二烯丙基二硫诱导人白血病HL-60细胞分化差异表达cDNA文库的构建

目的　应用抑制性消减杂交技术构建二烯丙基二硫(diallyldisulfide ,DADS)诱导人白血病细胞分化的消减杂交cDNA文库 ,以期克隆DADS诱导人白血病HL 6 0细胞分化的相关基因。方法　用DADS诱导人白血病HL 6 0细胞分化 ,提取polyA+ RNA ,反转录合成cDNA ,消化成短片段后分成两组 ,分别与两种不同的接头连接 ,再与未处理的白血病细胞cDNA进行两次消减杂交及两次抑制性PCR扩增 ,将PCR产物与pGEM T线性载体连接 ,转化大肠杆菌进行文库扩增 ,随机挑取克隆进行酶切鉴定。结果　成功地构建了具有高消减效率的DADS诱导白血病细胞分化的cDNA文库 ,随机挑取消 2 0 0个克隆制备质粒并酶切分析 ,其中 84 5 %的克隆均具有 1 0 0～ 6 0 0bp左右的插入片段 ,说明每一克隆中均含有特异性的目的片段 ,从而为大批量筛选、克隆DADS诱导人白血病细胞分化的相关未知新基因奠定了基础。结论　DADS能诱导人白血病HL 6 0细胞分化 ,并引起相关的基因发生改变 ,而抑制性消减杂交技术能有效地分离差异表达的基因。     

二烯丙基二硫对小鼠肾包膜下移植人胃癌细胞的生长抑制作用

二烯丙基二硫(diallyl disulfide,DADS)是大蒜的主要有效成分,对多种肿瘤均有明显的抑制作用[1].本室体外研究表明:DADS可明显抑制人胃癌MGC803细胞生长[2],其机制与G2/M期阻滞和信号传导通路ERK/AP-1途径有关[3,4].本研究采用人肿瘤细胞肾包膜下移植模型,探讨DADS对体内生长的MGC803细胞的抑制作用.     

Occupational exposure to low concentrations of carbon disulfide as a risk factor for hypercholesterolaemia

Objective: The objective of this study was to investigate the effect of occupational exposure to carbon disulfide (CS₂) concentrations below threshold limit value (TLV)-time-weighted average (TWA) (31 mg/m³) on total cholesterol, blood pressure and the prevalence of coronary heart disease (CHD). Methods: A cross-sectional study involving 141 viscose rayon workers (64 men), and 141 age- and gender-matched controls without occupational contact with noxious chemicals, was carried out. The probability for CHD was determined by means of the WHO questionnaire and was 12-lead electrocardiography-coded using Minnesota criteria. Blood pressure was measured by the standardized method of the WHO and blood was examined for total cholesterol. A cumulative exposure index (CS₂ index) was calculated for each worker by multiplying the number of years held in a particular job, by the CS₂ concentrations in that job-environment. According to the CS₂ index, the exposed workers were distributed into two groups: group 1 (CS₂ index <100) and group 2 (CS₂ index ≥100). Results: Depending on the job and specific work place the CS₂ concentrations were between 1 and 30 mg/m³. Cholesterol levels were significantly higher in the exposed group (4.9 ± 0.7) compared with the controls (4.6 ± 0.7). Adjustment for age, smoking, body-mass index (BMI) and gender showed the significant effect of the CS₂ index on the total cholesterol (P < 0.001). The prevalence of hypercholesterolaemia was significantly higher in the exposed group (42.6%), compared with the controls (26.2%); odds ratio (OR) (adjusted for potential confounders) was 2.56, 95% CI 1.47–4.46. Logistic regression showed a significantly increased risk for elevated cholesterol in group 2 (OR 5.52; 95% CI 2.81–10.83). No significant effect of CS₂ index on blood pressure and CHD prevalence was found. Conclusions: The results of our study show that occupational exposure to CS₂ concentrations below 31 mg/m³ and a CS₂ index >100 may increase total cholesterol. Our results imply that even the CS₂ concentrations below TLV-TWA may produce morbid changes, and suggest the mechanism of the effect of CS₂, leading to lipid metabolism disturbances and acceleration of atherosclerosis. Received: 1 February 2000 / Accepted: 24 June 2000     

溶剂解吸气相色谱法测定工作场所空气中乙酸乙烯酯

目的建立工作场所空气中乙酸乙烯酯的溶剂解吸气相色谱检测方法。方法根据乙酸乙烯酯的理化性质和在工作场所空气中存在的状态,依据《工作场所空气中有害物质监测的采样规范》(GBZ 159-2004)和《职业卫生标准制定指南》对工作场所空气中有害物质采样和测定方法的要求,以5%(V/V)对苯二酚-乙醇溶液浸渍的活性炭管采集空气中乙酸乙烯酯,二硫化碳解吸,毛细管色谱柱(DB-FFAP)分离,气相色谱-FID检测器测定;以色谱峰出峰时间定性,峰面积定量。结果乙酸乙烯酯在9.30~120.90μg/ml范围内显现线性关系,相关系数>0.999;检出限为0.15 mg/m3;解吸效率为94.97%~96.45%;相对标准偏差为0.30%~0.52%。结论该方法的样品前处理使用二硫化碳溶剂解吸代替热解吸,简化样品处理技术并缩减成本,适用于工作场所空气中乙酸乙烯酯的检测。     

Antigen delivery to dendritic cells by poly(propylene sulfide) nanoparticles with disulfide conjugated peptides: Cross-presentation and T cell activation

Vaccines aiming to activate cytotoxic T cells require cross-presentation of exogenous antigen by antigen-presenting cells (APCs). We recently developed a synthetic nanoparticle vaccine platform that targets lymph node-resident dendritic cells (DCs), capable of mounting an immune response to conjugated antigen. Here, we explore routes of processing and the efficiency of MHC I cross-presentation of OVA peptides conjugated using both reducible and non-reducible linkages, exploring the hypothesis that reduction-sensitive conjugation will lead to better antigen cross-presentation. Both clathrin and macropinocytic pathways were implicated in nanoparticle uptake by colocalization and inhibitor studies. Cross-presentation by DCs was demonstrated by direct antibody staining and in vitro stimulation of CD8(+) T cells from OT-I mice and was indeed most efficient with the reduction-sensitive conjugation. Similarly, we observed IFN-γ production by CD4(+) T cells from OT-II mice. Finally, immunization with the OVA peptide-bearing nanoparticles resulted in in vivo proliferation and IFN-γ production by adoptively transferred CD8(+) OT-I T cells and was also most efficient with reduction-sensitive linking of the peptide antigen. These results demonstrate the relevance of the poly(propylene sulfide) nanoparticle vaccine platform and antigen conjugation scheme for activating both cytotoxic and helper T cell responses.     

Sodium 2-propenyl thiosulfate derived from garlic induces phase II detoxification enzymes in rat hepatoma H4IIE cells

There is evidence that onions and garlic protect against cancer in humans. It has been suggested that this effect is partly due to the organosulfur compounds in Allium vegetables and that these substances act through induction of phase II detoxification enzymes. Here, we hypothesized that alk(en)yl thiosulfates, sodium n-propyl thiosulfate (NPTS), and sodium 2-propenyl thiosulfate (2PTS), which were identified in onions and garlic, respectively, may induce phase II enzymes. Therefore, rat hepatoma cells (H4IIE) were cultured with 1 to 100 μmol/L of NPTS or 2PTS for 48 hours at 37°C; and the activities and messenger RNA (mRNA) expression levels of phase II enzymes in H4IIE cells were investigated. The effects of diallyl trisulfide and tert-butylhydroquinone, known as phase II inducers, were also examined as positive controls and compared with the responses of NPTS and 2PTS. Quinone reductase (QR) activity and mRNA expression levels of QR and epoxide hydrolase 1 were significantly increased by 2PTS (P < .05-.005). In particular, QR activity was increased at a relatively low concentration of 2PTS (10 μmol/L). However, glutathione S-transferase activity and mRNA expression levels of glutathione S-transferase A5 and uridine diphosphate glucuronosyl transferase 1A1 were not changed by 2PTS. In contrast, NPTS did not affect the activities and mRNA expression levels of these phase II enzymes. These results show that 2PTS can induce phase II enzymes, and its inductive effect is comparable or superior to that of diallyl trisulfide and tert-butylhydroquinone.     

Serum miRNA profiling identifies miR-150/30a as potential biomarker for workers with damaged nerve fibers from carbon disulfide

As crucial small regulatory molecules, serum microRNAs (miRNAs) have been widely identified as potential noninvasive biomarkers. To survey and identify serum miRNAs associated with workers who had experienced injury to their nerve system from carbon disulfide (CS₂), we profiled abnormally expressed miRNAs using the microarray technique and further performed qRT-PCR validation in case and control samples (n=20). Microarray profiling in pooled RNA samples showed that many miRNAs in workers exposed to CS₂ were aberrantly expressed. Based on control samples exposed to CS₂, a great amount of abnormal miRNAs, including some miRNA gene clusters and families, were obtained from microarray datasets. Most of deregulated miRNAs were up-regulated, and almost all miRNAs showed consistent expression patterns between workers with different numbers of damaged nerve fibers. Functional enrichment analysis suggested that these abnormal miRNAs showed versatile roles by contributing to multiple biological processes. Some aberrantly expressed miRNAs were characterized as miRNA gene clusters or families, and they always showed consistent expression patterns. miR-150 and miR-30a were selected to be further validated by qRT-PCR as up-regulated species, and they could discern case samples from control samples. miR-150 and miR-30a may be potential noninvasive biomarkers for a damaged nervous system.     

Cytochrome P-450-dependent covalent binding of carbon disulfide to rat liver microsomal protein in vitro and its prevention by reduced glutathione

Carbon disulfide, a hepatotoxic solvent, is metabolized by liver microsomal enzymes to reactive sulfur atoms which get bound to the microsomal enzymes, causing inhibition of the enzyme system. These studies were carried out to examine whether glutathione can protect the liver enzymes from the sulfur binding and against carbon disulfide toxicity. When liver microsomes isolated from phenobarbital-pretreated rats were incubated with³⁵S-CS₂, NADPH and glutathione, almost 60% decrease in sulfur binding to microsomal protein was observed under the experimental conditions. It was further observed that the addition of glutathione to microsomal incubations resulted in almost complete recovery of the activity of the enzyme system as measured by cytochrome P-450 concentration and benzphetamine metabolism. The data suggest that the presence of glutathione in sufficient amount in the liver of subject exposed to CS₂ may significantly decrease the liver toxicity of this highly toxic compound.     

Cystine peptides. Spectroscopic studies on the conformations of a cyclic pentapeptide disulfide

The model cyclic pentapeptide disulfide Boc-Cys-Ala-Aib-Gly-Cys-NHMe 1, has been synthesized. ¹H n.m.r. studies in (CD₃)₂SO and CDCl₃-(CD₃)₂SO mixtures establish the solvent exposed nature of the Cys(l) and Aib NH groups, while a moderate degree of shielding is observed for the other four NH groups. Nuclear Overhauser effects between Cα₁H and N_i+1H protons provide evidence for extended or semi-extended conformations (ø ± 130° ± 30°) at the Cys(l), Ala(2), Gly(4) and Cys(5) residues. The n.m.r. results are supportive of an intramolecular antiparallel β-sheet conformation at these residues, nucleated by a γ-turn centered at Aib(3). This conformation is not stabilized by strong transannular hydrogen bonds. CD studies establish solvent dependent structural changes of the disulfide linkage in methanol-dioxane mixtures. An unusual CD pattern is observed for the peptide chromophore.     

Kinetic analysis of covalent binding between N-acetyl-L-cysteine and albumin through the formation of mixed disulfides in human and rat serum in vitro

Purpose. Covalent binding between N-acetyl-L-cysteine (NAC) and albumin was evaluated kinetically by conducting in vitro experiments. Methods. After ¹⁴C-NAC was incubated with human or rat serum, the solution was analyzed by anion-exchange HPLC. The albumin-bound ¹⁴C-NAC was quantified by measuring the radioactivity in the albumin fraction. Results. Ultraviolet chromatograms and/or radiochromatograms indicated the presence of a stable covalent bond between ¹⁴C-NAC and either human or rat albumin. By analyzing the time dependence of this protein binding in serum, the first-order binding and dissociation rate constants (k_on and k_off) were obtained. The serum was treated in a CO₂ incubator to avoid oxidative interference, and the initial rates were determined separately. The k_on values obtained were 0.33 (h^–1) and 0.48 (h^–1) for human and rat serum, respectively. L-Cysteine was required to initiate the dissociation of ¹⁴C-NAC bound to albumin. Following the addition of appropriate amounts of L-cysteine, the k_off values were determined to be 0.30-1.0 h^–1 and 0.54-1.4 h^–1 for human and rat serum, respectively. Conclusions. The k_on and k_off values obtained for rat serum were in good agreement with the in vivo plasma protein binding kinetics of NAC in rats, indicating the reliability of this in vitro method for evaluating protein binding. No species differences in protein binding kinetics were found between human and rat serum.