艾滋病相关痴呆机制研究：鼠海马脑片CA1区突触传递及可塑性与HIV-1包膜糖蛋白gp120的关系

背景：目前对艾滋病相关性痴呆(HIV—1 associated dementia，HAD)仍没有特效的治疗药物，主要因为对HIV—1感染引起的神经损伤和坏死的机制，仍没有完全阐明。目的：探讨人类免疫缺陷病毒Ⅰ型(human immunodeficiency vius I type，HIV—1)包膜糖蛋白gp120对鼠海马脑片CA1区的突触传递及可塑性的影响，以期阐明HAD的形成机制。设计：配对设计。单位：暨南大学医学院的病理生理教研室。材料：实验于2003—01／10在暨南大学医学院病理生理教研室[国家中医药管理局三级重点实验室(登记号：TCM-03-131)]完成。实验用2—5周龄雄性SD大鼠。干预：应用离体脑片灌流及记录技术。主要观察指标：记录大鼠海马CA1区的兴奋性突触后电位(excitatory postsynaptic potential，EPSP)，研究了gp120对高频电刺激Schaffer侧支引起的鼠长时程增强效应(long-term potentiation，LTP)的影响。结果：发现gp120对大鼠海马CA1区LTP产生抑制作用[LTP的平均幅度由正常的(216．1&;#177;140)％降到(90．8&;#177;6．0)％，n=12，P&;lt;0.01]，对其基础EPSP没有影响。PKA／PKC蛋白激酶抑制剂H7可以反转这种抑制效应[LTP的平均幅度为(198．8&;#177;16．2)％，n=8，P&;lt;0．01]。结论：gp120可能是通过抑制海马CA1区的LTP而参与HAD的形成。     

天泰1号对自发阿尔茨海默病鼠海马CA1区分子层突触可塑性的影响：超微结构定量研究

背景：中枢神经的突触可塑性是行为依赖性学习记忆的核心基础，治疗阿尔茨海默病的天然中药是否通过增加突触可塑性来改善学习记忆尚罕见报道。 目的：观察天泰1号对自发阿尔茨海默病模型学习记忆功能及神经突触可塑性的影响。 设计：随机对照实验。 单位：深圳市中西医结合研究所。 材料：实验在深圳市中西医结合临床研究所二级动物实验室完成。实验动物为昆明种小鼠。 方法：从21个月龄昆明种小鼠筛选出自发阿尔茨海默病（记忆障碍）鼠52只，随机分为4组即老年痴呆组、西药对照组、天泰1号6．80g/kg组、天泰1号20．41g／kg组，另设13只老年学习记忆正常鼠为老年正常组。西药对照组给以甲磺酸二氢麦角碱0．6mg／kg，天泰1号6．80g／kg，20．41g／kg组分别予天泰l号方6．80g／kg及20．41g／kg，以上均研配成0．5mL液体灌胃给药，连续60d，老年正常组和老年痴呆组均灌以等量双蒸水。用跳台实验检测学习记忆成绩；海马CA1区脑组织超薄切片，透射电镜观察，并全自动显微图像分析系统定量检测突触可塑性参数。 主要观察指标：①天泰1号不同剂量对自发阿尔茨海默病鼠学习记忆的影响。②突触电镜观察及体视学定量检测结果。 结果：实验过程中实验动物无脱失，均进入结果分析。①天泰1号不同剂量对自发阿尔茨海默病鼠学习记忆的影响：天泰1号6．80g／kg组，20．41g／kg组的学习、记忆错误次数均小于老年痴呆组，且天泰1号20．41g／kg组的学习、记忆错误次数小于天泰1号6．80g／kg组；天泰1号6．80g／kg组，20．41g／kg组的学习训练逃避潜伏期小于老年痴呆组，记忆测试安全平台潜伏期大于老年痴呆组，且天泰1号20．41g／kg组的记忆测试安全平台潜伏期大于天泰1号6．80g／kg组。（参突触电镜观察及体视学定量检测：与老年正常鼠相比，老年痴呆鼠出现突触数明显减少，部分突触间隙模糊不清，突触连接间断，突触小泡大小不等等变性现象，其余各组也可见突触变性改变，但程度较模型组轻。天泰1号可明显增高其海马CA1区分子层突触的数密度和面密度，且天泰1号20．41g／kg组所增高的幅度大于天泰1号6．80g／kg组。 结论：天泰1号可明显改善自发阿尔茨海默病模型鼠的学习记忆障碍，这一作用可能与其促进突触再生，改善其大脑的突触可塑性有关，其作用呈现出一定的量效关系。     

天泰1号对自发阿尔茨海默病鼠海马CA1区分子层突触可塑性的影响超微结构定量研究

背景中枢神经的突触可塑性是行为依赖性学习记忆的核心基础,治疗阿尔茨海默病的天然中药是否通过增加突触可塑性来改善学习记忆尚罕见报道.目的观察天泰1号对自发阿尔茨海默病模型学习记忆功能及神经突触可塑性的影响.设计随机对照实验.单位深圳市中西医结合研究所.材料实验在深圳市中西医结合临床研究所二级动物实验室完成.实验动物为昆明种小鼠.方法从21个月龄昆明种小鼠筛选出自发阿尔茨海默病(记忆障碍)鼠52只,随机分为4组即老年痴呆组、西药对照组、天泰1号6.80 g/kg组、天泰1号20.41 g/kg组,另设13只老年学习记忆正常鼠为老年正常组.西药对照组给以甲磺酸二氢麦角碱0.6 mg/kg,天泰1号6.80 g/kg,20.41 g/kg组分别予天泰1号方6.80 g/kg及20.41 g/kg,以上均研配成0.5 mL液体灌胃给药,连续60 d,老年正常组和老年痴呆组均灌以等量双蒸水.用跳台实验检测学习记忆成绩;海马CA1区脑组织超薄切片,透射电镜观察,并全自动显微图像分析系统定量检测突触可塑性参数.主要观察指标①天泰1号不同剂量对自发阿尔茨海默病鼠学习记忆的影响.②突触电镜观察及体视学定量检测结果.结果实验过程中实验动物无脱失,均进入结果分析.①天泰1号不同剂量对自发阿尔茨海默病鼠学习记忆的影响天泰1号6.80 g/kg组,20.41 g/kg组的学习、记忆错误次数均小于老年痴呆组,且天泰1号20.41 g/kg组的学习、记忆错误次数小于天泰1号6.80 g/kg组;天泰1号6.80 g/kg组,20.41 g/kg组的学习训练逃避潜伏期小于老年痴呆组,记忆测试安全平台潜伏期大于老年痴呆组,且天泰1号20.41 g/kg组的记忆测试安全平台潜伏期大于天泰1号6.80 g/kg组.②突触电镜观察及体视学定量检测与老年正常鼠相比,老年痴呆鼠出现突触数明显减少,部分突触间隙模糊不清,突触连接间断,突触小泡大小不等等变性现象,其余各组也可见突触变性改变,但程度较模型组轻.天泰1号可明显增高其海马CA1区分子层突触的数密度和面密度,且天泰1号20.41 g/kg组所增高的幅度大于天泰1号6.80 g/kg组.结论天泰1号可明显改善自发阿尔茨海默病模型鼠的学习记忆障碍,这一作用可能与其促进突触再生,改善其大脑的突触可塑性有关,其作用呈现出一定的量效关系.     

Structure and function of oligosaccharide chains of the envelope glycoprotein gp120 of human immunodeficiency virus type 1]

The surface of the human immunodeficiency virus (HIV-1), a causative agent for acquired immunodeficiency syndrome (AIDS), is covered with the major envelope glycoprotein gp120, of which the carbohydrate moiety accounted for 50% of the molecular mass. There is evidence that glycosylation of gp120 is prerequisite to the various stages of HIV infection. The oligosaccharide structures of gp120 have been determined using recombinant gp120 of HIV-1 (IIIB) produced in chinese hamster ovary cells and virus-derived gp120 isolated from H9 lymphocytes chronically infected with HIV-1 (IIIB). Three oligosaccharides have been suggested to be involved in the HIV-infection process. Occurrence of infection process which is independent of CD4 recognition and mediated by gp120 oligosaccharides, mannose-binding protein, and complement system has been suggested.     

A rat CD4 mutant containing the gp120-binding site mediates human immunodeficiency virus type 1 infection

CD4 is the primary receptor for the human immunodeficiency virus type 1 (HIV-1). Early mutational studies implicated a number of residues of CD4, centered in the region 41-59, in binding to gp120. However, further mutational analyses, together with studies using inhibitory antibodies or CD4-derived peptides, have suggested that other regions of CD4 are also involved in binding or postbinding events during infection. To resolve these ambiguities, we used rat CD4 mutants in which particular regions were replaced with the corresponding sequence of human CD4. We have previously shown that some of these are able to bind HIV-1 gp120, and here we test their ability to act as functional receptors. We find that the presence of human CD4 residues 33-62 is enough to confer efficient receptor function to rat CD4, and we conclude that it is unlikely that regions of CD4 outside this sequence are involved in specific interactions with HIV-1 during either infection or syncytium formation.     

Heparin-mimicking sulfonic acid polymers as multitarget inhibitors of human immunodeficiency virus type 1 Tat and gp120 proteins

Human immunodeficiency virus (HIV) Tat and gp120 intriguingly share the feature of being basic peptides that, once released by HIV(+) cells, bind to polyanionic heparan sulfate proteoglycans (HSPGs) on target uninfected cells, contributing to the onset of AIDS-associated pathologies. To identify multitarget anti-HIV prodrugs, we investigated the gp120 and Tat antagonist potentials of a series of polyanionic synthetic sulfonic acid polymers (SSAPs). Surface plasmon resonance revealed that SSAPs inhibit with a competitive mechanism of action the binding of Tat and gp120 to surface-immobilized heparin, an experimental condition that resembles binding to cellular HSPGs. Accordingly, SSAPs inhibited HSPG-dependent cell internalization and the transactivating activity of Tat. Little is known about the binding of free gp120 to target cells. Here, we identified two classes of gp120 receptors expressed on endothelial cells, one of which was consistent with an HSPG-binding, low-affinity/high-capacity receptor that is inhibited by free heparin. SSAPs inhibited the binding of free gp120 to endothelial cells, as well as its capacity to induce apoptosis in the same cells. In all the assays, poly(4-styrenesulfonic acid) (PSS) proved to be the most potent antagonist of Tat and gp120. Accordingly, PSS bound both proteins with high affinity. In conclusion, SSAPs represent an interesting class of compounds that bind both gp120 and Tat and inhibit their HSPG-dependent cell surface binding and pathological effects. As these activities contribute to both AIDS progression and associated pathologies, SSAPs can be considered prototypic molecules for the development of multitarget drugs for the treatment of HIV infection and AIDS-associated pathologies.     

Betulinic acid derivatives that target gp120 and inhibit multiple genetic subtypes of human immunodeficiency virus type 1

Betulinic acid (BA) derivatives can inhibit human immunodeficiency virus type 1 (HIV-1) entry or maturation depending on side chain modifications. While BA derivatives with antimaturation activity have attracted considerable interest, the anti-HIV-1 profile and molecular mechanism of BA derivatives with anti-HIV-1 entry activity (termed BA entry inhibitors) have not been well defined. In this study, we have found that two BA entry inhibitors, IC9564 and A43D, exhibited a broad spectrum of anti-HIV-1 activity. Both compounds inhibited multiple strains of HIV-1 from clades A, B, and C at submicromolar concentrations. Clade C viruses were more sensitive to the compounds than clade A and B viruses. Interestingly, IC9564 at subinhibitory concentrations could alter the antifusion activities of other entry inhibitors. IC9564 was especially potent in increasing the sensitivity of HIV-1 YU2 Env-mediated membrane fusion to the CCR5 inhibitor TAK-779. Results from this study suggest that the V3 loop of gp120 is a critical determinant for the anti-HIV-1 activity of IC9564. IC9564 escape viruses contained mutations near the tip of the V3 loop. Moreover, IC9564 could compete with the binding of V3 monoclonal antibodies 447-52D and 39F. IC9564 also competed with the binding of gp120/CD4 complexes to chemokine receptors. In summary, these results suggest that BA entry inhibitors can potently inhibit a broad spectrum of primary HIV-1 isolates by targeting the V3 loop of gp120.     

Estimating the risks of transfusion-associated acquired immune deficiency syndrome and human immunodeficiency virus infection

The risk of transfusion-associated acquired immunodeficiency syndrome (AIDS) has been difficult to estimate because of the long and variable incubation period. Mathematical modeling suggests there may eventually be 2100 cases among persons aged 13 to 65 who received transfusions between 1978 and 1984. An estimated 12,000 living transfusion recipients of all ages from these years are infected with the human immunodeficiency virus, the virus that causes AIDS. Secondary transmission might be prevented by testing and counseling recipients, but the likelihood of infection in any single recipient is small.     

Selective interactions of the human immunodeficiency virus-inactivating protein cyanovirin-N with high-mannose oligosaccharides on gp120 and other glycoproteins

The virucidal protein cyanovirin-N (CV-N) mediates its highly potent anti-human immunodeficiency virus activity, at least in part, through interactions with the viral envelope glycoprotein gp120. Here we dissect in further detail the mechanism of CV-N's glycosylation-dependent binding to gp120. Isothermal titration calorimetry (ITC) binding studies of CV-N with endoglycosidase H-treated gp120 showed that binding was completely abrogated by removal of high-mannose oligosaccharides from the glycoprotein. Additional ITC and circular dichroism spectral studies with CV-N and other glycoproteins as well showed that CV-N discriminately bound only glycoproteins that contain high-mannose oligosaccharides. Binding experiments with RNase B indicated that the single high-mannose oligosaccharide on that enzyme mediated all of its binding with CV-N (K(d) = 0.602 microM). A finer level of oligosaccharide selectivity of CV-N was revealed in affinity chromatography-liquid chromatography-mass spectrometry experiments, which showed that CV-N preferentially bound only oligomannose-8 (Man-8) and oligomannose-9 isoforms of RNase B. Finally, we biophysically characterized the interaction of CV-N with a purified, single oligosaccharide, Man-8. The binding affinity of Man-8 for CV-N is unusually strong (K(d) = 0.488 microM), several hundredfold greater than observed for oligosaccharides and their protein lectins (K(d) = 1 microM--1 mM), further establishing a critical role of high-mannose oligosaccharides in CV-N binding to glycoproteins.     

Inhibition of gp120-CD4 interaction and human immunodeficiency virus type 1 infection in vitro by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate and related compounds were tested for their ability to inhibit gp120-CD4 interaction and human immunodeficiency virus infection in vitro. The results show that pyridoxal 5'-phosphate is a unique CD4 antagonist whose antiviral potency derives from the presence of both lysine-reactive and anionic substituents.     

Wortmannin 对血管性痴呆大鼠海马 CA1区自噬相关蛋白 Beclin-1及凋亡相关蛋白Caspase-3表达的影响

目的探讨Wortmannin对血管性痴呆大鼠海马 CA1区的自噬及凋亡表达的影响及意义。方法将150只大鼠随机分为假手术组、模型组及Wortmannin组。每组又随机分为1周、2周、4周、8周和12周5个亚组（n＝10）。采用四血管阻断法制备血管性痴呆模型,应用Morris水迷宫试验检测大鼠的学习记忆能力,HE染色观察大鼠海马区神经元形态变化,免疫组化法检测Beclin-1及Caspase-3蛋白的表达。结果与假手术组比较,模型组出现明显学习记忆障碍,海马CA1区Beclin-1阳性表达在1周开始增多,4周达到高峰,8周开始下降,到12周仍增多;Caspase-3阳性表达在1周开始增多,2周达到高峰;4周开始下降,到12周仍增多,差异有统计学意义（ P＜0.05）;与模型组比较,Wortmannin组学习记忆障碍明显改善,海马CA1区Beclin-1及Caspase-3阳性表达减少,差异有统计学意义（ P＜0.05）。结论 Wortmannin可能通过减轻自噬及凋亡对神经细胞的损伤,从而达到保护神经细胞,增强和调节学习记忆能力的作用。这可能为血管性痴呆的治疗提供一个靶点。     

The relationship between knowledge about acquired immunodeficiency syndrome and the implementation of universal precautions by registered nurses

The relationship between the level of knowledge of registered nurses (RNs) concerning acquired immunodeficiency syndrome (AIDS)-related issues and the practical observance of universal precautions was studied. It was hypothesized that the more knowledge a nurse has concerning AIDS the more likely he or she is to implement universal precautions. All registered nurses who have direct patient contact (N = 400) and are employed at a Northeastern teaching medical center, were provided a packet of three questionnaires and encouraged to participate. Two hundred thirteen (53%) RNs returned completed questionnaires. Subgroups were examined for trends relating such parameters as age and the amount of AIDS knowledge, using analysis of variance. The major hypothesis was tested by correlating the overall scores for AIDS knowledge and the implementation score. Results indicated no relationship between knowledge and the implementation of universal precautions (r = -0.12). When evaluating scores according to work areas, those subjects with higher knowledge scores had lower practice scores. Other demographic variables showed no influence on either knowledge or implementation scores as measured by this study. Further study is needed to understand what factors will motivate RNs to implement universal precautions.     

脐血单个核细胞颈内动脉输注对血管性痴呆大鼠学习记忆能力和海马神经元突触可塑性的干预

目的:以主动回避反应和脑海马齿状回长时程增强为指标,观察颈内动脉输注脐血单个核细胞后血管性痴呆大鼠学习记忆能力和脑海马神经元突触可塑性的变化。方法:实验于2005-03/07在解放军第三军医大学生理教研室和大坪医院野战外科研究所完成。选取清洁级24月龄Wistar大鼠36只,随机分为正常对照组、模型对照组、脐血单个核细胞组,12只/组。选取解放军第三军医大学大坪医院产科的健康足月妊娠产妇(产妇均签署知情同意书),无菌条件下穿刺脐静脉,收集胎盘脐血后体外分离脐血单个核细胞。模型对照组、脐血单个核细胞组应用改良Pulsinellis四血管阻断法建立血管性痴呆大鼠模型,正常对照组不进行椎动脉烧灼和颈总动脉夹闭。造模后24h脐血单个核细胞组颈内动脉输注5-溴脱氧尿嘧啶核苷标记的脐血单个核细胞6×109L-1,正常对照组和模型对照组输注等量生理盐水。采用电脑控制的穿梭箱系统和海马齿状回长时程增强系统检测各组大鼠学习记忆能力及其电生理的改变。结果:实验选用36只大鼠,全部进入结果分析。①造模后4,8周各组大鼠主动回避反应率的比较:与正常对照组比较,模型对照组明显降低[(90.0±4.3)%,(42.5±5.0)%;(92.5±5.0)%,(45.8±3.6)%;P均<0.01];与模型对照组比较,脐血单个核细胞组显著提高(P均<0.01)。②造模后4,8周各组大鼠脑海马齿状回长时程增强诱导率的变化:模型对照组诱导率较正常对照组均明显降低(91%,17%,χ2=13.59,P<0.01;91%,25%,χ2=10.97,P<0.01);脐血单个核细胞组诱导率较模型对照组显著提高(χ2=4.44,P<0.05;χ2=4.20,P<0.05)。③各组造模后不同时间给予高频刺激后群体电位振幅的变化:造模后4,8周模型对照组较正常对照组均明显降低(P均<0.01);脐血单个核细胞组较模型对照组均显著提高(P均<0.01)。④各组高频刺激前后脑海马齿状回长时程增强潜伏期的变化:造模后4,8周模型对照组较正常对照组均明显延长(P均<0.01);脐血单个核细胞组较模型对照组均显著缩短(P均<0.05)。结论:慢性脑缺血可使大鼠脑海马齿状回长时程增强的诱导率、群体电位的振幅和潜伏期呈显著下降趋势,引起大鼠认知功能的损害,而颈内动脉输注脐血单个核细胞可显著易化脑海马齿状回神经元突触的可塑性,改善血管性痴呆大鼠的学习记忆能力。