Identification of the human salivary cystatin complex by the coupling of high-performance liquid chromatography and ion-trap mass spectrometry

Human salivary cystatins, five major (S, S1, S2, SA, SN) and two minor (C and D), are multifunctional proteins playing a different role in the oral environment. Salivary cystatin SN is able to effectively inhibit lysosomal cathepsins B, C, H and L and cystatin SA inhibits cathepsins C and L in vitro. These activities suggest, particularly for cystatin SN, an important role in the control of proteolytic events in vivo. Differently, cystatins S are involved, together with statherin, in the mineral balance of the tooth. Due to their distinct role, a reliable method for identification and quantification of the different cystatins, as well as of possible truncated and derived forms, could be helpful for the assessment of the status of the oral cavity. To this purpose high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI MS) was applied to the analysis of human saliva obtained from healthy subjects. All known salivary cystatins, with the exception of cystatin C, were detected. Strong evidence was also obtained for the presence in saliva of post-translational modified isoforms of cystatins, which may be related to donor habits. Cystatin SN and cystatins S, S1 and S2 were well separated by HPLC-ESI MS coupling from other components and thus this approach can be successfully applied to their quantification.     

The role of HIV replicative fitness in perinatal transmission of HIV

Perinatal transmission of Human immunodeficiency virus(HIV),also called mother-to-child transmission(MTCT),accounts for 90% of infections in infants worldwide and occurs in 30%-45% of children born to untreated HIV-1 infected mothers.Among HIV-1 infected mothers,some viruses are transmitted from mothers to their infants while others are not.The relationship between virologic properties and the pathogenesis caused by HIV-1 remains unclear.Previous studies have demonstrated that one obvious source of selective pressure in the perinatal transmission of HIV-1 is maternal neutralizing antibodies.Recent studies have shown that viruses which are successfully transmitted to the child have growth advantages over those not transmitted,when those two viruses are grown together.Furthermore,the higher fitness is determined by the gp120 protein of the virus envelope.This suggests that the selective transmission of viruses with higher fitness occurred exclusively,regardless of transmission routes.There are many factors contributing to the selective transmission and HIV replicative fitness is an important one that should not be neglected.This review summarizes current knowledge of the role of HIV replicative fitness in HIV MTCT transmission and the determinants of viral fitness upon MTCT.     

The human pituitary nitroproteome: detection of nitrotyrosyl-proteins with two-dimensional Western blotting, and amino acid sequence determination with mass spectrometry

Nitric oxide is an important mediator that participates in reduction-oxidation (redox) mechanisms and in cellular signal transduction pathways. Two types of post-translational modifications are induced by nitric oxide: S-nitrosylation of cysteine residues and nitration of tyrosine residues. Two-dimensional gel electrophoresis-based Western blotting was used to detect, and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) to determine the amino acid sequence of, several different nitrated proteins in the human pituitary. Proteins from several 2D gel spots, which corresponded to the strongly positive anti-nitrotyrosine Western blot spots, were subjected to in-gel trypsin-digestion and LC-MS/MS analysis. MS/MS, SEQUEST analysis, and de novo sequencing were used to determine the nitration site of each nitrated peptide. A total of four different nitrated peptides were characterized and were matched to four different proteins: synaptosomal-associated protein, actin, immunoglobulin alpha Fc receptor, and cGMP-dependent protein kinase 2. Those nitrotyrosyl-proteins participate in neurotransmission, cellular immunity, and cellular structure and mobility.     

YY1抑制效应的破坏可促进人乳头瘤病毒16型癌基因的转录

人乳头瘤病毒１６型（ＨＰＶ１６）癌基因的表达受病毒早期启动子Ｐ９７的控制。位于ＬＣＲ上ＹＹ１蛋白结合位点的破坏可明显提高Ｐ９７的活性。为了观测ＹＹ１位点破坏在全基因组范围内对病毒ｅ６／ｅ７基因转录的影响,将构建的带有ＬＣＲ特异性突变的重组ＨＰＶ１６全基因组ＤＮＡ和ＨＰＶ１６野毒株ＤＮＡ转染至培养细胞,同时组建ＨＰＶ１６Ｅ６反向序列ＲＮＡ体外转录质粒。ＲＮａｓｅ保护试验证实,突变ＨＰＶ１６ＤＮＡ在短     

利用单克隆抗体鉴定人甲胎蛋白(HAFP)抗原结构可分性

本文利用两株针对HAFP分子不同抗原决定簇的单克隆抗体,鉴定HAFP酶解片断的抗原抗体反应性质,并同完整HAFP分子进行比较。结果表明,酶解片断上失去了一株单克隆抗体所对应的分子部份,完整保留着另一株单克隆抗体所识别的抗原决定簇,从而证实HAFP分子某些抗原结构之间具有可分割性。     

Hepatocellular cancer therapy in patients with HIV infection: Disparities in cancer care,trials enrolment,and cancer-related research

In the highly active antiretroviral therapy (HAART) era, hepatocellular carcinoma (HCC) is arising as a common late complication of human immunodeficiency virus (HIV) infection, with a great impact on morbidity and mortality. Though HIV infection alone may not be sufficient to promote hepatocarcinogenesis, the complex interaction of HIV with hepatitis is a main aspect influencing HCC morbidity and mortality.Data about sorafenib effectiveness and safety in HIV-infected patients are limited, particularly for patients who are on HAART. However, in properly selected subgroups, outcomes may be comparable to those of HIV-uninfected patients. Scarce data are available for those other systemic treatments, either tyrosine kinase inhibitors, as well as immune checkpoint inhibitors (ICIs), which have been added to our therapeutic armamentarium. This review examines the influence of HIV infection on HCC development and natural history, summarizes main data on systemic therapies, offers some insight into possible mechanisms of T cell exhaustion and reversal of HIV latency with ICIs and issues about clinical trials enrollment. Nowadays, routine exclusion of HIV-infected patients from clinical trial participation is totally inappropriate, since it leaves a number of patients deprived of life-prolonging therapies.     

甲基毒死蜱对红细胞膜乙酰胆碱酯酶的抑制作用及其与膜脂相互关系

以人红细胞膜为材料,研究了甲基毒死蜱与膜上乙酰胆碱酯酶(AChE)的相互作用及其与膜脂的关系。结果显示,甲基毒死蜱对人红细胞膜AChE有明显的抑制作用,与膜温育30min,其半数抑制浓度约为0.10 mmol/L。动力学分析表明,其抑制作用为非竞争性。0.2％Triton X-100并不改变AChE对甲基毒死蜱的敏感性,亦即AChE上甲基毒死蜱的作用部位与其所处的脂质微环境无关。     

多药耐药基因Mdr1 RNA探针的构建及初步临床应用

检测Mdrl基因表达水平可预测白血病化疗效果,用原位杂交的方法可检测Mdrl在单个细胞的表达水平。本文用Rt-PCR的方法获得了一段特异的cDNA片段,将其克隆到PGEM4Z载体中,经DNA序列分析证明与文献报道一致,采用地高辛素（DIG）RNA标记试剂盒制备反义RNA探针,已初步用于临床骨髓涂片标本的原位杂交检测。     

T cell activation induces CuZn superoxide dismutase (SOD)-1 intracellular re-localization,production and secretion

Reactive oxygen species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H₂O₂), more stable and able to freely diffuse through cell membranes. Copper–zinc superoxide dismutase (CuZn-SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H₂O₂ and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H₂O₂ generation are involved in T cell receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the “non-T” cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors.