胆囊收缩素8对大鼠大脑皮质细胞钙调素和蛋白激酶C活性的影响

 为阐明中枢神经系统中胆囊收缩素 8(CCK8)受体的信号传递机制 ,以分离的大鼠大脑皮质神经细胞为材料 ,观察了CCK8对细胞内钙调素 (CaM)、3′ ,5′ 环腺苷酸 (cAMP)、蛋白激酶C(PKC)活性的影响 研究结果表明 ,CCK8可刺激大脑皮质细胞CaM、PKC活性的增加 ,并有剂量依赖关系 但CCK8在 10 -12 ～ 10 -6mol L范围内 ,细胞内cAMP含量无显著变化 利用受体亚型L 364,718和L 365,2 60的研究表明 ,两种拮抗剂均可抑制CCK8引起的CaM和PKC活性变化 ,但两者IC50 不同 对于CaM ,CCKB 受体拮抗剂L 365,2 60的IC50 比CCKA 受体拮抗剂L 364,718低 4 0倍 ;而对于PKC ,L 365,2 60的IC50 比L 364,718低 60倍 因此认为 ,CCK8主要是通过CCKB 受体介导了CaM和PKC活性的变化     

中华绒螯蟹眼柄MTXO细胞GABA受体通道研究

采用全细胞膜片钳技术测定了中华绒螯蟹（Eriocheir sinensis）眼柄视神经节端髓X器官（MTXO）三种类型神经内分泌细胞对0.01～5mmol/L γ氨基丁酸（γ-aminobutyric acid,GABA）的反应,并结合选择性拮抗剂和激动剂的使用进行了GABA受体研究.在电流钳模式下,依据不同Nernst Cl^－电位,三种类型细胞均对GABA产生去极化或超级化反应.在电压钳模式下,GABA激活Cl^－通道电流（I_GABA）.I_GABA在灌流GABA后约1 200 ms内激活,800 ms内达到峰值,没有明显的脱敏反应,反转电位接近Nernst Cl^－电位.I_GABA幅值呈浓度依赖性,激活阈值为0.01 mmol/L,约在0.5 mmol/L达到饱和.药理学实验结果表明,中华绒螯蟹眼柄神经内分泌细胞GABA受体是Cl^－通道蛋白,对Cl^－离子通道阻断剂Picrotoxin和Niflumic acid敏感,但是对GABA_A受体拮抗剂Bicuculline和GABA_C受体激动剂cis-4-aminocrotonic acid (CACA)和trans-4-aminocrotonic (TACA)均不敏感.     

尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征

将尖吻蝮蛇毒碱性磷脂酶A₂（A.aBPLA₂）基因克隆至温敏表达载体pBLMVL2,在大肠杆菌RR1中成功诱导表达.表达产物A.aBPLA₂约占细菌蛋白质总量的20％,并以包涵体的形式存在.纯化包涵体后,将产物变性、复性,然后用FPLC Superose^TM12纯化,产物经过SDS-聚丙烯酰胺凝胶电泳检测只有单一条带.对纯化后的表达A.aBPLA₂进行了酶活性、抑制血小板聚集活性和溶血活性的测定.结果显示,表达A.aBPLA₂的酶活性与变性后复性江浙蝮蛇酸性磷脂酶A₂酶活性相近,具有类似变性后复性江浙蝮蛇碱性磷脂酶A₂的溶血活性,没有抑制血小板聚集活性.最后对磷脂酶A₂的结构与这些活性的关系进行了讨论.     

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.     

苄基异喹啉类化合物 D20抑制钙调素激活磷酸二酯酶的研究

苄基异喹啉化合物是一类钙调素拮抗剂.对新合成的双苄基异喹啉化合物 D₂₀对钙调素依赖的磷酸二酯酶的抑制作用进行了研究,IC₅₀=5μmol/L,表明其拮抗作用大于三氟啦嗪,是强的拮抗剂.荧光分析表明,钙调素与化合物 D₂₀的结合常数为2.64(μmol/L)^-1,一个化合物 D₂₀分子与两个钙调素分子结合,并显示了结合方向性及空间位阻影响.     

胞浆型磷脂酶A2介导弱氧化修饰低密度脂蛋白诱导的人脐静脉内皮细胞凋亡

探讨弱氧化修饰低密度脂蛋白(MM-LDL)能否诱导人脐静脉内皮细胞(HUVECs)凋亡以及胞浆型磷脂酶A₂(cPLA₂)在此过程中的作用.MTT法测定细胞存活率;相差显微镜、荧光显微镜和流式细胞仪检测细胞凋亡;³H-花生四烯酸(³H-AA)预标法测定PLA₂活性;蛋白质印迹检测cPLA₂磷酸化;激光共聚焦显微镜检测单个细胞内钙离子浓度的变化.结果表明,MM-LDL(100～300 mg/L)作用后的HUVECs呈现凋亡典型的形态特征,凋亡率随MM-LDL浓度的增加而上升.MM-LDL能引起胞内钙离子浓度增加,cPLA₂的活化及磷酸化.15 μmol/L AACOCF₃和5 mmol/L EGTA在抑制cPLA₂活性的同时,部分抑制MM-LDL诱导的HUVECs凋亡.加入外源性AA(50 μmol/L)能逆转AACOCF₃引起的凋亡抑制.结果提示,cPLA₂参与了MM-LDL诱导HUVECs凋亡的信号传递.     

不同CO2浓度升高和氮肥水平对水稻叶绿素荧光特性的影响

为研究不同CO₂浓度升高和氮肥水平对水稻叶绿素荧光特性的影响,利用由开顶式气室（OTC）组成的CO₂浓度自动调控平台开展田间试验。以粳稻9108为试验材料,CO₂浓度设置CK（对照,环境大气CO₂浓度）、C₁（CO₂浓度比CK增加160 μmol/mol）和C₂（CO₂浓度比CK增加200 μmol/mol）3个水平;氮肥设置低氮（N₁：10 g/m²）、中氮（N₂：20 g/m²）和高氮（N₃：30 g/m²）3个水平。结果表明,在低氮条件下,与CK相比,C₁处理使拔节期的F_o上升4.8%（P=0.031）;C₂处理使拔节期的F_o上升6.3%（P=0.015）,F_v/F_m下降4.8%（P=0.003）,使孕穗期的F_o上升12.7%（P=0.039）,F_v/F_o下降18.2%（P=0.039）。在高氮条件下,与CK相比,C₂处理使灌浆期的F_m、F_v和F_v/F_m分别下降3.6%（P=0.039）、4.9%（P=0.013）和1.3%（P=0.039）。在中氮条件下,与CK相比,C₁和C₂处理的影响不明显。在整个生育期内,CO₂浓度升高与施氮处理交互作用对水稻叶绿素荧光特性的影响未到达显著水平。研究表明,大气CO₂浓度升高使水稻叶片光系统Ⅱ受损,抑制其电子传递能力、电子受体Q_A氧化还原情况、最大光化学效率和潜在活性,通过适量施氮可以有效地缓解其负面效应。     

微生物传感器测定维生素B12的研究

将大肠杆菌(Escherichia coli)215用吸附法固定于醋酸纤维素膜上,与氧电极配合组成微生物电极,建立了对维生紊B₁₂的快速测定系统。测定浓度范围5×10^-6mg—2．5×10^-5mg／ml;测定温度范围在28—39℃;最适Ph为6．7—7．8,测定一个样品所需时间为2h,比常用的生物学测定方法所需时间缩短10倍以上。该系统对维生素B₁₂重复测定的相对误差为±3％。固定化菌体在－25℃保存25天后再进行测定,应答电流不低于初始值的92%。     

邻二氮菲－Fe2＋氧化法检测H2O2／Fe2＋产生的羟自由基

报告检测H₂O₂/Fe^2＋所产生羟自由基的新方法. 羟自由基氧化反应后, 邻二氮菲-Fe^2＋的A₅₃₆明显下降, 且△A₅₃₆与邻二氮菲, FeSO₄及H₂O₂呈量效关系, 随反应时间延长, △A₅₃₆依幂函数规律上升. 此法试验结果表明, 甘露醇, 抗坏血酸及硫肥清除羟自由基作用呈明显的量效关系.     

基于宏基因组研究植物乳酸菌或博落回提取物对山羊回肠微生物合成维生素B12的影响

【目的】探索研究反刍动物胃肠道微生物合成维生素B₁₂的方法,并评估植物乳酸菌或博落回提取物对断奶山羊回肠食靡微生物合成维生素B₁₂的影响。【方法】选取体重相近年龄相仿的断奶黑山羊20只,随机分为对照组(CON, n=7)、乳酸菌组(LAC, n=7)和博落回组(MAC, n=6)。CON组饲喂普通的日粮,LAC组饲喂基础日粮+10 g/d的植物乳酸菌(Lactobacillus plantarum P-8 strains, 4.0×10⁹ CFU/g),MAC组饲喂基础日粮+0.3 g/d的博落回提取物(Macleaya cordata 3.75%)。试验结束后,采集回肠中段食靡样品。利用宏基因组测序技术,比对最新功能基因数据库VB12Path和公共数据库KEGG,分析植物乳酸菌和博落回提取物对山羊回肠食靡微生物合成维生素B₁₂的影响。【结果】结果显示,比对VB12Path数据库共注释到55个与维生素B₁₂合成相关的基因。与CON组相比,LAC组和MAC组中合成维生素B₁₂基因的丰富度和均匀度降低(P<0.05）。3组间基因的β多样性也有显著的差异(P<0.05);比对KEGG数据库共注释到49个与维生素B₁₂合成相关的基因,LAC组的多样性与CON组没有差异,但MAC组的α多样性显著降低(P<0.05)。值得注意的是,比对VB12Path数据库和KEGG数据库均发现LAC组和MAC组中参与前咕啉2合成途径、参与无氧合成途径、有氧合成途径、参与重排转换途径以及腺苷钴胺素合成途径的部分基因(gltX、cbiT、cobT和btuD等)的丰度均显著地高于CON组(P<0.05)。【结论】2个数据库比对后的相似结果表明博落回提取物在对断奶山羊回肠微生物合成维生素B₁₂相关代谢上与植物乳酸菌的作用相似,均可以通过改变其多样性和提高部分关键基因的丰度,从而影响微生物合成维生素B₁₂的潜能,为后期博落回提取物和植物乳酸菌在畜牧养殖中的运用提供一定的理论支撑。此外,2个数据库比对的差异提示未来研究胃肠道微生物维生素B₁₂相关代谢时,应用多个数据库比对,能更全面精确地进行评价,为后期分析过程奠定研究基础和提供新的思路。     

Cholecystokinin-8 activates myenteric neurons in 21- and 35-day old but not 4- and 14-day old rats

Cholecystokinin (CCK) activates the myenteric neurons of adult rats. The goal of this work is to determine the ontogeny of this activation by CCK-8 in the myenteric plexus of the duodenum (2 cm immediately following the pyloric sphincter aborally) and compare it with that of the dorsal vagal complex (DVC) - which occurs in 1-day old pups. Despite the existence of both of the CCK receptors, CCK₁ and CCK₂, in 4, 14, 21 and 35 day old rats, CCK-8 (0, 5, 10, 20 and 40 μg/kg, i.p.) increased Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) in the myenteric neurons of 21- and 35-day old rats but in the DVC of all age groups. As such, this belated activation of myenteric neurons by CCK-8 compared to the DVC may reflect a delayed role for these neurons in CCK-related functions.     

Localization of cholecystokinin receptor subtypes in the endocine pancreas.

This study was undertaken to clarify the controversy in the literature about pancreatic localization of the cholecystokinin (CCK) CCK(A) and CCK(B) receptors. With antibodies used by other investigators, we first established their specificity by Western blotting, indirect immunofluorescence, and confocal microscopy with each antibody's peptide antigen. Co-localization assays between the CCK receptors and the pancreatic hormones insulin, glucagon, and somatostatin revealed that the CCK(A) RAbs 1122 and R1-2 recognized insulin and glucagon cells in rat, pig, and human pancreas but not in the somatostatin cells. Conversely, the three CCK(B) RAbs tested, 9262, 9491, and GR4, identified the somatostatin cells. Abs 9491 and GR4 occasionally co-localized with glucagon, a feature that never occurred with Ab 9262. Finally, the specificity of Ab 9262 for the pancreatic CCK(B) R was confirmed in six different species. It co-localized with somatostatin but never with glucagon in these species. Our data suggest the use of Abs 1122 and 9262 to specifically identify and localize pancreatic CCK(A) and CCK(B) receptors, respectively. Confusion in the literature may result from the lack of specificity of most antibodies used, as established in this study.     

Enterostatin reduces serum cholesterol levels by way of a CCK(1) receptor-dependent mechanism

Enterostatin (APGPR), an anorectic pentapeptide derived from the amino terminus of procolipase, significantly reduced serum cholesterol levels after oral administration at a dose of 100 mg/kg for 3 days in mice fed a high-cholesterol-cholic acid diet. The hypocholesterolemic effect of APGPR was inhibited by pretreatment with lorglumide, an antagonist for cholecystokinin 1 (CCK(1)) receptor, even though APGPR does not have any affinity for CCK(1) receptors. Similarly, the hypocholesterolemic activity of VPDPR, an APGPR analogue, was blocked by lorglumide. These results suggest that the hypocholesterolemic effects of APGPR and VPDPR are mediated by a CCK(1) receptor-dependent mechanism.     

Rapakinin, Arg-Ile-Tyr, derived from rapeseed napin, shows anti-opioid activity via the prostaglandin IP receptor followed by the cholecystokinin CCK(2) receptor in mice

Rapakinin, Arg-Ile-Tyr, is a vasorelaxing, anti-hypertensive and anorexigenic peptide derived from rapeseed napin. In this study, we found that rapakinin intracerebroventricularly administered to mice inhibited the analgesic effect of morphine, evaluated by the tail-pinch test. The anti-opioid activity of rapakinin was blocked by LY225910, an antagonist of the cholecystokinin (CCK) CCK₂ receptor, but not by lorglumide, an antagonist of the CCK₁ receptor. The anti-opioid activity of rapakinin was also blocked by CAY10441, an antagonist of the prostaglandin (PG) IP receptor. These results suggest that the anti-opioid activity of rapakinin is mediated by the CCK₂ and IP receptors. The anti-opioid activity induced by ciprostene, an IP receptor agonist, was blocked by LY225910, while that of CCK-8 was not blocked by CAY10441. Thus, it is demonstrated that the CCK-CCK₂ system was activated downstream of the PGI₂-IP receptor system. Taken together, rapakinin shows anti-opioid activity via the activation of the PGI₂-IP receptor system followed by the CCK-CCK₂ receptor system.     

Valine-286 residue in the third intracellular loop of the cholecystokinin 2 receptor exerts a pivotal role in cholecystokinin 2 receptor mediated intracellular signal transduction in human colon cancer cells

Although expression of the gastrin/cholecystokinin-2 receptor (CCK2R) is widely reported in human colorectal cancer, little is known on its role in mediating mature amidated gastrin (gastrin-17 amide, G-17) induced intracellular signal transduction in colon cancer cells. The purpose of this study was to explore the intracellular events of colorectal cancer cells after gastrin binding to CCK2R. Meanwhile, the influence of a natural point mutation 286V-->F in the third intracellular loop of CCK2R on gastrin-envoked intracellular signal transduction was also investigated. Firstly, Colo320 cells were stably transfected with wild type (Colo320 WT) and mutant CCK2R (Colo320 M), respectively. The intracellular signal transduction events in response to gastrin were investigated in both Colo320 WT and Colo320 M cells. In Colo320 WT cells, G-17 induced formation of intracellular cyclic AMP and inositol 1,4,5-trisphosphate, and stimulated intracellular calcium mobilization. G-17 also stimulated tyrosine phosphorylation of ERKl/2, p38, FAK, and paxillin, and up-regulated the mRNA expression of early response gene c-Jun and c-Fos. However, G-17 inhibited proliferation and induced apoptosis in Colo320 WT cells. Mutation 286V-->F in the third intracellular loop of CCK2R blocked G-17 induced biological without affecting binding affinity of CCK2R to G-17. Our results suggest that activation of CCK2R by gastrin stimulates heterotrimeric G-protein Gq and G(12/13) mediated intracellular signal transduction pathway in colon cancer cells. The valine-287 residue in third intracellular loop of CCK2R plays a pivotal role in CCK2R mediated intracellular signal transduction.     

μ- and μ4-η coordination of A2H2 (A = C, Si, Ge, Sn and Pb) ligands with transition metals

The heavier analogs of C₂H₂ have been studied at the B3LYP level for their μ and μ₄-η² coordination properties with the transition metals. Based on known alkyne compounds, transition metal fragments [W₂(μ-NH)(Cp)₂(Cl)₂] and [Fe₄(CO)₁₂] have been chosen. The SBKJC relativistic effective core potentials and their associated basis sets were used on W, Fe, Sn and Pb, and the 6-31G(d) basis set was used on all other elements. All the complexes of Si₂H₂, Ge₂H₂, Sn₂H₂ and Pb₂H₂ are found to be local minima. The trans-twist nature of the ligand A₂H₂ (A = Si-Pb) is large in μ-coordinated complexes of W, and it is very small in μ₄-η² coordinated complexes of Fe. The electronic structure of these complexes was investigated using fragment molecular orbital method (FMO).     

Unambiguous through-bond sugar-to-base correlations for purines in 13C, 15N-labeled nucleic acids: The HsCsNb,HsCs(N)bCb,and HbNbCb experiments

Summary A set of three 3D (¹H, ¹³C, ¹⁵N) triple-resonance correlation experiments has been designed to provide H1-H8 intraresidue sugar-to-base correlations in purines in an unambiguous and efficient manner. Together, the H_sC_sN_b, H_sC_s(N)_bC_b, and H_bN_bC_b experiments correlate the H1 sugar proton to the H8 proton of the attached base by means of the {H1, C1, N9, C8, H8} heteronuclear scalar coupling network. The assignment strategy presented here allows for unambiguous H1-H8 intraresidue correlations, provided that no two purines have both the same H1 and C1 chemical shifts and the same C8 and N9 chemical shifts. These experiments have yielded H1-H8 intraresidue sugar-to-base correlations for all five guanosines in the [¹³C, ¹⁵N] isotopically labeled RNA duplex r(GGCGCUUGCGUC)₂.     

Crystal structure of the ternary FimC-FimF(t)-FimD(N) complex indicates conserved pilus chaperone-subunit complex recognition by the usher FimD

Type 1 pili, anchored to the outer membrane protein FimD, enable uropathogenic Escherichia coli to attach to host cells. During pilus biogenesis, the N-terminal periplasmic domain of FimD (FimD(N)) binds complexes between the chaperone FimC and pilus subunits via its partly disordered N-terminal segment, as recently shown for the FimC-FimH(P)-FimD(N) ternary complex. We report the structure of a new ternary complex (FimC-FimF(t)-FimD(N)) with the subunit FimF(t) instead of FimH(p). FimD(N) recognizes FimC-FimF(t) and FimC-FimH(P) very similarly, predominantly through hydrophobic interactions. The conserved binding mode at a "hot spot" on the chaperone surface could guide the design of pilus assembly inhibitors.     

Acetylcholinesterase/Butyrylcholinesterase inhibition activity of some new carbacylamidophosphate deriviatives

Eight newly synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl₂ with R = pCl–C₆H₄ 1a, pBr–C₆H₄ 2a, C₆H₅ 3a, and pMe–C₆H₄ 4a and RC(O)NHP(O)(NC₄H₈O)₂ R = pCl–C₆H₄ 1b, pBr–C₆H₄ 2b, C₆H₅ 3b, pMe–C₆H₄ 4b, were selected to compare the inhibition kinetic parameters, IC₅₀, K_i, k_p and K_D, on human erythrocyte acetylcholinesterase (hAChE) and bovine serum butyrylcholinesterase (BuChE), Also, the in vivo inhibition potency of compound 2a, 2b and 3a, were studied. The data demonstrates that compound 2a and compound 2b are the potent sensitive as AChE and BuChE inhibitors respectively, and the inhibition of hAChE is about 10-fold greater than that of BuChE.