牛脑皮层G_i的纯化及其与AC的偶联

用1％胆酸钠和20％饱和度的硫酸铵抽提牛脑皮层细胞膜得到含G蛋白和腺苷酸环化酶（AC）的制剂,通过Sepharose6B柱将两者分开,再将含G蛋白的级分用庚胺-Sepharose4B疏水柱、羟基磷灰石柱将其它亚型的G蛋白（主要是Gs和Go）从抑制型G蛋白（Gi）中除去,获得纯化的高活力的Gi,其GTP结合活力为17．6nmol／mg,比细胞膜Gi活力提高50倍;并具有较高的产率,从1g膜蛋白中可获得0．66mg的Gi,同时可获得无G蛋白污染的AC和少量的Gs蛋白．SDS-PAGE显示分子量为41000和36000的两条蛋白带,证实是Gi的α基和β亚基．进一步用重建脂酶体的方法检测Gi对AC的抑制作用,结果显示Gi对AC活力的抑制达40％左右,表明CAMP信息跨膜转导通路中Gi与AC之间具有较好偶联功能．     

神经节苷脂GM3对J6－2细胞蛋白质磷酸化的影响

通过研究神经节苷脂GM₃对国人单核样白血病细胞系J6-2细胞蛋白质磷酸化的影响,在[γ- ³²P]ATP,GM₃,ATP,Mg²⁺与J6-2细胞液及颗粒两部分共同反应,10min(30℃)体系中,观察到GM₃对两部分蛋白质磷酸化的调节作用.GM₃(100μmol/L)促进颗粒部分分子量为180 000,87 000,78 000,67 000,43 000及31 000的蛋白质磷酸化,促进胞液部分分子量为87 000及56 000的蛋白质磷酸化,而且能抑制70 000及43 000蛋白质磷酸化.由于GM₃已被前人证实能对J6-2细胞起分化作用,其作用时间长达4-6d,很可能GM₃对蛋白质磷酸化作用的调节是GM₃促分化作用的早期信号.     

重组人血管内皮细胞生长因子121 cDNA的基因克隆、表达和鉴定

应用RT-PCR方法,扩增人VEGF₁₂₁ cDNA基因片段,与酵母表达载体pPIC9K重组,获得表达质粒p9KVEGF₁₂₁.该质粒转化毕赤酵母菌GS115,用G418-YPD平板筛选高拷贝转化子,PCR鉴定VEGF₁₂₁ cDNA与酵母染色体整合状态,高拷贝转化子用甲醇诱导表达.工程菌用5 L发酵罐发酵,表达产物r-hVEGF₁₂₁占培养液中总蛋白量70%以上.纯化产物促进牛毛细血管内皮(BCE)细胞增殖,并强烈促进血管通透.     

黄土高原苹果园蒸腾导度大气驱动规律比较

植物蒸腾导度是表征土壤-植物-大气连续体（SPAC）中植物-大气间水汽传导过程、反映植物水分调控能力的一类重要变量,常见有冠层导度（G_c）、冠层气孔导度（G_s）与叶片气孔导度（g_s）,明确三者在反映冠层蒸腾过程时的异同或关联性对于理解植物水分利用机制具有重要意义。本研究基于对黄土高原果园苹果树生长季内树干液流（J_s）及环境因子的连续观测,计算了G_c、G_s及脱耦联系数（Ω）等变量,并与短期连续观测的叶片气孔导度（g_s）比较,分析了G_c、G_s和g_s在反映冠层蒸腾特征方面的异同及其关系。结果表明,日变化过程中G_s、g_s呈"单峰"型曲线,而G_c则呈"先增后减,午后抬升"的"双峰"型曲线。g_s与G_s存在较紧密的线性关系（R²=0.80）,但与G_c的线性关系较弱（R²=0.02）。G_c、G_s均随大气水汽压亏缺（VPD）的变化呈现确定的规律,其中,上边界函数呈递减的对数函数关系,平均值则符合先增后减的Log-Normal函数关系（R²>0.95）,拐点对应的VPD值分别为1.33和1.16 kPa。在一日内,G_s对VPD变化的响应过程与g_s对VPDL （基于叶片温度计算的水汽压亏缺）变化的响应过程总体一致,其一致性高于G_c对VPD变化的响应。整个生长季（4-10月）中果树的Ω平均值为0.12,随着Ω递减,G_c与G_s的线性相关性愈趋紧密,其斜率呈递增趋势,G_c越来越趋近于G_s。研究结果表明,在北方地区,基于树干液流的监测能较准确的推导整株并估算林分的冠层蒸腾导度。与实测g_s的变化过程比较,G_s比G_c具有更高的一致性,G_s可以作为描述苹果树水分利用过程响应大气驱动的更为恰当的变量。     

植物叶片中过氧化氢含量测定方法的改进

Ti（Ⅳ）-H₂O₂比色法因背景物质干扰而测得的植物叶片内H₂O₂含量偏高，5％三氯乙酸抽提，活性炭脱色，Ti（Ⅳ）-4-(2-吡啶偶氮)间苯二酚(PAR)比色法测得的H₂O₂含量偏低.萃取法有效地脱去丙酮提液中的色素，且H₂O₂的回收率在95％以上.用过氧化氢酶(CAT）处理作空白对照，利用H₂O₂与Ti（Ⅳ）-PAR的显色反应，建立了一种简便、快速、准确的植物叶片内的H₂O₂含量测定方法，H₂O₂的最低检测浓度为0.25 μmol·L^－1.用该方法测得多种植物叶片中H₂O₂的含量在0.1～0.8 μmol·g^－1.     

竹红菌乙素作猝灭剂研究Fo构象方法的建立

线粒体F₁F_o复合体F_o部分a亚基的色氨酸荧光可被竹红菌乙素(hypocrellin B, HB)猝灭.不同温度下测定Stern-Volmer图的结果显示,猝灭常数(K_sv)随温度的增加而加大,时间衰变荧光的结果显示,荧光寿命随HB浓度的增加而减小,加入不同浓度的HB, F₁F_o复合体的吸收峰没有位移.这些实验结果支持动态猝灭机理.HB还具有有效猝灭浓度低,不影响酶的活力;在脂相和水相的分布比率可高达16 560∶1;实验操作简便等优点.因此HB可作为理想的疏水相荧光猝灭剂,研究与膜结合的F₁F_o复合体中镶嵌于膜脂内F_o的构象变化.     

超临界CO2技术萃取蛋黄磷脂

采用新型物理分离技术──超临界CO₂萃取法,提取天然蛋黄粉中的磷脂.在40MPa,先去除蛋黄粉中甘油三酯和胆固醇,再萃取磷脂.结果显示,磷脂纯度为95%,N/P比值为1.003,λ_max=214nm,薄层层析显示磷脂着色点清晰,并去除了绝大部分甘油三酯和胆固醇.此法操作简单、产品质量高、安全和不污染环境,还可得到天然纯蛋黄油和蛋白.     

促甲状腺激素单克隆抗体的制备

获得了抗促甲状腺激素(TSH)单克隆抗体杂交瘤细胞20株,其中T₇₄A₁₀小鼠腹水滴度为1:50 000,亲和常数为7.15×10⁹L/mol,T₇₁B₁₁小鼠腹水滴度为1:150000,亲和常数为8.75×10⁹L/mol.两个抗体与人绒毛膜促性腺激素(HCG)、促卵泡激素(FSH)和促黄体生成激素(LH)的交叉反应分别小于1.1×10^-6%、0.01%和0.016%.将T₇₄A₁₀和T₇₁B₁₁应用于TSH免疫放射分析中,得到了满意的结果.     

一种改进的纯化和鉴定牛脑皮层Gs蛋白的方法

用１％胆酸钠和１５％孢和硫酸铵相结合的方法,从牛脑皮层细胞膜中抽提得到主要含激活型Ｇ－蛋白和腺苷酸环化酶两种蛋白组分的制剂,然后通过Ｓｅｐｈａｒｏｓｅ６Ｂ柱将两者分开,将含Ｇｓ高活力的级分用庚胺－Ｓｅｐｈａｒｏｓｅ４Ｂ柱进一步分离,即可获得高活力的Ｇｓ,ＳＤＳ－ＰＡＧＥ显示为分子量４５０００和３６０００的两条蛋白带,该法具简便,快速,重复性好、产率高等优点,且可同时获得无Ｇｓ污染的ＡＣ。用无Ｇｓ污     

过氧化氢对培养心肌细胞损伤作用的研究

氧化应激时产生大量的自由基,造成心肌细胞的损伤.过氧化氢(H₂O₂)是有机体氧化代谢产物,同时是一种活性氧.应用不同浓度的H₂O₂,分别于不同作用时间,动态观察其对心肌细胞的损伤作用.从实验结果看到,低浓度的H₂O₂（＜0.1 mmol/L）作用2 h,使心肌细胞产生早期的生物化学的改变,如MDA产生堆积和细胞周期时相改变（G1期细胞增加,G2期细胞减少）,此时心肌酶基本无泄漏,心肌细胞的死亡率很低,HE形态学观察基本无改变;随着H₂O₂浓度的增加（1～5 mmol/L）和作用时间的延长,进一步诱导细胞损伤加剧,LDH释放和MDA积累明显升高,细胞死亡率也明显增加,已具有统计学意义.同时可观察到其病理形态学的坏死性改变;当10 mmol/L H₂O₂作用时,细胞大量死亡,形态学可见细胞极度收缩、脱落,形成大面积的细胞脱失区.因此,H₂O₂作为一种活性氧自由基,依其浓度和作用时间不同可造成不同程度的心肌细胞的损伤.辣根过氧化物酶作为一种自由基清除剂,可明显减少H₂O₂活性氧自由基对心肌细胞的损伤作用.     

‘Cross-talk’ between phospholipase C and adenylyl cyclase involves regulation of G-protein levels in GH3 rat pituitary cells

We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the α-subunits of the stimulatory and inhibitory G-proteins of AC (G_sα and G_i−2α) in cultured prolactin-producing rat pituitary adenoma cells (GH₃ cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated G_sα levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G_sα levels by more than 50% at all time-points. G_i−2α levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G_s protein α-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways.     

亚洲玉米螟幼虫血清中酚氧化酶原的性

采用40%硫酸铵沉淀、Blue Sepharose CL-6B亲和层析和Phenyl Sepharose CL-4B疏水层析等方法,从亚洲玉米螟Ostrinia furnacalis (Guenée) 幼虫血清中分离纯化了酚氧化酶原。酚氧化酶原全酶相对分子量约为158 kD,亚基相对分子量约为80 kD和78 kD。酚氧化酶原为糖蛋白,该酶原易被0.1 mmol/L CPC (氯代十六烷基吡啶)、 50%甲醇、 1 mg/mL昆布多糖和1 mg/mL胰蛋白酶激活。该酶反应的最适pH为7.0,最适温度为25～30℃,Ca²⁺和Mg²⁺可增强该酶的活性。     

Specificity of heterotrimeric G protein regulation by human chorionic gonadotropin and low-molecular agonist of luteinizing hormone receptor

Luteinizing hormone (LH) and its homologue, human chorionic gonadotropin (hCG), are very important regulators of the reproductive system. These hormones stimulate various types of G proteins—primarily, G_s and G_q proteins—by binding to the specific LH-hCG receptor, which leads to the activation of adenylate cyclase (AC) and phospholipase C, respectively. It has been suggested that many side effects of LH and hCG are associated with low selectivity of their effect on G proteins. Low-molecular agonists of LH-hCG receptor developed on the basis of thienopyrimidine derivatives do not cause these side effects, and differences in the interaction with G proteins may be ones of the cause for this. To test this, a comparative study of the effect of hCG and synthesized by us thienopyrimidine derivative, 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP03) on the AC activity and GTP binding of G proteins in plasma membranes isolated from the rat ovaries and testes was performed. Cholera toxin (CT) and pertussis toxin (PT) were used to selectively switch off the signal transduction via G_s and G_i/o proteins, the peptide corresponding to the C-terminal segment 349–359 of the Gα_q subunit was used to suppress G_q-dependent cascades. It was shown that treatment of ovarian and testicular membranes with CT resulted in suppression of TP03 and hCG stimulatory effects on the AC activity, but in different ways influenced the GTP binding stimulation: it completely blocked the effect of 10^–6 M TP03 and reduced by 45–46% the effect of hCG (10^–8 M). Preincubation of membranes with the peptide 349–359 reduced the hCG stimulatory effect on GTP binding by 34 (ovaries) and 45% (testes), but did not affect the corresponding effect of 10^–6 M TP03. Preincubation with the peptide 349–359 also reduced the GTP stimulatory effect of 10^–4 M TP03, but to a small extent. The obtained data indicate that, in contrast to hCG, the targets of which in the ovaries and testes are G_s and G_q proteins, the action of TP03 is realized mainly via G_s proteins. Only at a concentration that exceeds EC₅₀ by two orders TP03 is capable to relatively weakly activate G_q proteins. The PT treatment of the membranes did not affect the effects of TP03 and hCG, which indicates the lack of their effective interaction with G_i/o proteins. Thus, the selectivity of activation of G_s-dependent cascades responsible for the synthesis and production of steroid hormones is a significant advantage of low-molecular agonists of LH-hCG receptor over gonadotropins.     

菜心和水稻绿叶中不同等电点的乙醇酸氧化酶

The proteins with glycolate oxidase activity from B.parachinensis Bailey and rice( Oryza sativa )green leaves were prepared respectively.From the second protein peak on DEAE\|Cellulose column,two glycolate oxidases,expressed as B.parachinensis Bailey GO Ⅲ(specific activity natove 13 2 U·mg -1 ·min -1 )and rice GOⅢ(specific activity 8 8 U·mg -1 ·min -1 ),could not migrate anywhere in 4%～20% native\|PAGE under a pH8.3 buffer system.GOⅢ's p I was about pH8.3. The protein containing B.parachinensis Bailey GOⅢ showed 67±2,43±2,and 38±2 kD in SDS PAGE,band 43±2 kD was the subunit of B.parachinensis Bailey GOⅢ.From the two proteins above,another group of glycolate oxidases,expressed as B.parachinensis Beiley GOⅠ(specific activity 5 U·mg -1 ·min -1 )and rice GOⅠ(specific activity 1 2 U·mg -1 ·min -1 ),showed only one 43±2 kD band in SDS\|PAGE,and was purified on the Sepharose\|6B column which migrated towards anode in the same native\|PAGE showing the M r about 420 kD,or 460 kD and 260 kD respectively.GOⅠ's p I was smaller than pH8.3.Antibody against B.parachinensis Bailey GOⅠ was prepared and its efficacy was about 1/1600 in ELISA.By native\|PAGE,Western blot and rocket immunoelectrophoresis,the third group of glycolate oxidases,expressed as B.parachinensis Bailey GOⅡ and rice G0Ⅱ,were confirmed in crude protein of green leaves and migrated towards cathode under the same native\|PAGE,so GOⅡ's p I was higher than pH8.3.The M r of B.parachinensis Bailey GOⅡ was about 669 kD determined by native\|PAGE Western blot.Rice GOⅠ,rice GOⅢ and rice GOⅡ showed different quantitation under different physiological conditions.Rice GOⅡ could be induced by glycolate.     

The Peptides Mimicking the Third Intracellular Loop of 5-Hydroxytryptamine Receptors of the Types 1B and 6 Selectively Activate G Proteins and Receptor-Specifically Inhibit Serotonin Signaling via the Adenylyl Cyclase System

The third intracellular loop (ICL3) of G protein-coupled receptors has, as a rule, a key role in their interaction with heterotrimeric G proteins. We synthesized peptides corresponding to the C-terminal region of the ICL3 (C-ICL3) of 5-hydroxytryptamine receptors of the type 1B (5-HT_1BR) and 6 (5-HT₆R) and studied their influence on the functional activity of adenylyl cyclase signaling system (ACSS) in synaptosomal membranes isolated from the rat brain. The 5-HT_1BR-peptide ARERKATKTL^307–316K-amide mimicking agonist-activated 5-HT_1BR reduced forskolin-stimulated adenylyl cyclase (AC) activity and activated pertussis toxin-sensitive G proteins. It lowered inhibitory effects of serotonin and 5-HT_1BR-agonists on forskolin-stimulated AC activity and their stimulating effects on GTP binding. This was not the case in the presence of 5-HT_1BR-antagonists. The 5-HT₆R-peptides mimicking 5-HT₆R activated both the basal AC activity and GTP binding of cholera toxin-sensitive G proteins. They lowered the stimulating effect of serotonin and 5-HT₆R-agonists on AC and G_s proteins, but in the presence of 5-HT₆R-antagonists their action was blocked. Of all the 5-HT₆R-peptides with linear and dimeric structure we studied the palmitoylated peptide KHSRKALKASL^258–268K(Pal)A-amide had a most pronounced effect both on the basal and 5-HT₆R-agonist-stimulated ACSS. The data was obtained indicating that the peptides corresponding to C-ICL3 of 5-HT_1BR and 5-HT₆R selectively activate G_i and G_s proteins, respectively, and in a receptor-specific manner reduce signal transduction via serotonin-sensitive ACSS in the rat brain. The results of the study give strong evidence in favor of active participation of C-ICL3 of these 5-HTRs in their coupling with the G proteins.     

Isolation and Purification of Cytokinin-Binding Protein from Wheat Chloroplasts

The extract of wheat chloroplast membrane proteins was precipitated by different saturation of (NH4)2SO4. Pellet of 0–30% saturation showing high binding activity to 3H-6BA wax loaded on the affinity chromatography column which was prepared by coupling 6BA to epoxy activated sepharose 6B. The CTK-binding protein was eluted from the BA-sepharose 6B column with Tris buffer containing 0.1 mmol/L 6BA. It showed a single protein band on PAGE and the apparent molecular weight was about 250kD. Two bands with molecular weight of 60kD and 66kD were detected on SDS-PAGE. It was supposed that the protomer of CTK-binding protein was a tetramer of two subunits.     

Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

Background and purpose

Despite the view that only β₂- as opposed to β₁-adrenoceptors (βARs) couple to G_i, some data indicate that the β₁AR-evoked inotropic response is also influenced by the inhibition of G_i. Therefore, we wanted to determine if G_i exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.

Experimental approach

We used the G_s-selective (R,R)- and the G_s- and G_i-activating (R,S)-fenoterol to selectively activate β₂ARs (β₁AR blockade present) in combination with G_i inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.

Key results

PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC₅₀ values of fenoterol matched published binding affinities. The PTX enhancement of the G_s-selective (R,R)-fenoterol-mediated responses suggests that G_i regulates AC activity independent of receptor coupling to G_i protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of G_i with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.

Conclusions and implications

Together, these data indicate that G_i exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G_i and G_s activity upon AC towards G_s, enhancing the effect of all cAMP-mediated inotropic agents.     

Anti-hypoxia activity of a polysaccharide extracted from the Sipunculus nudus L

A water-soluble polysaccharide, named as SNP, was extracted and fractioned from the body wall of Sipunculus nudus L. by DEAE-Sepharose anion exchange and Sepharose CL-6B column chromatography. The evaluation for anti-hypoxia activity demonstrated that SNP had significant anti-hypoxic activity on normobarie hypoxia, chemical intoxicant hypoxia and acute cerebral ischemia hypoxia models in mice. SNP also enhanced the number of red blood cell count (RBC) and the concentration of hemoglobin (HGB). The structural characteristics of SNP investigated by high performance size exclusion chromatography, Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry indicated that SNP was a homogeneous polysaccharide with a molecular mass of 350 kD and was composed of rhamnose (28%), fucose (16%) and galactose (56%). The results suggested that SNP could be explored as a novel potential anti-hypoxia agent.