Evidence for major differences in ribosomal subunit proteins from Plasmodium berghei and rat liver

Purified polysomes were isolated in high yield from the erythrocytic stages of the rodent malaria parasite, Plasmodium berghei, and from rat liver. Proteins extracted from the ribosomal subunits derived from these polysomes were fractionated and their number and molecular weights were estimated by two-dimensional polyacrylamide gel electrophoresis. Plasmodial small ribosomal subunits contained 30 proteins ranging in apparent molecular size from 11.7 to 40.7 kDa, while large subunits contained 35-36 proteins ranging from 12.1 to 42.6 kDa. None of these parasite proteins was shared by the two subunits nor altered in electrophoretic mobility by radioiodination. Rat liver 40 S ribosomal subunit proteins numbered 30 and ranged from 9.2 to 37.5 kDa, while liver 60 S subunits contained 41-43 proteins with apparent molecular sizes of 10.3-45.2 kDa. Coelectrophoresis of trace amounts of radioiodinated P. berghei ribosomal subunit proteins and stainable quantities of liver proteins demonstrated that most of these 139 parasite and host ribosomal proteins possessed different two-dimensional electrophoretic mobilities under the conditions of this study. Based upon a comparative analysis of P. berghei and rodent ribosomal RNA and these data, it was concluded that parasite and host ribosomes contain distinct ribosomal RNAs and ribosomal proteins.     

Modulation of specific T cell responses by concurrent infection with Leishmania major and LP-BM5 murine leukemia viruses

C57BL/6 mice infected with a murine leukemia virus (MuLV) mixturedesignated LP-BM5 develop an immunodeficiency syndrome termedMAIDS, characterized by a variety of T and B cell abnormalities,including elevated levels of IgE, suggesting that IL-4 expressionis increased in these animals. It has been suggested that theimmunodeficiency associated with MAIDS is caused by a conversionof immune responses normally characterized by T_h1 developmenttowards a T_h2- dominated response. Mice of the same strain,infected with Leishmania major, mount a protective T_h1 responsewith the induction of high levels of IFN- and undetectable IL-4.We therefore infected mice with L. major at differing time pointsbefore and after virus infection and assessed the effects onT cell responsiveness, cytokine production and survival to L.major, as well as the effect on MAIDS-associated pathology.We have also immunized C57BL/6 mice with trinitrophenol-keyholelimpet haemocyanln (TNP-KLH), which leads to a predominantlyT_h2 response, and compared the effects of MAIDS on the responseto TNP-KLH with the effect of MAIDS on L. major infection. Ourresults show that significant immunodeficiency with regard toinfection by L. major is only apparent after 8 weeks of LP-BM5MuLV infection, by which time T and B cell defects are welladvanced. Further, we have found that the strongly polarizedT_h1 response stimulated by L. major infection can modulate theeffect of MAIDS on T cells, leading to the survival of antigen-specificT cells. Our results suggest that the impairment of immune responsesto either TNP–KLH or L. major is due not to an alterationof the balance of T_h1/T_h2 subsets but to a general loss of reactivityin antigen-specific CD4⁺ cells. However, prior activation ofT_h1 but not T_h2 cells can inhibit the development of lymphoproliferationand immunodeficiency caused by MAIDS.     

Increased Fas antigen expression in murine retrovirus-induced immunodeficiency syndrome,MAIDS

The Fas antigen (Fas), which is a cell surface protein belonging to the tumor necrosis factor receptor family, mediates apoptosis. To assess the contribution of Fas to the pathogenesis of retrovirus-induced immunodeficiency, we examined the kinetics of Fas expression on the lymphocytes during the course of murine acquired immunodeficiency syndrome (MAIDS) induced by a defective LP-BM5 murine leukemia virus. The Fas-positive cells were increased in proportion both in αβ T cells and B cells with the progression of MAIDS. The appearance of Fas-positive cells in αβ T cells preceeded those in B cells during the course of MAIDS. Among αβ T cells, about half of the Thy1.2⁺ αβ T cells were positive for Fas, while almost all of Thy1.2^? CD4⁺ αβ T cells were of the Fas-positive phenotype. The Fas-positive cells in MAIDS mice, especially unique Thy1.2^?CD4⁺ αβ T cells, were easily rendered apoptotic by stimulation via Fas, indicating that Fas expressed on the lymphocytes is functional. Furthermore, concomitant infection with Mycobacterium avium in MAIDS mice caused a marked increase in Fas-positive cells accompanied by a severely impaired T cell reactivity to polyclonal stimuli. Taken together, these results suggest the possible participation of the Fas system in the pathogenesis of retrovirus-induced immunodeficiency.     

Apoptotic death of lymphocytes in murine acquired immunodeficiency syndrome: Involvement of Fas-Fas ligand interaction

Murine acquired immunodeficiency syndrome (MAIDS) is caused by a defective murine leukemia virus. The disease is characterized by abnormal lymphoproliferation, impaired T and B cell function and aberrant regulation of cytokines. Both T and B lymphocytes show activated phenotypes, but undergo apoptotic death with characteristic DNA fragmentation. These results indicate the presence of a continuous activation death pathway of the lymphocytes in MAIDS. Overexpression of the bcl-2 transgene in lymphocytes showed no effect on the apoptotic cell death or on the development of the disease. In contrast, mice carrying mutations in either Fas or Fas ligand exhibited accelerated progression of the disease upon infection with MAIDS virus. These results suggest the involvement of Fas-Fas ligand system in the pathogenesis of MAIDS.     

Deterioration in immunologic status of human immunodeficiency virus (HIV)-infected homosexual men with lymphadenopathy: Prognostic implications

Changes in immunologic parameters were followed in members of a cohort of human immunodeficiency virus (HIV)-positive homosexual or bisexual men with lymphadenopathy and were analyzed for differences between those who have and those who have not progressed to the acquired immunodeficiency syndrome (AIDS) (progressors, nonprogressors). T helpers and the Th/Ts ratio were lower in progressors than in nonprogressors both at entry into the study and at the latest visit. T suppressors were not different in the two groups at entry but were higher in nonprogressors at the latest visit. Evaluation of the patterns of change over time showed that T helpers and Th/Ts ratios tended to decrease over time in both nonprogressors and progressors, while T suppressors increased in nonprogressors and decreased in progressors. Although progressors had a greater deterioration in immunologic parameters over time, nonprogressors also had significant deterioration when compared with controls. Based on the respective percentages of men with abnormal or normal T helpers or Th/Ts ratio at entry who have already progressed to AIDS, we would conservatively estimate, considering their latest T helpers and Th/Ts ratio, that at least an additional 16 (32%) of our nonprogressors will develop AIDS in the next 5 years.     

H22肝癌小鼠血清差异蛋白质的质谱鉴定

目的 分析正常小鼠和H22肝癌小鼠血清之间差异表达的蛋白并对部分差异蛋白进行鉴定分析,筛选可能与肝癌发生密切相关的蛋白质.方法 利用双向凝胶电泳技术（2-DE）与基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）,分析正常组和肿瘤组筛选并鉴定部分差异表达的蛋白,再进行生物学分析.结果 通过2-DE图谱研究发现,与正常组相比,肿瘤组存在明显差异的蛋白质点.选择其中可能为关键功能蛋白的蛋白点进行质谱分析,获得肽指纹图谱（PMF）,运用Mascot软件查询NCBI数据库分析并鉴定得出其中2个蛋白：假尿苷合成酶和线粒体核糖体大亚基蛋白.结论 肝癌的发生机制与假尿苷合成酶和线粒体核糖体大亚基蛋白2个差异表达蛋白密切相关,其参与肝癌的发生发展.     

脑脊液蛋白质组学高丰度蛋白去除方法的建立与评价

目的建立和评价脑脊液蛋白质组学中高丰度蛋白的去除方法。方法使用Oasis HLB（hydrophilic—lipophilic balance）过滤柱。运用反相固相法，根据蛋白质不同的疏水性分馏脑脊液，去除高丰度蛋白和盐。将含有低丰度蛋白的过滤液冻干，利用双向凝胶电泳（2-DE）分析评价脑脊液蛋白层析去除高丰度蛋白的效果。结果通过Oasis HLB柱处理可将大部分高丰度蛋白去除。2-DE图谱中低丰度蛋白质分辨率明显增加，点数增多59．86％。结论本研究建立的脑脊液高丰度蛋白去除方法可有效地应用于疾病的脑脊液蛋白质组学研究。     

慢性间歇性缺氧大鼠肾脏组织蛋白质组的变化

目的:比较慢性间歇性缺氧大鼠与正常大鼠肾脏组织蛋白质双向电泳图谱,寻找和鉴定慢性间歇性缺氧大鼠肾脏组织中的差异蛋白表达谱。方法:以固相pH梯度等电聚焦为第一向和垂直SDS-PAGE为第二向,分别对正常对照大鼠和慢性间歇性缺氧大鼠的肾脏组织蛋白质样品进行二维分离,2-DE图谱经ImageMaster 2D Platinum V5.0软件分析,选取4个差异蛋白点用基质辅助激光解吸附离子化飞行时间质谱(MALDI-TOF-MS)进行鉴定。结果:通过对2-DE图谱蛋白斑点的匹配及对比分析,与慢性间歇性缺氧相关的差异表达蛋白斑点为112个;经质谱鉴定的4个差异表达的蛋白斑点,慢性间歇性缺氧组量下调的差异点1个,即线粒体ATP合成酶δ亚基;上调的差异点3个,分别为己糖激酶、儿茶酚-O-甲基转移酶、脱嘌呤/脱嘧啶核酸内切酶/氧化还原因子-1。结论:大鼠肾脏组织慢性间歇性缺氧损伤相关的差异蛋白表达变化可能通过多种途经影响肾脏功能。     

Expression of hypothetical proteins in human fetal brain: increased expression of hypothetical protein 28.5kDa in Down syndrome,a clue for its tentative role

Major advances have been made in annotation of sequences of the human genome, although elucidating the functions of these newly discovered genes remains to be a strong challenge. In an effort to give insight into how triplication of chromosome 21 leads to mental retardation in Down syndrome, we have constructed a two-dimensional protein map from control and Down syndrome fetal brain and identified hypothetical proteins with no known functions. Subsequent quantitative analysis of these proteins revealed no apparent change in expression of hypothetical proteins DKZp564P0562.1 (fragment), 16.6, 21.4, 39.5, and 40kDa as well as putative 55kDa protein between controls and Down syndrome fetuses. By contrast, hypothetical protein 28.5kDa was significantly elevated (P<0.05) in fetal Down syndrome. This finding offers an important clue that a hypothetical protein might be involved in the pathomechanisms of brain abnormality in Down syndrome.     

Altered protein patterns in brains of children with neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative diseases characterized by massive intralysosomal accumulation of storage materials. We have studied the protein patterns in 5 NCL, 5 control, and one Alzheimer disease brains. When protein patterns in NCL and control brain gray matter homogenates were examined by SDS-PAGE, NCL brains showed an absence or greatly reduced amounts of the M_r 160–180 kDa component and reduced amounts of the M_r 29–36 kDa component. Concomitantly, an increase in several components with M_rs of 45–50 kDa was noted. The 180 kDa polypeptide appears to be a glycoprotein because it was bound to the lectins concanavalin A and Ulex europaeus. Recently, the abnormal processing of amyloid protein precursor (APP) and its potential role in NCL have been suggested. Possible defects in tissue proteases and protease inhibitors may be considered responsible for the presence of these amyloid beta protein precursor fragments. To examine this possibility we are using polyclonal antibodies to the C terminal 672–695 (APP) and monoclonal antibodies to inter-α-trypsin inhibitor. Polypeptides with molecular weights of approximately 35–38 kDa were detected in the NCL brain, but not in controls in both cases. These findings suggest abnormal protein processing in NCL brain tissue, disturbances in protein and glycocon-jugate metabolism, impaired lysosomal function (i.e., metabolic enzyme and/or proteases/ proteinase inhibitor abnormalities), and the involvement of improperly processed APP.     

mRNA population in the liver, kidney and brain of young and senescent mice: analysis of in vitro translation products

Possible alterations in the population of poly(A)(+)mRNA during ageing were investigated by translation in vitro of poly(A)(+)mRNA from the liver, kidney and brain of male ddY mice of different ages. [35S]Methionine-labeled translation products were analysed by two-dimensional polyacrylamide gel electrophoresis followed by fluorography. A protein product with a molecular weight of 30 000 and isoelectric point of 6.5 was reproducibly observed only in the fluorograms of translation products of poly(A)(+)mRNA derived from the livers of senescent mice (24.5 months old). However, no age-related change was detected in the translation products of the kidney and brain. These results suggest that gene expression in liver cells changes at the level of the population of cytoplasmic poly(A)(+)mRNA during ageing.     

High viral suppression and low attrition in healthy HIV-infected patients initiated on ART with CD4 above 500 cells/µL in a program setting in Uganda

BackgroundThe World Health Organization recommends antiretroviral therapy (ART) for all HIV-infected patients at all CD4 counts. However, there are concerns that asymptomatic patients may have poorer viral suppression and high attrition.ObjectivesWe sought to determine attrition and viral suppression among healthy HIV-infected patients initiated on ART in program settings.MethodsThis cross-sectional study enrolled ART-experienced patients attending two PEPFAR-supported, high-volume clinics in Kampala, Uganda. Eligible patients were >18 years and had completed at least six months on ART. Participants were interviewed on socio-demographics, ART history and plasma viral load (VL) determined using Abbott Real-time. Predictors of viral suppression (<75 copies/ml) were determined using multivariate logistic regression.ResultsOverall, 267 participants were screened, 228 were eligible and 203 (89%) retained in care (visit within 90 days). Of the 203 participants, 115 (56.7%) were key-populations. Viral suppression was achieved in 173 patients (85%; 95% CI, 80.3%–90.1%). The factors associated with viral suppression were prior VL tests (AOR 6.98; p-value <0.001) and receiving care from a general clinic (AOR 5.41; p=0.009).ConclusionAsymptomatic patients initiated on ART with high baseline CD4 counts, achieve high viral suppression with low risk of attrition. VL monitoring and clinic type are associated with viral suppression.     

bFGF诱导的心肌微血管内皮细胞蛋白质组变化

目的:研究碱性成纤维细胞生长因子(bFGF)对培养的心肌微血管内皮细胞(MMEC)蛋白质表达变化的影响。方法:将培养的MMEC随机分为二组(n均=3):(1)bFGF诱导组(bFGF组):向培养瓶内加入终浓度10ng/ml的bFGF,置细胞培养箱内常氧培养24h。(2)对照组:常规37℃培养箱孵育。二组均提取细胞总蛋白质双向凝胶电泳后选取8个差异表达蛋白点进行胶内酶切、肽质量指纹图谱分析。结果:双向凝胶电泳可分离出约500±10个蛋白质。与对照组相比,bFGF组MMEC特异表达8种蛋白,上调1种蛋白表达,下调3种蛋白表达(bFGF组和对照组蛋白点的Volume值相差≥2.0倍)。结论:bFGF能诱导MMEC蛋白质差异表达。     

Elevated plasma glutamate concentrations in HIV-1-infected patients may contribute to loss of macrophage and lymphocyte functions

We tested the hypothesis that the loss of immunological reactivity in HIV-1-infected persons may result partly from a virus-induced metabolic disorder. Patients who are infected with the acquired immunodeficiency syndrome (AIDS)-associated human immunodeficiency virus 1 were found to have, on average, markedly elevated and highly variable plasma glutamate concentrations. A similar elevation of the extracellular glutamate concentration was found to inhibit DNA synthesis in cultures of mitogenically stimulated lymphocytes. An even stronger inhibition was seen with the structural analogue quisqualate, and a moderate inhibition was seen with N-methyl-D-aspartate and kainate, i.e. with well established pharmacological probes for the excitatory glutamate receptors in the vertebrate central nervous system. The inhibitory effect of glutamate was compensated by adding cysteine or relatively large numbers of 'splenic adherent cells' to the culture. Elevated extracellular glutamate levels were also found to reduce the capacity of murine macrophages, human blood monocytes, and murine fibroblastoid cells (L929 cells) to release acid-soluble thiol (cysteine) into the extracellular space. The three cell types differed, however, with respect to their sensitivity against the three structural analogues of glutamate. The elevated glutamate concentration was not non-specifically toxic for cultured macrophages, since glycolytic activity and arginase activity were not inhibited. Implications of these observations for the pathogenetic mechanism of AIDS are discussed.     

强直性脊柱炎的外周血CD4+T淋巴细胞双向电泳技术

目的 优化双向凝胶电泳的实验步骤,总结出一套适用于强直性脊柱炎的外周血CD4+ T细胞的双向凝胶电泳技术.方法 对双向电泳实验中的关键环节:样本处理,上样方法,聚焦条件等进行调整与优化,硝酸银染色后进行凝胶图像比较.结果 采用pH4-7胶条,用免疫磁珠分离外周血CD4+ T 淋巴细胞,Cleaning-up试剂盒沉淀蛋...     

Biopsy pathology of the gastrointestinal tract in human immunodeficiency virus-associated disease: a 5 year experience in Zürich

Patients with human immunodeficiency virus (HIV)-associated disease are subject to a wide variety of unusual opportunistic infections and to the development of malignant tumours. These complications frequently manifest themselves within the gastrointestinal tract and endoscopic biopsies may contribute to their diagnosis. The results of 63 such biopsies from 28 patients are reviewed in this study. The diagnoses made included: upper gastrointestinal tract candidiasis (n = 6); cytomegalovirus infection of large intestine (n = 2); cryptosporidiosis (n = 1); spirochaetosis (n = 2); Kaposi's sarcoma (n = 4); malignant lymphoma (n = 3); and anal carcinoma (n = 2). Many of the specimens also showed inflammatory changes with no demonstrable aetiological agent. No specific pattern could be recognized for HIV infection per se.     

Quantitation of antigen-specific immune responses in human immunodeficiency virus (HIV)-infected individuals by limiting dilution analysis

The lymphocyte proliferative response to recall antigens is lost following HIV infection. We sought to devise a means by which the functional immune status of persons in the early stages of HIV infection could be monitored quantitatively. The response to tetanus toxoid was examined in 45 HIV-infected individuals and 11 controls using conventional lymphocyte proliferative assays concurrently with limiting dilution analysis utilizing the secretion of interleukin-2 as the measure of a response. Our data show that the limiting dilution analysis detects tetanus toxoid-reactive T cells in 80% of those tested, as compared to only 44% by proliferation. However, the frequency of tetanus-reactive T cells in HIV-infected individuals (median frequency = 1/59,156) is decrease five-fold as compared to seronegative controls (median frequency = 1/11,599). Longitudinal studies demonstrated a time-dependent decrease in the frequency of tetanus-specific T cell responses in the HIV-infected individuals. Thus, the limiting dilution analysis is a quantitative approach for detecting antigen-specific T cells in HIV-infected individuals, and may be used to monitor changes in T cell function in HIV infection.     

Time-course of synthesis,transport and incorporation of a protein identified in purified membranes of host erythrocytes infected with a knob-forming strain of Plasmodium falciparum

Membranes from host erythrocytes infected with a knob-positive strain of Plasmodium falciparum were purified by free-flow electrophoresis or gradient centrifugation. In these membranes the main parasite-derived protein was a 92 000 Da protein which was not present after infection of erythrocytes with the corresponding knob-negative strain. The protein is synthesized between 9–21 h after merozoite invasion and then synthesis ceases. At least 6 h elapses between the start of synthesis and the appearance of the protein in the erythrocyte membrane. No precursor proteins for the 92 000 Da protein were found. Since the purified erythrocyte membranes were free from contamination with whole parasites or parasite membranes, the 92 000 Da protein is clearly present in the host erythrocyte membrane.     

北京鸭腔上囊发育过程中淋巴细胞中间纤维－核纤维－核内骨架体系的蛋白质成分分析

生物化学分析表明，在北京鸭腔上囊发育过程中，淋巴细胞胞质内波形蛋白含量逐渐减少，而核内骨架成分变得丰富。ＩＥＦ－ＳＤＳ－ＰＡＧＥ双向电泳分析显示，２１６日胚腔上囊淋巴细胞核内骨架成分含２０多种多肽，生后３周龄腔上囊淋巴细胞核内骨架成分含４０多种多肽，并出现了分子量为３５ＫＤ和３７ＫＤ、等电点约为５．２和５．５的两个含量丰富的多肽。生后１２周龄退化期的腔上囊的淋巴细胞核内骨架成分也趋于简单化，仅显示出１０多种多肽。腔上囊淋巴细胞核纤层的主要成分是ＬａｍｉｎＢ，而ＬａｍｉｎＡ和Ｃ含量很少。腔上囊发育过程中淋巴细胞ＬａｍｉｎＢ的含量呈渐增趋势。以上表明，在腔上囊发育过程中，淋巴细胞的中间纤维－核纤层－核内骨架体系蛋白质成分和含量有明显变化，其与腔上囊功能的关系值得深入探讨。     

Molecular characterization of the complement activating protein in the venom of the Indian cobra (Naja N. siamensis)

The cobra venom factor (CVF) from Naja n. siamensis was isolated from crude freeze-dried venom by a combination of ion-exchange chromatography and gel filtration (Bio-Rex 70, Sephadex and QAE-Sephadex). The yield of isolated product was 6–8 mg per g starting material. CVF appeared as a homogenous protein band in polyacrylamide gel electrophoresis with and without SDS present. In equilibrium sedimentation analysis the protein was homogenous with a molecular weight of 133,000. Under reducing conditions three protein bands appeared in polyacrylamide gel electrophoresis in SDS with molecular weights of 71,000, 48,000 and 28,000, corresponding to a total molecular weight of 147,000. All of the bands stained with basic fuchsin, suggesting the presence of carbohydrate. Gel filtration in 6 M quanidine hydrochloride on Sepharose 4B of reduced and alkylated material gave three peaks, each corresponding to one of the bands visible on polyacrylamide gel electrophoresis in SDS. The amino acid composition and N-terminal amino acid sequence of each peak were determined. Using a monospecific antiserum to CVF, molecules immunologically related to CVF were detected in several other elapid venoms.