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61.
Summary A DNA segment carrying the full-length, intronless firefly luciferase gene was inserted into the high expression secretion vector, pIN-III -ompA. Upon induction of gene expression, luciferase activity was detected in extracts prepared from periplasmic fractions. The results indicated that the OmpA signal peptide was able to direct secretion of firefly luciferase across the cytoplasmic membrane. This has important implications for using this luciferase as a reporter in studying protein export and targeting. 相似文献
62.
Jian Wang C. Bruce Verchere Christopher H. S. McIntosh John C. Brown 《Cell communication & adhesion》1994,1(4):343-353
The intracellular free Ca2+ ion concentration ([Ca2+]i) was measured using fura-2 microspec-trofluorimetry in individual rat pancreatic β-cells prepared by enzymatic digestion and fluorescence-activated cell sorting. The mean basal concentration of [Ca2+]i in β-cells in the presence of 4.4 mM glucose and 1.8 mM Ca2+ was 112±1.6 nM (n=207). The action of acetylcholine (ACh) was concentration-dependent, and raising the concentration resulted in [Ca2+]i spikes of increasing amplitude and duration in some, but not all of the β-cells. In addition, the β-cells demonstrated variable sensitivity to ACh. The increases in [Ca2+]i were rapid, transient and were blocked by atropine at 10-6M. A brief exposure to 50 mM K+ resulted in a transient increase in [Ca2+]i similar to that induced by ACh, but resistant to atropine. A high concentration of ACh (100μL 10-4M or 10-3M) induced [Ca2+]i oscillations in 11 out of 57 β-cells in the presence of 4.4 mM glucose. Using calcium channel blockers and Ca2+ free medium, the source of the increase in [Ca2+]i was deduced to be from extracellular spaces. Changing the temperature from 22 to 37°C did not affect the action of ACh on [Ca2+]i. These data strongly suggest that ACh exerted a direct action on [Ca2+]i in normal rat pancreatic β-cells and support a role for Ca2+ as a second messenger in the action of ACh. 相似文献
63.
Since cytoplasmic Ca2+ levels are reported to regulate neurite elongation, we tested whether calcium-activated kinases might be necessary for growth cone motility and neurite elongation in explant cultures of goldfish retina. Kinase inhibitors and activators were locally applied by micropipette to retinal growth cones and the responses were observed via phase-contrast videomicroscopy. In some cases, growth rates were also quantifed over several hours after general application in the medium. The selective inhibitors of protein kinase C, calphostin C (0.1–1 μM) and chelerythrin (up to 50 μM), caused no obvious changes in growth cones or neurite elongation, and activators of PKC (phorbols, arachidonic acid, and diacylglycerol) also were generally without effects, although phorbols slowed the growth rate. Inhibitors of protein kinase A and tyrosine kinases also produced no obvious effects. The calmodulin antagonists, calmidazolium (0.1 μM), trifluoperazine (100 μM), and CGS9343B (50 μM), however, caused a reversible growth cone arrest with loss of filopodia and lamellipodia. The growth cone became a club-shaped swelling which sometimes moved a short distance back the shaft, leaving evacuated filaments at points of strong filopodial attachments. A similar reversible growth cone arrest occurred with the general kinase inhibitors: H7 at 200 but not at 100 μM, and staurosporine at 100 but not 10 nM, suggesting possible involvement of a calmodulin-dependent kinase (camK) rather than PKC. The selective inhibitor of camKII, KN-62 (tested up to 50 μM), produced no effects but the specific myosin light-chain kinase (MLCK) inhibitors ML-7 (3–5 μM) and ML-9 (5–10 μM) reversibly reproduced the effect, suggesting that MLCK rather than camKII is necessary for growth cone motility. The MLCK inhibitors' effects both on growth cone morphology and on F-actin filaments (rhodamine-phalloidin staining) were similar to those caused by cytochalasin D (5 μM), and are discussed in light of findings that inhibiting MLCK disrupts actin filaments in astrocytes and fibroblasts. 1994 John Wiley & Sons, Inc. 相似文献
64.
RGDS肽对大鼠主动脉球囊内膜剥脱后血管壁增殖的影响 总被引:1,自引:0,他引:1
在大鼠主动脉球囊内膜剥脱术后血管壁细胞过度增殖模型上,用合成的血小板膜纤维蛋白原受体(glycoproteinⅡb/Ⅲacomplex,GPⅡb/Ⅲa)拮抗剂RGDS(Arg-Gly-Asp-Ser,50μmol·kg-1·d-1)治疗可有效地抑制损伤血管壁的细胞计数增加和内膜增厚以及血管平滑肌细胞增殖,显著降低其血管组织3H-TdR和3H-Leu的参入增加程度。实验结果提示RGDS肽作为血管成型术的辅佐剂,对于防治血管再狭窄可能具有潜在的临床应用前景。 相似文献
65.
岩豆凝集素的圆二色性与生物学活性关系的研究 总被引:1,自引:0,他引:1
岩豆凝集素(MDL)的远紫外圆二色性谱(CD谱)显示216-217nm处的单一负峰。此时MDL分子含有16.2%的α螺旋,46.3%的β折叠和37.5%的无规卷曲。pH9.0时负峰红移至220nm,且在217-222nm处的峰值几乎相同;在20-40℃范围内,CD谱的变化甚微;60℃时谱峰蓝移;在80℃或100℃时,212nm处出现一大负峰。1mol/L或2mol/L脲时,MDL的CD谱已发生明显变化,二级结构单元也有变化,凝集兔红细胞的活性也随之减弱;随脲浓度的增加,MDL的谱峰蓝移,最终在212nm处出现大负峰。当胍浓度为0.75mol/L时,MDL的CD谱即有明显变化和活性丧失;胍浓度继续增加,CD谱逐渐成为特征的无规卷曲的谱形。在pH9.0、温度超过80℃、脲或胍浓度分别高于2mol/L和0.75mol/L时,MDL的CD谱发生显著变化的同时,其凝集兔红细胞的生物学活性全部丧失,分子的二级结构单元也发生很大改变。 相似文献
66.
采用核酸分子杂交Southern印迹法,以32P标记的HBVDNA为探针,检测HBsAg阳性母亲引产的40例胎儿的肝、肾组织。结果有2例胎肝和1例胎肾细胞DNA出现大于3.2kb的杂交带,表明HBVDNA已处于整合状态。胎肾细胞基因组中查出HBVDNA整合为首次报道。 相似文献
67.
黄花棘豆的喹诺里西定生物碱 总被引:5,自引:0,他引:5
豆科棘豆属有毒植物含多种有毒生物碱,有关棘豆属中生物碱成分的研究,早在1929年Couch由兰伯氏棘豆(Oxytropis lambertii DC.)中分离到一种多羟基化的含氮有机化合物,这是最早报道由棘豆属植物中分离出的生物碱成分,由于当时条件限制未能确定其结构。1982年美国农业部西部研究中心的植物化学家Molyneux从绢毛棘豆(Oxytropis sericea)中分离到苦马豆碱(swainsonine)等吲哚里西定生物碱(indolizidinealkaloids);1989年沈阳药学院于荣敏等由小花棘豆(Oxytropis glabra DC.)中分离出多种喹诺里西定生物碱。喹诺里西定生物碱具有多种生理活性,对试验动物中枢神经系统产生抑制,呼吸抑制或兴奋,致幻、流产和致畸等作用。本文报道由黄花棘豆(Oxytropis ochrocephala Bunge)中测得4种喹诺里西定生物碱。 相似文献
68.
Zhi Jian Yu Changbae Jin Robin William Rockhold Beth Hoskins Ing Kang Ho 《Neurochemical research》1993,18(11):1203-1209
Wistar-Kyoto and spontaneously hypertensive rats received i.v. infusions of cocaine hydrochloride (60 mg/kg per day) for 3, 7, and 14 days, or saline for 7 days. Acute cocaine challenge (40 mg/kg, s.c.) was given to treated and control rats 24 hr after the termination of each infusion period. There were no strain differences in brain levels of cocaine during cocaine infusion, nor after cocaine challenges. There were no strain differences in resting levels of [3H]dopamine release. Release of [3H]dopamine decreased in nuclei accumbens of 7- and 14-day cocaine-infused animals. Release of [3H]dopamine was maximal in both brain regions 2 hr after acute cocaine challenge. After 14 days of cocaine infusion, cocaine challenge in both strains reduced [3H]dopamine release in the nucleus accumbens, but not in the striatum; the reduction being greater in Wistar-Kyoto rats. The behavioral tolerance which accompanies similar cocaine infusion regimens may be related to striatal tolerance to cocaine-induced dopamine release. 相似文献
69.
中华硬蜱和二棘血蜱的交叉免疫反应 总被引:3,自引:1,他引:2
本文首次比较了经中华硬蜱(Ixodes sinensis)叮咬三次后再经二棘血蜱(Haemaphysalisbispinosa)叮咬的家兔与仅经二棘血蜱叮咬的家兔的交叉免疫抗性。二棘血蜱叮咬被中华硬蜱致敏的家兔时,吸血增重为:143.12±32.67mg,但二棘血蜱在正常家兔体上寄生,初次吸血增重为:181.30±44.35mg,两者之间有显著性差异(P<0.01)。中华硬蜱和二棘血蜱唾液腺提取物(SGE)经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),显示两者分别有24条和22条电泳带,中华硬蜱主带有6条,分子量分别为142、105、94、66/65、64和56kD,而二棘血蜱主带有5条,分子量分别为:215、114、105、66/65和58kD,经中华硬蜱叮咬致敏的家兔血清和经二棘血蜱叮咬致敏的家兔血清作免疫印渍,均显示出105kD这一电泳带。该实验表明中华硬蜱和二棘血蜱叮咬家兔两者之间存在着交叉免疫反应,提示105kD蛋白质抗原可能是两者的共同抗原。 相似文献
70.
硫酸盐引起的生态学效应已得到了越来越多的关注,但目前关于硫酸盐对养殖池塘底泥微生物的影响还知之甚少。【目的】探究不同浓度硫酸盐对养殖池塘底泥微生物的影响规律及可能的机制。【方法】本研究利用采集自养殖池塘的底泥和表层水构建了试验系统,研究了加入约0 mg/L (对照组)、30 mg/L (T1处理组)、150 mg/L (T2处理组)、500 mg/L (T3处理组) Na2SO4后表层底泥微生物的丰度、多样性、组成和共生网络的变化规律,并分析了环境影响因素。【结果】孵育第30天前,各实验组底泥微生物变化不大;但到第50天时,T2和T3处理组微生物丰度和多样性相比对照组均明显下降。相比其他实验组,T1处理组酸杆菌门(Acidobacteriota)、拟杆菌门(Bacteroidota)相对丰度出现显著升高(P<0.05),T3处理组变形菌门(Proteobacteria)和放线菌门(Actinobacteriota)相对丰度出现显著升高(P<0.05)。与对照组相比,T1处理组增加了较多差异类群(62种),而T3处理组差异类群大量减少(45种)。共生网络图分析显示硫酸盐浓度的增加引起了底泥微生物网络复杂性的增加,说明微生物群落可能通过自身的调节来响应硫酸盐引起的环境改变。冗余分析(redundant analysis,RDA)和相关性分析揭示底泥总有机碳、总氮和氧化还原电位是影响底泥微生物的主要环境因素,提示底泥微生物可能受到硫酸盐和有机质作用的影响。【结论】较长时间的高浓度硫酸盐会对池塘底泥微生物群落造成重要影响,微生物群落自身的转变和硫酸盐引起的有机质分解改变可能是造成微生物群落变化的关键因素。 相似文献