酶活性抑制实验对3种有机提取物的抗糖尿病活性进行评价,结果显示,乙酸乙酯提取物对α-葡萄糖苷酶有较强的抑制活性。采用柱层析技术从乙酸乙酯提取物中分离纯化出10种化合物,经核磁等方法鉴定为：（1）顺,顺-9,12-十八(碳)二烯酸;（2）顺式-9-十八烯酸;（3）(22E,24R)-麦角甾烷-5,7,22-三烯-3β醇;（4）3β-5α-6α-22E-麦角甾-7,22-双烯-3,5,6-三醇-6-亚油酸酯;（5）3β-5α-6α-22E-麦角甾-7,22-双烯-3,5,6-三醇-6-油酸酯;（6）邻苯二甲酸二异丁酯;（7）对羟基苯乙醇;（8）4-羟基苯乙基乙酸酯;（9）3-(4-羟基-3-甲氧苯基)败脂酸;（10）N-反式-3,4亚甲二氧基肉桂酰基-3-甲氧基酪胺。对分离化合物的α-葡萄糖苷酶活性抑制实验结果显示,N-反式-3,4亚甲二氧基肉桂酰基-3-甲氧基酪胺对α-葡萄糖苷酶具有较强的抑制活性,其IC₅₀值为4.17mg/mL。     

青弋江鱼类分类群和功能群的α和β多样性纵向梯度格局

确定溪流鱼类多样性的时空分布格局可为鱼类多样性保护与管理提供科学基础。尽管溪流鱼类分类群多样性的纵向梯度格局已有大量报道, 但以鱼类生物学特征为基础的功能多样性研究较少。本文基于2009-2010年4个季度对青弋江1-5级溪流共15个样点的调查数据, 利用形态特征数据和食性构建了鱼类复合功能群, 研究了不同级别溪流间鱼类分类群和功能群组成及多样性的异同, 着重探讨了鱼类分类群和功能群的α和β多样性沿溪流纵向梯度的变化规律。采集到的56种鱼类可分为4个营养功能群和5个运动功能群, 共计14个“营养-运动”复合功能群。双因素交互相似性分析结果显示, 鱼类分类群和功能群组成都随河流级别显著变化, 但季节动态不显著; 双因素方差分析后发现, 鱼类分类群和功能群α、β多样性都随河流级别显著变化, 但受季节影响不显著。经回归分析, 分类群和功能群α多样性与河流级别大小呈显著的线性正相关, 但最大分类群α多样性出现于4级河流, 最大功能群α多样性在4级和5级河流间一致; 分类群和功能群β多样性与河流级别大小呈显著的二项式关系, 呈U型分布。分类群β多样性的空间变化主要取决于物种周转, 而功能群β多样性主要由嵌套所驱动。本研究表明, 沿着“上游-下游”的纵向梯度, 河流鱼类的α和β多样性的空间变化规律不同, 分类群和功能群α多样性的空间格局基本一致, 但分类群(主要是物种周转)和功能群β多样性(主要是功能嵌套)的空间变化过程的驱动机制不同。     

不同处理方法对灵芝β-葡聚糖降解效果的比较研究

本研究采用酸法、碱法、酶法和微波法对灵芝β-葡聚糖进行降解,通过降解率、产物分子量变化、产物聚合度分布等指标比较了不同方法的降解效果。结果表明,微波法降解率高达94%,处理后产物的分子量明显降低,寡糖产物聚合度分布广。酶法降解率约为40%,寡糖产物中含有DP2-5的成分。酸法及碱法降解率低于20%,寡糖产物少。研究表明,与其他3种方法相比,微波法降解率高、产物丰富、操作条件易于控制,是一种简单、高效的降解灵芝β-葡聚糖、制备灵芝β-葡寡糖的方法。     

致幻毒蘑菇卵囊裸盖菇化学成分研究初探

从一种采集于贵州省的致幻毒蘑菇——卵囊裸盖菇Psilocybe ovoideocystidiata中首次分离得到3种化合物,分别是3β-羟基-5α,8α-桥二氧麦角甾-6,22E-二烯(化合物1)、β-D-葡萄糖(化合物2)和腺苷(化合物3)。基于高分辨质谱与核磁共振谱数据以及相关文献比对确定以上3种化合物的结构,并首次推导出化合物2和3质谱裂解规律,其中重排与中性丢失在质谱裂解过程中起主导作用。利用UPLC-MS/MS法对卵囊裸盖菇的干燥子实体和新鲜子实体中的裸盖菇素和脱磷裸盖菇素进行检测,在干燥子实体中检测到裸盖菇素和脱磷裸盖菇素,但在-80 ℃保存6个月的新鲜子实体中未检测到裸盖菇素和脱磷裸盖菇素,推测可能是由于保存方法和提取方法的原因导致化合物发生变化。     

固相萃取-超高效液相色谱-串联质谱法测定新鲜块菌子实体中α-雄烷醇

本研究建立了一种固相萃取-超高效液相色谱-串联质谱的检测方法,用于检测新鲜块菌子实体中α-雄烷醇(5α-雄甾-16-烯-3α-醇)的含量。新鲜块菌样品经无水乙醇提取,Qasis HLB柱萃取富集后,采用超高效液相色谱-串联质谱进行分析定量。方法学验证结果表明该方法的回收率为88.49%-92.22%;检出限为0.120 9 ng/mL,定量限为0.398 9 ng/mL。该方法简便、精确,适用于新鲜块菌中α-雄烷醇含量的测定。     

提高源自Bacillus circulans 251的β-CGTase对麦芽糖亲和性及其在生产海藻糖中的应用 *

将B. circulans 251 β-CGTase应用于海藻糖制备,海藻糖转化率从50.4%提高至71.9%。为进一步提高底物的转化率,运用易错PCR-高通量筛选技术筛选对以麦芽糖为歧化反应受体的亲和性提高的B. circulans 251 β-CGTase突变体。利用低底物浓度的96孔板4,6-亚乙基-对硝基苯-α-D-麦芽七糖苷(EPS)显色法,最终筛选得到了一株对麦芽糖亲和性提高的突变体M234I。将野生型β-CGTase和突变体酶M234I进行蛋白质纯化,测定其酶学性质。结果表明,突变体的比活为345.25U/mg,野生型则为357.63U/mg;突变体M234I对麦芽糖的K_m为0.258 2mmol/L,仅为野生型(0.474 9mmol/L)的54.4%,对麦芽糖的亲和性显著提高;突变体的最适温度、最适pH较野生型未发生较大变化。以麦芽糊精(DE值16)为底物,将突变体M234I用于多酶复配体系生产海藻糖,酶反应结果表明海藻糖的转化率最高达74.9%,较野生型β-CGTase提高约3%。     

Biotechnological advances and perspectives of gamma-aminobutyric acid production

Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid that is widely distributed among various organisms. Since GABA has several well-known physiological functions, such as mediating neurotransmission and hypotensive activity, as well as having tranquilizer effects, it is commonly used as a bioactive compound in the food, pharmaceutical and feed industries. The major pathway of GABA biosynthesis is the irreversible decarboxylation of l-glutamate catalyzed by glutamate decarboxylase (GAD), which develops a safe, sustainable and environmentally friendly alternative in comparison with traditional chemical synthesis methods. To date, several microorganisms have been successfully engineered for high-level GABA biosynthesis by overexpressing exogenous GADs. However, the activity of almost all reported microbial GADs sharply decreases at physiological near-neutral pH, which in turn provokes negative effects on the application of these GADs in the recombinant strains for GABA production. Therefore, ongoing efforts in the molecular evolution of GADs, in combination with high-throughput screening and metabolic engineering of particular producer strains, offer fascinating new prospects for effective, environmentally friendly and economically viable GABA biosynthesis. In this review, we briefly introduce the applications in which GABA is used, and summarize the most important methods associated with GABA production. The major achievements and present challenges in the biotechnological synthesis of GABA, focusing on screening and enzyme engineering of GADs, as well as metabolic engineering strategy for one-step GABA biosynthesis, will be extensively discussed.     

GABA signalling during development: new data and old questions

In addition to being the major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) is thought to play a morphogenetic role in embryonic development. During the last decade, considerable progress has been made in elucidating the molecular mechanisms involved in GABA synthesis and biological action. The present review is an attempt to summarise recent results on the ontogeny of the different components of embryonic GABA signalling with an emphasis on the synthesis of GABA by different molecular forms of glutamic acid decarboxylase (GAD).     

全反式维甲酸通过γ-氨基丁酸途径促进小鼠胚胎腭板间充质细胞的凋亡

维甲酸(RA)是一种能够诱导腭裂发生的致畸物．研究显示γ-氨基丁酸(GABA)在腭板的发育过程中发挥重要作用．而GABA是否参与了RA诱导的腭裂发生还不清楚．本研究以小鼠胚胎腭板间充质细胞(MEPM)为研究对象,观察全反式维甲酸(atRA)(0.2、0.67、2.0和 6.7 μmol/L)对MEPM细胞增殖和凋亡的影响,并探讨GABA信号通路在其中的可能作用．结果显示,atRA(2.0 μmol/L和6.7 μmol/L)显著性抑制了MEPM的增殖,并促进了细胞凋亡．atRA(0.67、2.0和 6.7 μmol/L)显著性降低了GABA合成的关键酶谷氨酸脱羧酶(GAD67)mRNA和蛋白质的表达,但对γ-氨基丁酸A型受体-β3(GABAAR-β3)mRNA和蛋白质的表达没有影响．1.0 μmol/L的GABA逆转了atRA(6.7 μmol/L)对MEPM细胞增殖和凋亡的影响．以上结果表明,atRA通过下调GAD67的表达,减少GABA的产生,抑制MEPM的增殖和促进MEPM的凋亡,从而可能影响腭板的发育,诱导腭裂形成．     

Presence of γ-Aminobutyric Acid in Rat Ovary

Abstract: As γ-aminobutyric acid (GABA) was first discovered as the free acid in the mammalian central nervous system, it has been assumed that GABA is generally to be found in significant amounts only in the brain, in spite of reports of its presence in a number of non-neuronal tissues. In this study, GABA was detected amongst the free amino acids in most rat tissues that were examined. The highest concentration outside the brain was in the ovary (0.59 μmol/g fresh tissue). It is concluded that the synthesis of the GABA is intragonadal and probably of metabolic importance.     

富含γ-氨基丁酸食品的研究进展

γ-氨基丁酸(gamma-aminobutyric acid,GABA)是哺乳动物中枢神经系统主要的抑制性神经递质,并具有调节血压与心率、调节情绪、抗焦虑、抗抑郁、抗肿瘤、保肝护肾、调节激素分泌等生理功能。目前主要通过厌氧等技术处理植物原料、微生物发酵以及外源添加的方式加工富含GABA的食品,涉及富含GABA的茶叶、粮食、豆类制品、乳制品、糖果、饮料等食品的研究、专利及部分产品,但产品较少,今后需加强产业化。     

Conversion of 4-aminobutyraldehyde to γ-aminobutyric acid in striatum treated with semicarbazide and kainic acid

4-Aminobutyraldehyde (ABAL) has been shown to cross the blood-brain barrier and to be converted rapidly to -aminobutyric acid (GABA) in various regions of the brain. In this paper, the formation of GABA from ABAL was studied with striatum that had suffered a lesion to GABA synthesis via glutamic acid decarboxylase (GAD). The GABA formation from ABAL was invariably observed in striatum in which GAD was severely inhibited by semicarbazide or kainic acid. Thus, this is another pathway for GABA formation.     

Importance of Glutamine for γ-Aminobutyric Acid Synthesis in Rat Neostriatum In Vivo

This work was carried out to evaluate the importance of glial cells in providing precursors for the in vivo synthesis of gamma-aminobutyric acid (GABA). Fluorocitrate, which selectively inhibits the tricarboxylic acid cycle in glial cells, was administered locally in rat neostriatum. Inhibition of the glial cell tricarboxylic acid cycle led to a decrease both in glutamine level and in gamma-vinyl GABA (GVG)-induced GABA accumulation, an observation indicating reduced GABA synthesis. The role of glutamine, which is synthesized in glial cells as a precursor for GABA, was further investigated by inhibition of glutamine synthetase with intrastriatally administered methionine sulfoximine. In this case, the glutamine level was reduced to near zero values, and the GVG-induced GABA accumulation was only half that of normal. The results show that glutamine is an important precursor for GABA synthesis, but it cannot be the sole precursor because it was not possible to depress the GVG-induced GABA accumulation completely.     

Gamma-aminobutyrate: defense against invertebrate pests?

Gamma-aminobutyrate (GABA) is a ubiquitous four-carbon, non-protein amino acid. In plants, stress-induced GABA accumulation is well documented. However, the role(s) of GABA accumulation is contentious. In this Opinion article, we argue that wounding due to herbivory and crawling by insect larvae causes rapid GABA accumulation via the disruption of cellular compartmentation and the release of the acidic vacuolar contents to the cytosol. The activity of glutamate decarboxylase, the cytosolic enzyme responsible for GABA synthesis, has an acidic pH optimum. Subsequent GABA ingestion has a plant defense function by directly acting on GABA-regulated invertebrate neuromuscular junctions. Plants with an enhanced GABA-producing capacity reduce herbivory by invertebrate pests. These findings suggest that GABA accumulation is a rapidly deployed, local resistance mechanism that constitutes a first line of defense in deterring herbivory.     

Synthesis of neurotransmitter GABA via the neuronal tricarboxylic acid cycle is elevated in rats with liver cirrhosis consistent with a high GABAergic tone in chronic hepatic encephalopathy

Hepatic encephalopathy (HE) is a neuropsychiatric complication to liver disease. It is known that ammonia plays a role in the pathogenesis of HE and disturbances in the GABAergic system have been related to HE. Synthesis of GABA occurs by decarboxylation of glutamate formed by deamidation of astrocyte-derived glutamine. It is known that a fraction of glutamate is decarboxylated directly to GABA (referred to as the direct pathway) and that a fraction undergoes transamination with formation of alpha-ketoglutarate. The latter fraction is cycled through the neuronal tricarboxylic acid cycle, an energy-generating pathway, prior to being employed for GABA synthesis (the indirect pathway). We have previously shown that ammonia induces an elevation of the neuronal tricarboxylic acid cycle activity. Thus, the aims of the present study were to determine if increased levels of ammonia increase GABA synthesis via the indirect pathway in a rat model of HE induced by bile-duct ligation and in co-cultures of neurons and astrocytes exposed to ammonia. Employing (13) C-labeled precursors and subsequent analysis by mass spectrometry, we demonstrated that more GABA was synthesized via the indirect pathway in bile duct-ligated rats and in co-cultures subjected to elevated ammonia levels. Since the indirect pathway is associated with synthesis of vesicular GABA, this might explain the increased GABAergic tone in HE.