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%。     

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。     

副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 *

为了实现糖苷类物质的高效转化,将来源于副干酪乳杆菌(Lactobacillus paracasei)TK1501 β-葡糖苷酶基因连接于表达载体pET28a(+)上,在E. coli BL21中表达,重组酶经镍离子亲和层析分离得到纯酶,其分子质量和比酶活分别为86.63kDa和675.56U/mg。最适作用温度和pH分别为30℃和6.5。 Mg ²⁺和Ca ²⁺对β-葡糖苷酶酶活抑制作用最小,Cu ²⁺几乎使其丧失催化活性。其底物特异性较宽泛,对大豆异黄酮、栀子苷、水杨苷、七叶苷、虎杖苷、熊果苷均有降解作用。以β-pNPG为底物时,该酶的K_m和V_max分别为1.44mmol/L和58.32mmol/(L·s),催化系数k_cat为3 982/s。结果与分析表明,来源于副干酪乳杆菌TK1501 β-葡糖苷酶对水解大豆异黄酮和合成糖苷将会发挥重要作用。     

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。     

鳞杯伞α-半乳糖苷酶的提取纯化及酶学性质研究

采用离子交换层析和凝胶过滤层析对鳞杯伞子实体中的α-半乳糖苷酶进行纯化,得到了一种分子量为50 kDa的α-半乳糖苷酶,命名为CSG。纯化后的CSG纯化倍数为891.46倍,比活力为54.78 U/mg,得率为0.71%。通过BLAST比对液相色谱-串联质谱(LC-MS/MS)获得其肽段,发现其为GH27家族的α-半乳糖苷酶。CSG的最适pH为3.0,最适温度为50 ℃。在酸性范围pH 2.2-7.0和温度范围4-30 ℃有较好的稳定性。Mn²⁺、Cd²⁺、Cu²⁺对CSG有较强的抑制作用。半乳糖和蜜二糖对CSG的抑制类型为混合型抑制。化学修饰剂N-溴代琥珀酰亚胺显著降低CSG的活力,碳二亚胺对CSG具有显著的激活作用。该酶具有良好的蛋白酶抗性,且对棉子糖家族寡糖(RFOs)、瓜尔豆胶和赤槐豆胶均表现出良好的水解作用。     

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。     

农杆菌介导Txpam基因转化烟曲霉TMS-26及其产紫杉醇效果评价

为研究红豆杉紫杉醇合成途径限速酶基因功能及其对内生真菌烟曲霉TMS-26发酵产紫杉醇的影响,以曼地亚红豆杉愈伤组织制备cDNA作为模板扩增苯丙氨酸氨基变位酶基因（Txpam）,构建重组质粒pGEX-4T-1-Txpam,转入大肠杆菌中进行异源诱导表达,经亲和层析纯化,获取重组酶TxPAM并验证其酶活性。构建pCAMBIA1302-Txpam质粒,转化农杆菌感受态细胞,利用农杆菌介导的转化体系获得转化子并优化转化条件,结合插入片段携带的分子标记和目的基因进行转化子验证,同时培养转化菌株并检测紫杉醇产量。结果表明：纯化获取的重组酶TxPAM,经HPLC检测具有将α-苯丙氨酸催化为β-苯丙氨酸的功能;在最优转化条件下,转化子数目达到471个/10⁶个孢子;根据基因hyg和Txpam的克隆以及测序结果,说明成功构建了基因工程菌株,通过对其发酵条件进行优化,紫杉醇产量达到721.87μg/L。     

神经退行性疾病动物模型的建立与分析

为进一步分析突变α-synuclein蛋白的作用机理,该研究组将表达人α-synuclein(A53T)的病毒载体注射入不同年龄的猕猴脑黑质部位,首次证明了老龄化能够促进突变α-synuclein蛋白在灵长类动物脑组织中的聚集和神经毒性作用,也为突变α-synuclein介导的神经病变提供一个新的治疗靶点。相关结果将于2015年5月在线发表于The Journal of Neuroscience杂志。     

儿童孤独症相关基因NRXN1β启动子功能分析

Neurexins是神经特异性突触蛋白,Neurexin1β结构的异常与孤独症密切相关。为分析孤独症相关基因NRXN1β最小启动子和调节基因转录的功能元件,本文构建了含NRXN1β基因上游调控区不同区域的荧光素酶报告基因质粒,转染HEK293细胞后,利用检测双荧光素酶报告基因的转录活性以确定NRXN1β基因最小启动子区,进而筛选出相应的显著增强或抑制报告基因活性的功能区;同时,为鉴定顺式作用元件,利用基因定点突变技术对基因功能区内和临近DNA序列进行连续的碱基突变;最后,采用转录因子预测工具对启动子功能区内的转录调控元件进行分析。结果首次发现NRXN1β最小启动子区位于-88~+156 bp,-88~-73 bp和+156~+149 bp可增强启动子活性,+229~+419 bp可抑制启动子活性,且-84~-63 bp为能够显著性增强启动子活性的顺式作用元件,该区域可能存在DBP(Albumin D-site-binding protein,DBP)和ABF1(Autonomously replicating sequence-binding factor 1,ABF1)两个转录因子结合位点。     

地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

从地衣芽孢杆菌(Bacillus licheniformis)中克隆到耐高温α-淀粉酶基因全长, 构建了原核表达载体, 转入大肠杆菌(Escherichia coli)中, 使用IPTG于28°C诱导6小时后, 通过SDS-PAGE检测到目的蛋白, 分子量约为55 kDa, 并通过酶活力检测实验证明该蛋白具有耐高温α-淀粉酶活性。同时构建了该基因融合GFP的植物表达载体, 通过农杆菌(Agro- bacterium tumefaciens)介导瞬时转化烟草(Nicotiana tabacum)下表皮细胞并在荧光显微镜下观察, 发现在烟草下表皮细胞的细胞质和液泡中均有绿色荧光。使用I₂-KI溶液对乙醇脱色后的烟草叶片进行染色, 显色反应表明在烟草中表达的耐高温α-淀粉酶具有酶活性。最后, 采用农杆菌介导的花蕾浸泡法将重组载体转化到拟南芥(Arabidopsis thaliana)中, 筛选到稳定遗传的耐高温α-淀粉酶基因的拟南芥纯合子。研究结果为后期开展表达耐高温α-淀粉酶的转基因植物的相关研究奠定了实验基础。     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

Estrogen-like effect of a Cimicifuga racemosa extract sub-fraction as assessed by in vivo, ex vivo and in vitro assays

Black cohosh (Cimicifuga racemosa) is used in the treatment of painful menstruation and menopausal symptoms. Data about the nature of the active compounds and mechanism(s) of action are still controversial, chiefly with respect to its estrogenic activity.

This work aimed to assess the possible estrogenic activity of a commercial dry hydro-alcoholic extract of C. racemosa and its hydrophilic and lipophilic sub-fractions on in vivo, ex vivo, and in vitro assays.

In a yeast estrogen screen, only the lipophilic sub-fraction was able to activate the human estrogen receptor , with a lower potency but comparable efficacy to that of 17 β-estradiol.

Neither the total extract nor the lipophilic sub-fraction showed an in vivo uterotrophic effect in 21-day-old rats. Uterine tissues obtained ex vivo from C. racemosa treated animals were generally much less sensitive to oxytocin, prostaglandin F_2, and bradykinin than tissues obtained from estradiol valerate treated rats.

The lipophilic sub-fraction, instead, induced a dose-dependent inhibitory activity on the in vitro response to oxytocin, prostaglandin F_2, and bradykinin of uterine horns from naïve 28-day-old rats, with a potency rate close to 1:30 of that of 17 β-estradiol.

Reported results confirm the effectiveness of C. racemosa in menstrual distress and further emphasize the possibility that lipophilic constituents bind to an as yet not identified estrogen receptor, likely inversely involved in inflammation.     

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration to guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17-hydroxylase and 21-hydroxylase activities were decreased to 20–25% of control values by the higher dose of ABT. Mitochondrial 11β-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3β-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.     

DSIP affects adrenergic stimulation of rat pineal N-acetyltransferase in vivo and in vitro

The natural occurrence, sleep, and extra-sleep effects of delta sleep-inducing peptide (DSIP) have been shown by different laboratories. However, neither an in vitro assay system nor a probable mechanism of action of the peptide have been conclusively demonstrated so far. The recent finding that DSIP influences the nocturnal rise of N-acetyltransferase (NAT) activity in rat pineal led us to investigate a possible effect on pharmacologically induced NAT activity in vivo and in vitro. Stimulation of the enzyme with adrenergic drugs such as isoproterenol and phenylephrine was reduced by DSIP at doses of 150 and 300 μg/kg injected subcutaneously. In vitro, 6, 150 and 300 nM DSIP attenuated isoproterenol stimulation of the enzyme in cultured pineals, whereas 150 nM DSIP effectively reduced stimulation induced by a combination of the two drugs. The peptide alone did not influence NAT activity in vitro, but produced a slight stimulation in vivo. To our knowledge, these results represent the first report of a direct interaction of DSIP with adrenergic transmission. The in vitro system could prove useful for establishing possible mechanism(s) of action of the ‘sleep peptide.’     

In vitro steroid metabolic studies in human testes II: Metabolism of cholesterol, pregnenolone, progesterone, androstenedione and testosterone by testes of an estrogen-treated man

The effect of long-term in vivo estrogen treatment on in vitro steroidogenesis by the testes of a young man was investigated. In vitro incubation of testicular tissue of this man with ³H-pregnenolone, ³H-progesterone, ³H-androstenedione and ³H-testosterone demonstrated suppression of 17-hydroxylase activity, with little or no effect of the treatment on Δ⁵-3β-hydroxysteroid oxidoreductase, 5a-reductase and aromatase. Increased 20-hydroxysteroid oxidoreductase activity was observed. Determination of intratesticular steroid concentrations led to similar conclusions.     

11β-Amidoalkyl estradiols, a new series of pure antiestrogens

In order to find new antiestrogens, devoid of any agonistic activity, a series of 11β-amidoalkyl estradiols were prepared. These compounds have been studied in comparison with tamoxifen (TAM): in vitro, for their relative binding affinities (RBA) for mouse and MCF-7 estrogen receptors (ER) and for their antiproliferative effect on MCF-7 (estradiol or EGF/PDGF stimulated) and Ly2 human breast cancer cell lines; in vivo, for their uterotrophic/antiuterotrophic activities in the mouse and for their antitumoral activities on MCF-7 tumors implanted in nude mice.

The most representative compounds are N-methyl-N-isopropyl-(3,17β-dihydroxy-estra-1,3,5(10)-trien-11β-yl)-undecanamide (RU 51625) and its 17-ethynyl derivative (RU 53637). They showed good RBAs for ER and a stronger antiproliferative effect than TAM in vitro. Unlike TAM, these compounds inhibited growth factor stimulated MCF-7 proliferation, and the growth of the TAM resistant cell line Ly2. In vivo, they were completely devoid of uterotrophic activity, when given subcutaneously in mice, but exhibited a slight agonistic effect when administered orally. They showed interesting antitumor activities in nude mice by the percutaneous route, but RU 53637 was significantly more potent than RU 51625 when given orally.     

Effects of Calpain on Antioxidant Enzyme Activities

The activities of superoxide dismutase, catalase and glutathione reductase were not affected by in vitro incubation with the intracellular proteinase calpain, suggesting that these enzymes are not in vivo substrates of calpain. In contrast, the activity of another important antioxidant enzyme, glutathione peroxidase, is stimulated in vitro by calpain. This may explain the correlation between elevations in glutathione peroxidase activity and calpain activity which occur in aging, exercised and dystrophic muscle. Calpain treatment in vitro caused a large decrease in the activity of carnosine synthetase which is involved in the synthesis of the putative antioxidant carnosine. This may be the reason for the in vivo correlation between elevated calpain and diminished carnosine levels in aging, hypertensive, denervated and dystrophic muscles.     

Glycyrrhizae Radix attenuates peroxynitrite-induced renal oxidative damage through inhibition of protein nitration

We investigated the protective effects of Glycyrrhizae Radix extract against peroxynitrite (ONOO^-)-induced oxidative stress under in vivo as well as in vitro conditions. The extract showed strong ONOO^- and nitric oxide (NO) scavenging effects under in vitro system, in particular higher activity against ONOO^-. Furthermore, elevations of plasma 3-nitrotyrosine levels, indicative of in vivo ONOO^- generation and NO production, were shown using a rat in vivo ONOO^--generation model of lipopolysaccharide injection plus ischemia-reperfusion. The administration of Glycyrrhizae Radix extract at doses of 30 and 60 mg/kg body weight/day for 30 days significantly reduced the concentrations of 3-nitrotyrosine and NO and decreased inducible NO synthase activity. In addition, the nitrated tyrosine protein level and myeloperoxidase activity in the kidney were significantly lower in rats given Glycyrrhizae Radix extract than in control rats. However, the administration of Glycyrrhizae Radix extract did not result in either significant elevation of glutathione levels or reduction of lipid peroxidation in renal mitochondria. Moreover, the in vivo ONOO^- generation system resulted in renal functional impairment, reflected by increased plasma levels of urea nitrogen and creatinine, whereas the administration of Glycyrrhizae Radix extract reduced these levels significantly, implying that the renal dysfunction induced by ONOO^- was ameliorated. The present study suggests that Glycyrrhizae Radix extract could protect the kidneys against ONOO^- through scavenging ONOO^- and/or its precursor NO, inhibiting protein nitration and improving renal dysfunction caused by ONOO^-.     

4. Progress and prospects in plant metabolism

This review considers some of the landmarks in our investigation of plant metabolism: the merging of work in vitro with that in in vivo, the use of labelled substrates, inter- and intra-cellular compartmentation. Progress in the study of the control of metabolism is reviewed from Blackman's Law of Limiting Factors via the concept of ‘Pacemaker’ reactions to determination of Control Coefficients. The tremendous opportunities afforded by the availability of mutant and transgenic plants are extolled.