Chemical Constituents from the Leaves and Stems of Schisandra lancifolia

A new nortriterpenoid, 20-hydroxymicrandilactone D (1) and a novel lignan glycoside, lancilignanside A (2) were isolated from leaves and stems of Schisandra lancifolia, together with three known nortriterpenoids (3—5) and nine known phenolics (6—14). The structures of new compounds 1 and 2 were determined by detailed analysis of their 1D and 2D NMR spectra, and chemical evidences. In addition, compounds 1—2, 6—7, and 9—11 showed anti-human immunodeficiency virus (HIV)-1 activities with 50% effective concentration (EC50) in the range of 3.0—99.0m g/ml. Compound 12 was not bioactive in this assay with EC50 more than 200m g/ml.

Immune Responses to Six Synthetic Peptides of Capsid Protein Immune Responses to Six Synthetic Peptides of Capsid Protein

Many B cell epitopes within p24 of human immunodeficiency virus type 1 (HIV-1) were identified, while most of them were determined by using murine monoclonal antibodies reacting with overlapping peptides of p24. Therefore these epitopes may not represent the actual epitopes recognized by the HIV-1 infected individuals. In the present study, immune responses of 67 HIV-1 positive sera from Yunnan Province, China to five peptides on p24 of HIV-1 and one of HIV-2 were analyzed. All of 67 sera did not recognize peptide GA-12 on HIV-1 and peptide AG-23 on HIV-2, which indicated that GA-12 was not human B cell epitope and AG-23 did not cross-react with HIV-1 positive serum. Except 13 sera (19.4%), all remaining sera did not recognize peptides NI-15, DR-16, DC-22 and PS-18, which indicated that these four peptides represented B cell linear epitopes of HIV-1 p24 in some HIV-1 infected individuals but not the immuno-dominant epitopes in most individuals. Cellular & Molecular Immunology. 2005;2(4):289-293.

TRIM5α基因的分子进化及AIDS 免疫治疗相关腺病毒载体的构建

本文包括两方面的研究工作。第一部分对TRIM5α基因在灵长类动物中的分 子进化进行了详尽的研究，希望能从分子进化的角度认识该基因在灵长类动物抵 抗病毒感染尤其是在限制HIV-1 感染过程中所起的作用，并希望能够从中寻找适 合用于AIDS 基因治疗的基因。第二部分构建了艾滋病治疗性DC 疫苗相关的腺 病毒载体，包括表达HIV-1 gp120 基因、表达HIV-1 gp41 基因、表达HSP70 基 因以及仅表达绿色荧光蛋白基因的重组腺病毒，为利用HIV-1 抗原刺激的DC 免 疫治疗AIDS 的研究奠定了基础。 在长期的HIV-1 和AIDS 研究中，人们发现很多种旧大陆猴不能被HIV-1 感 染，而旧大陆猴中的平顶猴却对HIV-1 有易感性，并且对SIV 的感染也表现出较 其它旧大陆猴更为严重的症状，TRIM5α基因的发现解释了这一“历史之谜”。人 们发现旧大陆猴的TRIM5α基因具有很强的抗HIV-1 的功能，是旧大陆猴抵抗 HIV-1 感染必须的免疫因子；并且进一步证明TRIM5α是灵长类动物中普遍存在 的、具有抗多种逆转录病毒功能的天然免疫因子。我们通过对TRIM5α基因在灵 长类动物中的分子进化研究发现，证明该基因在进化过程中表现出很强的达尔文 正向选择作用，尤其是其负责识别逆转录病毒衣壳蛋白的SPRY 结构域。TRIM5 α基因表现出的正向选择很可能是灵长类动物在长期的进化过程中反复被病毒 感染的选择压力造成的，这也从另一个方面证明TRIM5α基因是一个普遍存在的、 具有抗病毒功能的天然免疫因子。对不同灵长类动物的TRIM5α基因序列分析发 现，该基因的序列变化很大，这可能是不同灵长类动物被HIV-1 感染后表现不同 症状的原因；基于密码子的中性检测证明，TRIM5α基因SPRY 结构域上第347、 354、552 位氨基酸表现出很强的正向选择，说明这些位点可能与其抗病毒功能 关系紧密。另外，我们发现平顶猴的TRIM5α基因与CypA 基因以一种新的方式 融合，这种融合方式不同于鹰猴中该基因与CypA 基因的融合，而且与具有较强抗HIV-1 功能的恒河猴的TRIM5α基因比较，平顶猴的TRIM5α基因在几个与其 抗病毒功能紧密相关的位点上的氨基酸也发生了变化，这可能是其被HIV-1 和 SIV 感染后表现出较其它旧大陆猴更为严重症状的原因。 将经过HIV-1 抗原刺激的DC 回输到HIV-1 感染者体内可以诱导产生较强的 抗HIV-1 细胞免疫反应，这种免疫反应理论上可以治疗AIDS，而且对HARRT 治 疗能够产生很好的协同作用；HSP70 蛋白可以提高DC 对抗原的加工和呈递。为 了检测经HIV-1 抗原刺激的DC 免疫治疗AIDS 的实际效果，同时检验通过HSP70 增强DC 对HIV-1 抗原的加工和呈递功能，发现并解决这一治疗方案存在的问题， 我们首先从HIV-1 的基因组及带有HSP70 基因的质粒上克隆了gp120、gp41 和 HSP70 基因，测序及序列分析证明所克隆的基因没有发生影响表达的突变。然后 我们分别构建了表达有关基因的重组腺病毒载体。在荧光显微镜下可以观察到感 染后的细胞内绿色荧光蛋白报告基因的表达，证明成功构建了四种重组腺病毒， 而PCR 扩增也证明在其中三种重组腺病毒的基因组上确实分别带有gp120、gp41、 HSP70 基因。同时我们还构建了不表达外源基因的重组腺病毒，作为未来实验中 的对照载体。未来的研究工作包括目的基因表达检测，重组腺病毒感染DC 及腺 病毒感染DC 功能分析等。

丙型肝炎病毒(HCV)株的全基因组序列测定: 完成HCV 基因型6 全套17 个亚型基因组的测序

丙型肝炎病毒（Hepatitis C virus, HCV）的全基因组序列测定，曾经由于许多方面 的条件限制而难于完成。但是，其对于研究HCV 分子病毒学、流行病学、进化和致 病性却至关重要，特别是在临床应用中，不同序列的基因型决定α-干扰素治疗的不同 效果。在本研究完成之前，HCV 基因型6 仅有6 个亚型有其全基因组序列。因此， 本研究的主要目的在于，测定HCV 变异株代表基因型6 其余的11 个亚型和新亚型的 全基因组序列，并深入分析。 本研究从样品分别来自于中国、泰国，和在美国及加拿大生活的东南亚国家移民 的HCV 感染者。因为样品有限，改良传统的PCR 方法，摸索出“桥”和“岛”DNA 全序列扩增法，从每例样品100μl 血清或从100μl 血清中获得的cDNA 中测定了13 个HCV 全基因组核苷酸序列。 以来源于Genbank 的已知基因型6 的七个全长序列为参考对所测定的13 个亚型 全序列进行共同分析显示，这些全基因组核苷酸的两两比较相似率变化范围为 71.9%--82.7%，著地， 这四对序列间的相同率高于标准定义的HCV 基因亚型之间的 范围值75%-80%。为了进一步理解和证实这些亚型间的遗传相似性，本研究还测定 了代表这4 对亚型的病毒原型株的全基因序列，结果显示了相同的核苷酸水平上的变 异范围，这为HCV 基因亚型的分类提供了新的认识，亦强调了全长序列对于分类的 重要性。 从系统发育方面的分析证实，本研究所测得的13 个分离株都属于基因型6。在系 统发育树上，每个病毒株代表一个独立的枝。并形成了高度分化的HCV 基因型6 分 枝，从而清楚显示，各亚型的独立分布。本研究至此完成了基因型6 中17 个亚型的 全序列测定，而km41 和gz52557 因缺乏其临床上和流行病学上的多个感染病例的证 实，而继续保留其亚型未命名状态。结合来源于Los Alamos HCV database 的基因型6 的已知部分序列的变异株进一步分析，发现各相近亚型变异株均来自东南亚或东南亚国家移民，这提示了这些HCV 的相同感染源。 为了探讨HCV 夫妻间传播的可能性，本研究还测定了来自于泰国的两位感染 HCV 的献血员及其感染HCV 的配偶。这4 个基因序列C-0044 和C-0046 之间核苷 酸相同率为98.1%，而C-0185 和 C-0192 之间为97.8%。文献研究感染HCV 的夫妇 间的部分亚基因序列的相同率为96.3%至100%，本研究结果与此范围相符，并第一 次用全基因组序列提示了HCV 在夫妻间传播的可能性。 本研究还测定了基因型6 的另一个变异株的全基因组序列：HK6554，香港的某患 者，与上文中的GX004 一起，均为静脉吸毒者，并共同感染了HCV 和HIV-1。分析 结果还表明了一种趋势，即是在中国南方，基因亚型6e 有从以前的地方性传播方式 转为现有的流行性传播方式。这种转变可能由于静脉吸毒感染HCV 的人群的网络传 播而加快。 综上，本研究用传统PCR、简并引物结合链特异引物的方法有效地测定了共21 个病毒株的全基因序列。该方法也可用于其它分子流行病学的研究，特别在测定珍贵 的病毒序列然而样品量又受限时。本研究所测定的全基因组序列代表HCV 中最古老、 分化最多、地方性传播、又可能动物源性的基因型6 的全套17 个亚型。这有助于进 一步理解HCV 基因亚型的分类意义、更准确评价HCV 的进化和起源，亦有助于发 现HCV 新的变异株和提高临床诊断、治疗，为将来HCV 的流行及公众健康的预测、 预防和疫苗的制备奠定了坚实的分子遗传学基础。

Multifunctional G-Quadruplex Aptamers and Their Application to Protein Detection

Two significant G-quadruplex aptamers named AGRO100 and T30695 are identified as multi functional aptamers that can bind the protein ligands nucleolin or HIV-1 integrase and hemin. Besides their strong binding to target proteins, both AGRO100 and T30695 exhibit high hemin-binding affinities comparable to that of the known aptamer (termed PS2M) selected by the in vitro evolution process. Most importantly, their corresponding hemin-DNA complexes reveal excellent peroxidase-like activities. higher than that of the reported hemin-PS2M DNAzyme. This enables these multifunctional aptamers to be applied to the sensitive detection of proteins. which is demonstrated by applying AGRO100 to the chemiluminescence detection of nucleolin expressed at the surface of HeLa cells. Based on the specific AGRO100-nucleolin interaction, the surface-expressed nucleolin of HeLa cells is labeled in situ with the hemin-AGRO100 DNAzyme, and then determined in the luminol-H2O2 system.

Structural features of the L-argininamide-binding DNA aptamer studied with ESI-FTMS

The 24-mer DNA aptamer of Harada and Frankel ( Harada, K.; Frankel, A. D. EMBO J. 1995, 14, 5798-5811) that binds L-argininamide (L-Arm) was studied by electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). This DNA folds into a stem and loop such that the loop is able to engulf L-Arm. As controls, two derivatives of the same base composition, one with the same stem but a scrambled loop and the other with no ability to form a secondary structure, were studied. The two DNAs that could fold into stem-loop structures showed a more negatively charged distribution of ions than the linear control. This tendency was preserved in the presence of ligand; complexes expected to have more secondary structure had ions with more negative charges. Distinct species corresponding to no, one, and two bound L-Arm molecules were observed for each DNA. The fractional peak intensities were fit to a straightforward binding model and binding constants were obtained. Thus, ESI-FTMS can provide both qualitative and quantitative data regarding the structure of DNA and its interactions with noncovalent ligands.

Triplex selective 2-(2-naphthyl)quinoline compounds: Origins of affinity and new design principles

A novel competition dialysis assay was used to investigate the structural selectivity of a series of substituted 2-(2-naphthyl)quinoline compounds designed to target triplex DNA. The interaction of 14 compounds with 13 different nucleic acid sequences and structures was studied. A striking selectivity for the triplex structure poly dA:[poly dT](2) was found for the majority of compounds studied. Quantitative analysis of the competition dialysis binding data using newly developed metrics revealed that these compounds are among the most selective triplex-binding agents synthesized to date. A quantitative structure-affinity relationship (QSAR) was derived using triplex binding data for all 14 compounds used in these studies. The QSAR revealed that the primary favorable determinant of triplex binding free energy is the solvent accessible surface area. Triplex binding affinity is negatively correlated with compound electron affinity and the number of hydrogen bond donors. The QSAR provides guidelines for the design of improved triplex-binding agents.

Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor

A surface plasmon resonance biosensor has been used to determine antibody activity in serum. As a model system, the interaction of mouse IgG and sheep anti-mouse IgG polyclonal antibody was investigated in real time. The factors, including pH value, ionic strength, protein concentration, influencing electrostatic adsorption of mouse IgG protein onto carboxylated dextran-coated sensor chip surface, were studied. The procedures of mouse IgG protein immobilization and immune reaction were monitored in real time. The regeneration effect using the different elution reagents was also investigated. The same mouse IgG immobilized surface can be used for 100 cycles of binding and elution with only 0.38% loss per regeneration in reactivity. The results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification. (C) 2000 Elsevier Science B.V. All rights reserved.

注射用低分子量褐藻多糖硫酸酯的制备及理化性质研究

褐藻多糖硫酸酯（Fucoidan Polysaccharide of Sulfated, FPS）是褐藻细胞间的一类硫酸化多糖，组成复杂，在不同褐藻中变化很大，典型的结构是岩藻多糖硫酸酯，也有由甘露糖、半乳糖、糖醛酸、木糖、葡萄糖、褐藻糖等多种单糖组成的结构复杂的杂多糖。研究发现FPS具有抗凝血、降血脂、抗肿瘤和抗HIV的作用，成为当今天然药物主攻的重点之一。由中国科学院海洋研究所所主持研究的海洋新药——褐藻多糖硫酸酯，用于临床治疗肾病综合症和中、早期肾衰具有很好的疗效。本论文研究的注射用褐藻多糖硫酸酯是用过氧化氢-抗坏血酸（1：1）体系降解所得的低分子量FPS，并用琼脂糖凝胶（DEAE）色谱柱进行分级制得，在治疗肾脏疾病方面，具有意想不到的良好效果。 本论文模拟植物生长发育过程中的氧化还原反应，分别用抗坏血酸-过氧化氢和Fe2+-过氧化氢-抗坏血酸两种体系降解FPS。各降解试剂的比例和浓度是影响降解效果的主要因素。分别用抗坏血酸-过氧化氢体系和Fe2+-过氧化氢-抗坏血酸体系降解所得样品的硫酸根、褐藻糖和糖醛酸含量与原料多糖无明显差别，红外光谱与原料多糖一致，表明多糖在降解过程中化学组成和结构并未发生改变。而所得样品多分散指数在1.49-1.77之间，表明用此法能获得分子量分布范围较窄的低分子量多糖。 本论文建立了柱前衍生化高效液相色谱法分析褐藻多糖硫酸酯中的单糖组成，将多糖样品用2 mol•L-1三氟乙酸降解成单糖后，以核糖为内标，用1-苯基-3-甲基-5-吡唑啉酮（PMP）进行柱前衍生化，优化分析条件，采用反相高效液相色谱，245nm紫外检测和使用梯度洗脱，分离测定各单糖。方法学验证实验，在浓度0.10 mmol•L-1～2.0 mmol•L-1各单糖衍生物都有良好的线性关系（r2≥0.999）；除葡萄糖醛酸外，各单糖衍生物都有很好的重现性和稳定性；甘露糖、鼠李糖、葡萄糖醛酸、葡萄糖、半乳糖、木糖、褐藻糖平均加样回收率分别为86.2%，95.1%，62.5%，102.0%，94.8%，66.6%，105.1%。此法可以用于FPS中褐藻糖、半乳糖、葡萄糖主要单糖进行含量测定。 本论文对注射用褐藻多糖硫酸酯的理化性质进行研究，包括性状、鉴别、检查和含量测定,同时做了原料药的强制降解实验，为原料药质量标准的制定和制 剂研究提供了依据。

Human papillomavirus detection in women with and without human immunodeficiency virus infection in Colombia

Background: HIV infection leads to a decreasing immune response, thereby facilitating the appearance of other infections, one of the most important ones being HPV. However, studies are needed for determining associations between immunodeficiency caused by HIV and/or the presence of HPV during the course of cervical lesions and their degree of malignancy. This study describes the cytological findings revealed by the Papanicolaou test, laboratory characteristics and HPV molecular profile in women with and without HIV infection. Methods: A total of 216 HIV-positive and 1,159 HIV-negative women were invited to participate in the study; PCR was used for the molecular detection of HPV in cervical samples. Statistical analysis (such as percentages, Chi-square test and Fisher's exact test when applicable) determined human papillomavirus (HPV) infection frequency (single and multiple) and the distribution of six types of high-risk-HPV in women with and without HIV infection. Likewise, a logistic regression model was run to evaluate the relationship between HIV-HPV infection and different risk factors. Results: An association was found between the frequency of HPV infection and infection involving 2 or more HPV types (also known as multiple HPV infection) in HIV-positive women (69.0% and 54.2%, respectively); such frequency was greater than that found in HIV-negative women (44.3% and 22.7%, respectively). Statistically significant differences were observed between both groups (p = 0.001) regarding HPV presence (both in infection and multiple HPV infection). HPV-16 was the most prevalent type in the population being studied (p = 0.001); other viral types had variable distribution in both groups (HIV-positive and HIV-negative). HPV detection was associated with <500 cell/mm(3) CD4-count (p = 0.004) and higher HIV-viral-load (p = 0.001). HPV-DNA detection, <200 cell/mm(3) CD4-count (p = 0.001), and higher HIV-viral-load (p = 0.001) were associated with abnormal cytological findings. Conclusions: The HIV-1 positive population in this study had high multiple HPV infection prevalence. The results for this population group also suggested a greater association between HPV-DNA presence and cytological findings. HPV detection, together with low CD4 count, could represent useful tools for identifying HIV-positive women at risk of developing cervical lesions.

A transcriptase reversa como alvo terapêutico em doenças retrovirais

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Similar T-cell immune responses induced by group M consensus env immunogens with wild-type or minimum consensus variable regions.

Consensus HIV-1 genes can decrease the genetic distances between candidate immunogens and field virus strains. To ensure the functionality and optimal presentation of immunologic epitopes, we generated two group-M consensus env genes that contain variable regions either from a wild-type B/C recombinant virus isolate (CON6) or minimal consensus elements (CON-S) in the V1, V2, V4, and V5 regions. C57BL/6 and BALB/c mice were primed twice with CON6, CON-S, and subtype control (92UG37_A and HXB2/Bal_B) DNA and boosted with recombinant vaccinia virus (rVV). Mean antibody titers against 92UG37_A, 89.6_B, 96ZM651_C, CON6, and CON-S Env protein were determined. Both CON6 and CON-S induced higher mean antibody titers against several of the proteins, as compared with the subtype controls. However, no significant differences were found in mean antibody titers in animals immunized with CON6 or CON-S. Cellular immune responses were measured by using five complete Env overlapping peptide sets: subtype A (92UG37_A), subtype B (MN_B, 89.6_B and SF162_B), and subtype C (Chn19_C). The intensity of the induced cellular responses was measured by using pooled Env peptides; T-cell epitopes were identified by using matrix peptide pools and individual peptides. No significant differences in T-cell immune-response intensities were noted between CON6 and CON-S immunized BALB/c and C57BL/6 mice. In BALB/c mice, 10 and eight nonoverlapping T-cell epitopes were identified in CON6 and CON-S, whereas eight epitopes were identified in 92UG37_A and HXB2/BAL_B. In C57BL/6 mice, nine and six nonoverlapping T-cell epitopes were identified after immunization with CON6 and CON-S, respectively, whereas only four and three were identified in 92UG37_A and HXB2/BAL_B, respectively. When combined together from both mouse strains, 18 epitopes were identified. The group M artificial consensus env genes, CON6 and CON-S, were equally immunogenic in breadth and intensity for inducing humoral and cellular immune responses.

Multivalent benzoboroxole functionalized polymers as gp120 glycan targeted microbicide entry inhibitors.

Microbicides are women-controlled prophylactics for sexually transmitted infections. The most important class of microbicides target HIV-1 and contain antiviral agents formulated for topical vaginal delivery. Identification of new viral entry inhibitors that target the HIV-1 envelope is important because they can inactivate HIV-1 in the vaginal lumen before virions can come in contact with CD4+ cells in the vaginal mucosa. Carbohydrate binding agents (CBAs) demonstrate the ability to act as entry inhibitors due to their ability to bind to glycans and prevent gp120 binding to CD4+ cells. However, as proteins they present significant challenges in regard to economical production and formulation for resource-poor environments. We have synthesized water-soluble polymer CBAs that contain multiple benzoboroxole moieties. A benzoboroxole-functionalized monomer was synthesized and incorporated into linear oligomers with 2-hydroxypropylmethacrylamide (HPMAm) at different feed ratios using free radical polymerization. The benzoboroxole small molecule analogue demonstrated weak affinity for HIV-1BaL gp120 by SPR; however, the 25 mol % functionalized benzoboroxole oligomer demonstrated a 10-fold decrease in the K(D) for gp120, suggesting an increased avidity for the multivalent polymer construct. High molecular weight polymers functionalized with 25, 50, and 75 mol % benzoboroxole were synthesized and tested for their ability to neutralize HIV-1 entry for two HIV-1 clades and both R5 and X4 coreceptor tropism. All three polymers demonstrated activity against all viral strains tested with EC(50)s that decrease from 15000 nM (1500 microg mL(-1)) for the 25 mol % functionalized polymers to 11 nM (1 microg mL(-1)) for the 75 mol % benzoboroxole-functionalized polymers. These polymers exhibited minimal cytotoxicity after 24 h exposure to a human vaginal cell line.

Impact of simian immunodeficiency virus infection on chimpanzee population dynamics.

Like human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus of chimpanzees (SIVcpz) can cause CD4+ T cell loss and premature death. Here, we used molecular surveillance tools and mathematical modeling to estimate the impact of SIVcpz infection on chimpanzee population dynamics. Habituated (Mitumba and Kasekela) and non-habituated (Kalande) chimpanzees were studied in Gombe National Park, Tanzania. Ape population sizes were determined from demographic records (Mitumba and Kasekela) or individual sightings and genotyping (Kalande), while SIVcpz prevalence rates were monitored using non-invasive methods. Between 2002-2009, the Mitumba and Kasekela communities experienced mean annual growth rates of 1.9% and 2.4%, respectively, while Kalande chimpanzees suffered a significant decline, with a mean growth rate of -6.5% to -7.4%, depending on population estimates. A rapid decline in Kalande was first noted in the 1990s and originally attributed to poaching and reduced food sources. However, between 2002-2009, we found a mean SIVcpz prevalence in Kalande of 46.1%, which was almost four times higher than the prevalence in Mitumba (12.7%) and Kasekela (12.1%). To explore whether SIVcpz contributed to the Kalande decline, we used empirically determined SIVcpz transmission probabilities as well as chimpanzee mortality, mating and migration data to model the effect of viral pathogenicity on chimpanzee population growth. Deterministic calculations indicated that a prevalence of greater than 3.4% would result in negative growth and eventual population extinction, even using conservative mortality estimates. However, stochastic models revealed that in representative populations, SIVcpz, and not its host species, frequently went extinct. High SIVcpz transmission probability and excess mortality reduced population persistence, while intercommunity migration often rescued infected communities, even when immigrating females had a chance of being SIVcpz infected. Together, these results suggest that the decline of the Kalande community was caused, at least in part, by high levels of SIVcpz infection. However, population extinction is not an inevitable consequence of SIVcpz infection, but depends on additional variables, such as migration, that promote survival. These findings are consistent with the uneven distribution of SIVcpz throughout central Africa and explain how chimpanzees in Gombe and elsewhere can be at equipoise with this pathogen.