HIV-1 gp120对鼠海马长时程增强效应的影响

为了探讨人类免疫缺陷病毒Ⅰ型(HIV-1)的包膜糖蛋白gp120对鼠海马脑片CA1区的突触传递及可塑性的影响,应用离体脑片记录技术,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,对其基础EPSP没有影响,而且这种抑制效应随着gp120浓度增大而增强,即具有剂量依赖性.PKA/PKC蛋白激酶抑制剂H7可以反转这种抑制效应.提示:gp120可能是通过抑制海马CA1区的LTP而参与艾滋病相关性痴呆(HIV-1 associated dementia,HAD)的形成.     

携带HIV-1抗原的单纯疱疹病毒载体疫苗的构建及鉴定

利用细菌人工染色体技术将串联的HIV-1 gp160、gag和protease基因以及表达元件插入1型单纯疱疹病毒(Herpes simplex virus type 1,HSV-1)内部反向重复序列区,以获得携带HIV-1抗原的单纯疱疹病毒载体疫苗。首先将HIV-1 gp160(B型和C型)、gag和protease基因串联克隆入pc DNA3获得重组质粒pc DNA/g Bgp和pc DNA/g Cgp,重组质粒转染293FT细胞,Western blotting检测HIV抗原表达。继而将pc DNA/g Bgp和pc DNA/g Cgp中包括HIV-1抗原基因和表达元件的表达框克隆入p KO5/BN获得重组穿梭质粒p KO5/BN/g Bgp和p KO5/BN/g Cgp,穿梭质粒电转含BAC-HSV的大肠杆菌,筛选重组菌,提取重组DNA并转染Vero细胞,挑取病毒蚀斑纯化重组病毒,Southern blotting鉴定重组病毒DNA,Western blotting检测重组病毒感染细胞中HIV抗原表达,并分析病毒的增殖特性。结果表明,Western blotting在pc DNA/g Bgp和pc DNA/g Cgp转染的293FT细胞中检测到表达的gp160和gag蛋白。p KO5/BN/g Bgp和p KO5/BN/g Cgp分别电转获得重组菌,并从重组DNA转染的Vero细胞中纯化获得重组HSV,Southern blotting检测表明重组HSV基因组发生特异性重组,重组病毒感染细胞中检测到gp120和gp41,且重组HSV保留了在哺乳动物细胞中的复制能力。本研究获得携载HIV-1 gp160、gag和protease基因的重组HSV,并保留了在哺乳动物细胞中的复制能力,可作为HIV-1复制型病毒载体疫苗。     

HIV-1 CRF07 B/C亚型gp41的表达、纯化和初步结构分析

HIV-1跨膜蛋白gp41是HIV-1包膜与靶细胞膜融合过程中的关键蛋白,而且序列保守,是理想的HIV-1作用靶点。为获得HIV-1中国流行株CRF07 B/C gp41蛋白的晶体结构来指导疫苗设计及药物开发,采用CRF07 B/C gp160基因序列为模板,经PCR、酶切、连接,将gp41 helix-bundle区域克隆到pET30-his表达载体中,经表达、纯化和结晶筛选,获得了gp41 helix-bundle的晶体并解析了结构,为针对中国艾滋病病毒流行株疫苗的设计及药物开发提供了结构参考。     

人类免疫缺陷病毒1型gp140包膜蛋白三聚体在哺乳动物细胞中的高效表达及其性质鉴定

本研究目的是通过优化1型人类免疫缺陷病毒(HIV-1)gp140编码基因的密码子和克隆设计,从而实现在293T哺乳动物细胞中高效表达,并对纯化获得的gp140蛋白进行抗原性质鉴定。在本研究中选择HIV-1B亚型NL4-3全基因序列为模板进行gp140克隆构建,通过密码子优化、信号肽替换、增加柔性linker、三聚体折叠序列等方法优化设计。通过HIV-1转录反式激活因子tat共转HEK293T细胞进行gp140蛋白表达,采用镍柱纯化。SDS-PAGE、Western blot、ELISA、负染电镜等结果显示目的蛋白纯度高于70%,每升培养基可获得0.5mg gp140蛋白,并且具有良好的抗原活性,电镜下呈现三聚体结构。通过弗氏佐剂与目的蛋白混合免疫Balb/c小鼠,检测小鼠免疫血清显示gp140蛋白能有效刺激机体产生免疫应答。本研究通过优化表达获得B亚型HIV-1NL4-3gp140蛋白,为HIV-1病毒包膜蛋白结构和重组疫苗研究奠定基础。     

HIV-1 gp41 NC六螺旋的结构与功能研究

AIDS是严重危害人类健康的疾病,而HIV是导致这种疾病的病毒。gp41六螺旋在介导HIV-1病毒与靶细胞间的膜融合过程中起着重要作用。因此,对于gp41结合蛋白的研究有助于深入了解gp41在HIV-1感染整个过程中扮演的角色,解释gp41对靶细胞的调控机制,为寻找新的抗艾滋病药物靶点以及艾滋病抑制剂的设计提供有益的思路。作者的实验室相继发现了一批与gp41六螺旋结构相互作用的蛋白质,进而对HIV-1 gp41六螺旋介导的膜融合过程和HIV-1感染机理有了更深入的了解。     

HIV-1B亚型gp120基因密码子优化前后免疫原性的比较

对HIV-1B亚型gp120基因按照哺乳动物优势密码子的使用原则进行优化,以Westem blot方法比较其体外表达量.将优化前的野生型gp120基因和改造后的modgp120基因插入重组腺伴随病毒载体,构建了重组病毒rAAV-wtgp120和rAAV-modgp120,比较两者免疫Balb/C小鼠后的抗体和CTL应答.Western blot检测结果显示:优化后基因的体外表达量明显高于野生型基因,rAAV-modgp120与rAAV-wtgp120相比可更好地诱导Balb/C小鼠的CTL应答,但检测不到明显的抗体反应.由此得出结论,优化后gp120基因的体外表达量明显高于野生型基因,并且可以诱导更强的特异性CTL应答,但检测不到gp120抗体.     

HIV-1 C亚型密码子优化gp120基因重组腺病毒载体的构建及表达

目的构建含有HIV-1C亚型gp120基因重组腺病毒载体,并在293细胞中表达gp120蛋白。方法PCR扩增,获得HIV-1C亚型gp120片段,定向克隆人腺病毒转移载体pTrack-CMV,线性化后转化至含有腺病毒骨架载体pAd-easy-1的大肠埃希菌BJ5183,获得重组子prAd—gp120,PacI酶切纯化后转染293细胞,包装成复制缺陷型重组腺病毒vAd—gp120。结果经PCR、酶切及DNA测序,插入片段大小、方向正确,获得了具有感染力的vAd—gp120重组腺病毒;通过Western印迹检测,重组腺病毒在293细胞中表达出分子量为120kD的蛋白。结论成功构建了含有HIV-1C亚型gp120基因重组腺病毒载体,并获得该基因的表达。     

共表达HIV-1中国流行株gp120与IL-18重组鸡痘病毒的构建及其免疫原性观察

为筛选我国的HIV-1疫苗候选株,以鸡痘病毒282E4中国疫苗株为载体,构建了共表达中国流行株HIV-1外膜蛋白gp120和IL-18的重组鸡痘病毒,并将该重组鸡痘病毒免疫BALB/c小鼠,检测免疫小鼠脾特异性CTL杀伤活性和血清抗体水平。结果显示,HIV_1外膜蛋白gp120和IL-18不但可在重组鸡痘病毒感染的鸡胚成纤维细胞中表达,而且可在重组鸡痘病毒感染的哺乳动物细胞中表达。重组病毒具有良好的免疫原性,可诱导小鼠产生特异性抗体和脾特异性CTL反应,且IL_18发挥了免疫佐剂的作用。本研究结果为制备安全、有效的HIV-1基因工程活载体疫苗奠定了基础。     

重组HIV-1 gp120 AAV2/1在小鼠和恒河猴体内的免疫原性

本文旨在研究表达HIV-1中国流行株gp120基因的重组腺相关病毒疫苗在小鼠和恒河猴体内的免疫原性。用rAAV2/1-gp120免疫BALB/c小鼠和恒河猴,分别用ELISA和HIV-1假病毒中和试验检测免疫动物血清中HIV-1特异性IgG抗体水平和中和抗体水平,用ELISPOT方法和体内杀伤实验检测HIV-1特异性细胞免疫应答水平。结果显示在小鼠体内用rAAV2/1-gp120仅免疫一次就能诱导较高水平的IgG抗体,IgG抗体至少能持续21周,但没有检测到中和抗体。rAAV2/1-gp120在小鼠体内诱导微弱水平的HIV-1特异性细胞免疫应答。rAAV2/1-gp120在恒河猴体内诱导的HIV-1特异性细胞和抗体反应均很弱,且检测不到中和抗体。提示rAAV2/1载体在诱导抗体反应方面具有特殊优势,但若希望诱导HIV-1特异性中和抗体,则需要改造env基因以提高其免疫原性。     

复杂的正选择压力驱动不同细胞嗜性HIV-1的进化

对HIV-1细胞嗜性的研究是理解HIV-1传播和发病机制的关键.通过对HIV-1B和C亚型的R5和X4型病毒的全基因组进行适应性进化分析,发现R5和X4病毒经历了不同的进化方式,并且不同的HIV-1基因受到不同的正选择压力,意味着复杂的自然选择压力驱动HIV-1的进化.分析HIV-1Gp120超变区上的正选择位点,发现相对于其他超变区,更多的正选择位点发生在B亚型X4病毒的V3区,B亚型R5病毒的V2区以及C亚型X4病毒的V1和V4区域.因为这些区域通常影响和决定HIV-1的细胞嗜性,更多的正选择发生在这些特定的超变区意味着作用于Gp120上的选择压力与Gp120的受体识别和结合功能有关.值得注意的是,无论是B和C亚型还是R5和X4型病毒,显著更多的正选择位点发生在Gp120的C3区域（33.3%～55.6%,P〈0.05）,意味着C3区对HIV-1的生存和适应比先前认识的更为重要.另外,在R5和X4病毒的env基因中,约有一半的正选择位点是相同的,尤其是Gp41上的第96,113和281位正选择位点均出现在所分析的4种病毒类型中.这些共同的正选择位点不仅意味着对病毒生存和适应的重要性,也意味着R5和X4存在交叉免疫源性位点的可能性,这对于AIDS疫苗的发展具有重要意义.     

High-affinity glycine and glutamate transport in pig forebrain white and gray matter: a quantitative study

High-affinity uptake of glycine and glutamate modulates glutamatergic neurotransmission in gray matter. N-Methyl-D-aspartate (NMDA) receptors were recently described on white matter oligodendrocytes, therefore uptake of glutamate and glycine in white matter may also modulate NMDA receptor function. We found that glycine uptake in white structures of pig forebrain (corpus callosum, fimbria, subcortical pyramidal tracts, and occipital subcortical white matter) was similar to that in gray structures (frontal and temporal cortices and hippocampus), and that it was sensitive to sarcosine, a GLYT1 inhibitor (IC(50) 15 microM). Glutamate uptake in white matter was approximately 10% of that in gray; it was sensitive to dihydrokainate, an EAAT2 inhibitor. The levels of glycine and its precursor serine were similar in white and gray matter: approximately 2 and 1 nmol/mg tissue, respectively. The white matter level of glutamate was approximately 7.6 nmol/mg tissue, or approximately 74% of gray matter levels. The activity of serine hydroxymethyl transferase, which converts serine into glycine, was similar in white and gray matter (11-18 pmol/(mg tissue)min), whereas the white matter activity of phosphate-activated glutaminase, which converts glutamine into glutamate, was approximately 100 pmol/(mg tissue)min, or approximately 34% of gray matter activity. The white matter activity of glutamine synthetase, the glial enzyme that converts glutamate into glutamine, was 20-40 nmol/(mg tissue)min in neocortex and 5-6 nmol/(mg tissue)min in white matter. The data show that forebrain white matter is equipped to regulate extracellular levels of glycine and glutamate, functions that may modulate white matter NMDA receptor function.     

Assessment of trace elements in human brain using inductively coupled plasma mass spectrometry

Recent brain research reveals a major role of trace elements in various diseases such as multiple sclerosis, Alzheimer's and Wilson's disease. The majority of published tissue concentrations dates back decades, and was assessed with various methods. Little is known about hemispherical differences, the correlation of trace elements or age-dependent changes in the human brain. Thus, the aim of this study was to examine trace element concentrations in different human brain regions after whole brain formalin fixation.549 samples of 13 brain regions were investigated in 11 deceased subjects without known history of brain pathology. Regional wet-to-dry mass ratios and concentrations of iron, copper, magnesium, manganese, calcium and zinc were determined using inductively coupled plasma mass spectrometry.Cortical gray matter revealed higher water content (wet-to-dry mass ratios 5.84–6.40) than white matter regions (wet-to-dry mass ratios 2.95–3.05). Element concentrations displayed specific regional differences. Good linear correlation of concentrations between elements was found for iron/copper as well as for manganese/magnesium (Spearman's rank correlation coefficient 0.74 and 0.65, respectively). Significant inter-hemispherical differences were found for copper in occipital white matter, for magnesium and calcium in putamen and for iron and copper in temporal white matter. An age dependent increase was seen in cortical gray matter for calcium, for magnesium in all regions except in cortical gray matter, for copper in substantia nigra and for zinc in occipital cortex.The presented trace element concentrations can serve as a fundamental basis for further brain research. Wet-to-dry mass ratios allow a comparison with reference data from other studies.     

Glutathione peroxidase activity in surgical and autopsied human brains

Glutathione peroxidase (GSHPx) activity was assayed in normal cerebral gray and white matter samples obtained from frontal, temporal, occipital and parietal lobes during surgical approach to an underlying lesion, and also in normal autopsied human frontal gray and white matter. GSHPx was assayed by a 2 step enzyme reaction which was monitored by following the oxidation of NADPH at 340 nm. It was found that all the brain samples studied contained GSHPx activity. Parietal lobe appeared to have the lowest GSHPx activity compared to temporal, occipital or frontal lobes. Mean enzyme activity in autopsied samples was comparable to that in surgical material. However, considerable loss of activity was observed after 10 years of tissue storage at –80°C.This investigation was supported by the Verterans Administration.     

Regional distribution of adenosine deaminase in the human neuraxis

Adenosine deaminase was determined in 28 different areas of the human neuraxis in 5 adult male cadavers, with no known disease of the nervous system, using a very sensitive colorimetric method. The enzyme was highest in the frontal lobe white matter, and lowest in the medulla and all levels of the spinal cord. Enzyme content was about twice as great in the white matter of the frontal and temporal lobes and cerebellum as it was in the cortical gray matter of these areas, but only slightly higher in the white matter of the parietal and occipital lobes as compared to gray. Average values of the enzyme were found in the remaining areas of the brain, with the exception of the pons and cerebellar white matter, where a higher than average value was noted.     

High-affinity GABA uptake and GABA-metabolizing enzymes in pig forebrain white matter: a quantitative study

GABA receptor activation in central nervous white matter may be protective during white matter hypoxia in the adult, and it may modify axonal conduction, especially in the developing brain. GABA uptake is important for the shaping of the GABA signal, but quantitative data are lacking for GABA uptake and GABA-metabolizing enzymes in central nervous white matter. We report that high-affinity uptake of GABA in adult pig corpus callosum, fimbria, subcortical pyramidal tracts, and occipital white matter is approximately 20% of that in temporal cortex gray matter. Tiagabine (0.1 microM), an antiepileptic drug that specifically inhibits the GAT-1 GABA transporter inhibited GABA uptake 50% in temporal cortex and 60-68% in white structures. This finding indicates that GAT-1 is an important GABA transporter in white matter and suggests that white matter GABA uptake is inhibited during tiagabine therapy. GABA transaminase activity in white structures was approximately 20% of neocortical values. Glutamate decarboxylase (GAD) activity in white structures was only 4% of that in neocortex (7-12 pmol/mg tissue x min(-1) versus approximately 200 pmol/mg tissue x min(-1)). Since white matter activity of citrate synthase of the tricarboxylic acid cycle was approximately 25% of neocortical values ( approximately 0.4 nmol/mg tissue x min(-1) versus approximately 1.5 nmol/mg tissue x min(-1)), the low GAD activity suggests a slower metabolic turnover of GABA in white than in gray matter.     

Protein oxidation and enzyme susceptibility in white and gray matter with in vitro oxidative stress: relevance to brain injury from intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating stroke sub-type with high mortality and morbidity. ICH frequently occurs in subcortical white matter generating hematomas that contain high heme iron levels. In this study, we examined the consequences of iron-induced oxidation (1-100 microM Fe2+ for 30 min. or 50 microM Fe2+ for 1-120 min.) on the activities of two oxidatively sensitive enzymes, creatine kinase (CK) and glutamine synthetase (GS), and on an oxidative stress marker, protein carbonyl formation, in porcine cerebral cortical white and gray matter. In vitro iron oxidation produced time and concentration dependent decreases in both CK [maximum decreases of 49.3+/-1.2% and 44.3+/-4.1% (average +/- SEM, N=3) for white and gray matter, respectively] and GS activities (maximum decreases of 16.9+/-1.7% and 13.2+/-1.0% for white and gray matter, respectively) and increases in protein carbonyl formation. Interestingly, protein carbonyl concentrations were significantly greater (p<0.05) in white vs. gray matter at 100 microM iron (30 min.) and 50 microM iron (120 min.). Additionally, CK and GS activities were lower for white versus gray matter at several time points and iron concentrations. It is our hypothesis that iron induced oxidative stress contributes to the pathogenesis of perihematomal brain injury following ICH.     

Family Poverty Affects the Rate of Human Infant Brain Growth

Living in poverty places children at very high risk for problems across a variety of domains, including schooling, behavioral regulation, and health. Aspects of cognitive functioning, such as information processing, may underlie these kinds of problems. How might poverty affect the brain functions underlying these cognitive processes? Here, we address this question by observing and analyzing repeated measures of brain development of young children between five months and four years of age from economically diverse backgrounds (n = 77). In doing so, we have the opportunity to observe changes in brain growth as children begin to experience the effects of poverty. These children underwent MRI scanning, with subjects completing between 1 and 7 scans longitudinally. Two hundred and three MRI scans were divided into different tissue types using a novel image processing algorithm specifically designed to analyze brain data from young infants. Total gray, white, and cerebral (summation of total gray and white matter) volumes were examined along with volumes of the frontal, parietal, temporal, and occipital lobes. Infants from low-income families had lower volumes of gray matter, tissue critical for processing of information and execution of actions. These differences were found for both the frontal and parietal lobes. No differences were detected in white matter, temporal lobe volumes, or occipital lobe volumes. In addition, differences in brain growth were found to vary with socioeconomic status (SES), with children from lower-income households having slower trajectories of growth during infancy and early childhood. Volumetric differences were associated with the emergence of disruptive behavioral problems.     

High-affinity choline uptake and acetylcholine-metabolizing enzymes in CNS white matter. A quantitative study

The presence of nicotinic and muscarinic receptors suggests the occurrence of cholinergic neurotransmission in white matter; however no quantitative information exists on acetylcholine formation and breakdown in white matter. We compared white structures of pig brain (fimbria, corpus callosum, pyramidal tracts, and occipital white matter) to gray structures (temporal, parietal and cerebellar cortices, hippocampus, and caudate) and found that sodium-dependent, high-affinity choline uptake in white structures was 25–31% of that in hippocampus. White matter choline acetyltransferase activity was 10–50% of the hippocampal value; the highest activity was found in fimbria. Acetylcholine esterase activity in white structures was 20–25% of that in hippocampus. The caudate, which is rich in cholinergic interneurons, gave values for all three parameters that were 2.8–4 times higher than in hippocampus. The results suggest a certain capacity for cholinergic neurotransmission in central nervous white matter. The white matter activity of pyruvate dehydrogenase, which provides acetyl-CoA for acetylcholine synthesis, ranged between 33 and 50% of the hippocampal activity; the activity in the caudate was similar to that in hippocampus and the other gray structures, which was true also for other enzymes of glucose metabolism: hexokinase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. Acetylcholine esterase activity in white matter was inhibited by the nerve agent soman, which may help explain the reported deleterious effect of soman on white matter. Further, this finding suggests that acetylcholine esterase inhibitors used in Alzheimer's disease may have an effect in white matter.     

The efficacy of T cell-mediated immune responses is reduced by the envelope protein of the chimeric HIV-1/SIV-KB9 virus in vivo

Gp120 is a critical component of the envelope of HIV-1. Its role in viral entry is well described. In view of its position on the viral envelope, gp120 is a part of the retrovirus that immune cells encounter first and has the potential to influence antiretroviral immune responses. We propose that high levels of gp120 are present in tissues and may contribute to the failure of the immune system to fully control and ultimately clear the virus. Herein, we show for the first time that lymphoid tissues from acutely HIV-1/SIV (SHIV)-KB9-infected macaques contain deposits of gp120 at concentrations that are high enough to induce suppressive effects on T cells, thus negatively regulating the antiviral CTL response and contributing to virus survival and persistence. We also demonstrate that SHIV-KB9 gp120 influences functional T cell responses during SHIV infection in a manner that suppresses degranulation and cytokine secretion by CTLs. Finally, we show that regulatory T cells accumulate in lymphoid tissues during acute infection and that they respond to gp120 by producing TGFbeta, a known suppressant of cytotoxic T cell activity. These findings have significant implications for our understanding of the contribution of non-entry-related functions of HIV-1 gp120 to the pathogenesis of HIV/AIDS.     

Catalytic nitric oxide synthase activity in the white and gray matter regions of the spinal cord of rabbits

The latest research reveals that nitric oxide as a gas messenger may diffuse into the surrounding extracellular fluid and act locally upon neighboring target cells. However, several observations raise the possibility that nitric oxide may also be released at a greater distance from the neuronal cell body. The catalytic nitric oxide synthase (cNOS) activity was therefore studied in the cervicothoracic and lumbosacral segments of the spinal cord of rabbits, including the white matter of dorsal columns (DC), lateral columns (LC) and ventral columns (VC), as well as the gray matter of dorsal horns (DH), intermediate zone (IZ) and ventral horns (VH). Lower cNOS activity was found in the white matter of both cervicothoracic (47%) and lumbosacral (30%) regions, whereas that detected in the gray matter of the lumbosacral part of the spinal cord was considerably higher (70%). Enzyme activity varied from 43.4 to 77.2 dpm/microg protein in the cervicothoracic segments of the gray matter in the descending order: DH>VH>IZ. Similar cNOS activity was found in the white matter of the cervicothoracic segments (42.1-62.8 dpm/microg protein). When the activity of cNOS was compared in the lumbosacral segments, the highest enzyme activity was found in DH of the gray matter (198.7 dpm/microg protein) and the lowest cNOS in DC (45.8 dpm/microg protein) of the white matter. It was concluded that the white matter of the spinal cord contains similar cNOS activity in comparison to the gray matter.