Hypoxia-activated Ca2+ currents in pacemaker neurones of rat rostral ventrolateral medulla in vitro.

We examined the effects of brief periods of hypoxia or application of cyanide on the discharge and membrane properties of medullary pacemaker neurones in slices of the rostral ventrolateral reticular nucleus (RVL) of the medulla oblongata of rats. Stable intracellular recordings were obtained from seventy-nine neurones within the RVL which exhibited spontaneous rhythmic discharge in the absence of excitatory postsynaptic potentials (EPSPs). The membrane potential cycles of these neurones could be reset with an evoked spike without eliciting EPSPs or inhibitory postsynaptic potentials and hence met criteria of RVL pacemaker neurones. Hypoxia, produced by reducing O2 from 95 to 20% for 40 s or exposure to cyanide (30-300 microM for 40 s), reversibly increased neuronal discharge 1.6-fold (20% O2) or 2.6-fold (300 microM cyanide), respectively, in association with membrane depolarization and a significant fall in membrane resistance. The membrane responses to hypoxia and cyanide were observed in the presence of tetrodotoxin (TTX) at a concentration (10 microM) which eliminated spontaneous spikes or spikes evoked by intracellular depolarization. When recorded at a holding potential of -70 mV by single-electrode voltage clamp, hypoxia or cyanide (300 microM) elicited inward currents of 0.44 +/- 0.06 and 0.58 +/- 0.08 nA, respectively, which are attenuated by reducing the concentration of extracellular Ca2+ ions, and abolished by 2 mM CoCl2 and 100 microM NiCl2, but not affected by 50 microM CdCl2, replacement of 83% extracellular Na+, or adenosine deaminase (2U ml-1). We conclude that hypoxia and cyanide directly excite RVL pacemaker neurones in vitro by a common mechanism: activation of Ca2+ channel conductance.     

Wg3h细胞系转染PSV_2-neo质粒前后生长特性及表面超微结构改变的研究

本文对转染PSV_2-neo质粒后的Wg3h细胞系(Wg3h-neo)在长期传代中的生长特性和表面超微结构与母系Wg3h细胞进行了比较研究。结果发现:转染后Wg3h细胞的DNA合成及生长速度明显高于母系Wg3h细胞,生长饱和密度增大。在软琼脂培养中不形成集落,接种在裸鼠不长肿瘤。在扫描电镜下,细胞表面的微绒毛较母系Wg3h细胞丰富。Southern印迹杂交实验证明PSV_2-neo质粒已整合到宿主细胞的基因组中。转染后Wg3h细胞的生长特性和表面超微结构均发生了某些转化特征。     

当归对大鼠肺β-受体数目的影响

建立了用放射性配基测定肺组织β-受体数目的方法,并探讨了当归对其影响。结果表明:当归与~3H-DHA没有竞争性结合,对正常大鼠、低氧大鼠肺组织β-受体数均无明显影响。提示当归缓解低氧性肺动脉高压不是通过改变肺β-受体数目而实现的。     

胰高血糖素衍生物在大肠杆菌的克隆表达和纯化

 目的 通过基因工程途径获得胰高血糖素衍生物。 方法 根据胰高血糖素基因及凝血酶酶切位点序列，分段合成引物行 PCR 扩增，以 PCR 扩增得到的胰高血糖素-甘氨酸基因序列和 pET-30a 质粒转化 E.coli DH5α，获得重组质粒 pET-G。将重组质粒 pET-G 转化至 E.coli BL21(DE3)，重组菌株命名为 E.coli BL21[pET-G]。以异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，对表达产物进行SDS-Tricine- PAGE[十二烷基硫酸钠-三（羟甲基）甲基甘氨酸-聚丙烯酰胺凝胶电泳]分析和蛋白质印迹分析。对表达产物行Ni2+-NTA亲和层析纯化、凝血酶酶切和高效液相色谱（HPLC）纯化后，行 SDS-Tricine-PAGE分析和飞行质谱分析，并以 ELISA 方法检测其免疫活性。 结果 PCR 扩增获得的条带与预期的DNA表达片段大小一致，重组质粒 pET-G 测序结果与预期完全一致。SDS- Tricine-PAGE 和蛋白质印迹分析显示 E.coli BL21（DE3）的表达产物相对分子质量与预期相符。经亲和层析纯化、凝血酶酶切和 HPLC 纯化后得到了完整的重组胰高血糖素-甘氨酸衍生物，SDS-Tricine-PAGE 分析显示其相对分子质量约为 3500，飞行质谱分析相对分子质量为 3531，二者基本一致。ELISA 检测表明重组胰高血糖素-甘氨酸衍生物具有胰高血糖素免疫活性。 结论 采用基因工程技术在大肠杆菌中成功表达了胰高血糖素-甘氨酸衍生物，为通过体外酰胺化途径研制酰胺化胰高血糖素奠定了基础。     

旋毛虫Ts87重组蛋白诱导的小鼠黏膜免疫保护性研究

本实验探讨旋毛虫Ts87重组蛋白灌胃免疫小鼠诱导的黏膜免疫保护性作用。实验分对照组、佐剂组(霍乱毒素B亚单位组,CTB)和免疫组(CTB Ts87重组蛋白),灌胃免疫,间隔1周共免疫3次。末次免疫后第7天用400条旋毛虫感染期幼虫攻击,比较3组小鼠肠道成虫数、雌虫生殖力和肌幼虫数。且于末次免疫后第7天刮取肠黏液、取血检测特异性sIgA、IgG抗体水平。结果显示免疫组小鼠成虫减虫率、新生蚴减虫率、肌幼虫减虫率分别是81·34%、67·02%、84·49%;小鼠肠黏液sIgA水平及血清IgG水平显著高于对照组和佐剂组。结果表明Ts87重组蛋白黏膜免疫能够诱导小鼠产生抗旋毛虫的保护性免疫。灌胃免疫能显著提高肠黏液特异性sIgA水平,对促进肠道成虫的排出有明显作用。     

利用表型筛选法测定整合子对耐药性基因盒的整合频率

目的 建立一种利用表型筛选的方法来测定整合子对耐药性基因盒的整合频率. 方法 将整合子和aadA2耐药性基因盒克隆到同一质粒pACYCl84的不同位点,该重组质粒和高表达整合酶的重组质粒分别转化大肠杆菌BL21(DE3),挑选阳性克隆过夜培养后,取适量菌液涂布含有链霉素的LB琼脂平板,同时取适量菌液涂布不含链霉素的LB琼脂平板,过夜培养后计数菌落个数,用以计算整合频率.同时以链霉素平板上的阳性克隆为模板,进行PCR扩增.对扩增产物切胶回收纯化,然后进行测序,以确定aadA2耐药性基因盒整合位点. 结果 在大肠杆菌BL21(DE3)宿主中,整合子对aadA2耐药性基因盒的整合频率为1.1×103,主要的整合位点为attI. 结论 该系统可以用于整合子对基因盒捕获频率的测定.     

17βE2对切除卵巢兔动脉粥样硬化与血液流变学的作用

目的: 探讨17βE₂对卵巢切除(OVX)兔的AS斑块、血脂代谢及血液流变学的影响。方法: 34只成熟未孕雌兔分为4组:A为NC;B为Sham+CHO;C为OVX+CHO;D为OVX+CHO+17βE₂。喂养12周,喂养前与12周后测定血脂TC、TG、HDL-C、LDL-C、ApoA1、ApoB;测定全血粘度、血浆粘度、AIRC及纤维蛋白原。喂养12周后处死,测定AS斑块面积与主动脉总面积百分比。结果: ①AS斑块面积与主动脉总面积之比,A、B、C、D组分别为0.02±0.00、0.42±0.15、0.67±0.23、0.12±0.11,B组小于C组(P＜0.05)、D组明显小于C组(P＜0.01)。②血脂TC、TG、LDL-C、ApoB,B、D组明显小于C组(P＜0.01)。③血液流变学:全血粘度、血浆粘度AIRC与纤维蛋白原D组明显低于C组(P＜0.01)。结论: 17βE₂能抑制脂质斑块沉积,改善血脂代谢,减轻高脂血症,改善血液流变学。这可能是雌激素抑制AS形成的部份机理。     

Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia, South Africa, and the United States

One of the major limitations of the use of adenoviruses as gene therapy vectors is the existence of preformed immunity in various populations. Recent studies have linked failure of adenoviral gene therapy trials to the presence of antiadenoviral neutralizing antibodies (NAb). Understanding the distribution and specificity of such antibodies will assist in the design of successful recombinant adenoviral gene therapies and vaccines. To assess the prevalence of NAb to adenovirus serotypes 5 and 35 (Ad5 and Ad35), we analyzed serum samples from adult immunocompetent individuals living in The Gambia, South Africa, and the United States by using a neutralization assay. Serum samples were incubated with A549 lung carcinoma cells and adenoviruses encoding enhanced green or yellow fluorescent proteins; results were analyzed by fluorescence microscopy and flow cytometry. Using this technique, we found a high prevalence of NAb against Ad5 in Gambian, South African, and U.S. subjects at both low and high titers. Conversely, all subjects displayed a low prevalence of NAb to Ad35; when present, anti-Ad35 NAb were seen at low titers. Because of the ability of adenoviruses to elicit systemic and mucosal immune responses, Ad35 with its low NAb prevalence appears to be an attractive candidate vector for gene therapy applications.     

兔急性脑缺氧时脑及脑脊液内腺嘌呤核苷含量的变化

本文报告了由低张性低氧血症所致兔急笥脑缺氧时脑组织内腺苷、次黄嘌呤核苷及次黄嘌呤水平分别从正常对照的５３．３±２．９、１１５．６±１１．８及１８６．５±１０．３增至８１６．４±５９．０、１０４９．７±３７．５及７０４．４±５５．３μＭ／ｇ（Ｘ±ＳＤ），各组间Ｐ值均＜０．０１至０．０５。同步测定的脑脊液中三种腺嘌呤核苷水平分别从正常对照的１．６±０．８、５．１±１．０及１３３．９±５０．８增至７．０     

Phosphonate-phospholipid analogues inhibit human phospholipase A2

A phosphonate-containing phospholipid (PL) analogue (Compound 1) designed as a transition-state inhibitor competively inhibits non-human extracellular PLA₂ at a mole fraction of 0.003 in the kinetic scooting mode (Jain et al., Biochem. 284135 (1989)). To further profile the activity of Compound 1, we examined its activity with purified human enzyme and in whole cell systems. Compound 1 effectively inhibited a 14 kDa human PLA₂ purified from joint synovial fluid of patients with rheumatoid arthritis using³H-AA labeledE. coli as substrate (IC₅₀=1.7M) and a high MW PLA₂ (110 kDa) isolated from the cytosol of a human monocytic cell line, U-937, which selectively hydrolyzes AA-containing PL (IC₅₀=165M). It failed to reduce A23187-induced PGE₂ or LTC₄ production by human adherent monocytes or LTB₄ release from human neutrophils which may be due, in part, to poor membrane partitioning.