Cellular energetics and glutathione status in NRK-52E cells: toxicological implications

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including gamma-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of gamma-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [14C]alpha-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na(+)+K(+))-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.     

CS2对原代星形胶质细胞的毒性研究

为探讨ＣＳ２的神经毒性机制及体外评价方法,我们研究了ＣＳ２对原代星形胶质细胞的毒性,采用常规培养方法,观察ＣＳ２染毒（０,１０,１００,１０００,１００００μｍｏｌ／Ｌ对星形胶质细胞脱落、细胞形态学、及Ｎａ＋－Ｋ＋－ＡＴＰ酶活性的影响。结果表明：ＣＳ２染毒可使细胞脱落数增加,且与接触剂量和接触时间有关;细胞形态学明显异常,电镜下观察表现为髓样小体的形成及空泡样改变;Ｎａ＋－Ｋ＋－ＡＴＰ酶活性下降。提示ＣＳ２对于星形胶质细胞有明显的毒性,此酶活性的下降可作为ＣＳ２毒性的一种标志物     

二烯丙基三硫对胃癌MGC-803细胞的生长抑制作用

目的　研究二烯丙基三硫 (DATS)在体外对胃癌MGC 80 3细胞的生长抑制作用。方法　采用MTT法、集落形成率及生长曲线绘制分别观察不同浓度DATS对胃癌MGC 80 3细胞生长的影响。结果　DATS处理后 ,培养的MGC 80 3细胞增殖抑制而稀少。不同浓度DATS对胃癌MGC 80 3细胞的作用 ,4、8、12、16、2 4mg·L-1的细胞生长抑制率分别为 2 6 %、4 6 %、6 5 %、76 %、89% ,其半数抑制浓度(IC50 )为 8 2mg·L-1。 8、12、16、2 4mg·L-1的细胞集落形成率和集落形成相对数各为 32 4 %和 5 8 7%、2 4 8%和4 2 5 %、19%和 33 5 %、8 8%和 15 1% ,皆具有明显的量效关系 ,且随着浓度增高 ,细胞生长曲线趋于低平。结论　DATS在体外对胃癌MGC 80 3细胞具有良好的抑制作用     

Chemical synthesis of β‐defensins and LEAP‐1/hepcidin

Abstract: A large and steadily growing subfamily of antimicrobially active peptides of animals and plants is formed by the defensins, which are highly disulfide‐bonded, cationic peptides with a molecular mass of about 4 kDa. The synthesis of the human β‐defensins 1 and 2 (hBD‐1, hBD‐2) as well as of the novel murine β‐defensins 7 and 8 (mBD‐7 and mBD‐8) is reported. The peptides were synthesized by solid‐phase peptide synthesis using fluorenylmethoxycarbonyl chemistry. The linear products were oxidized in the presence of the cysteine/cystine redox system to the biologically active molecules. The correct disulfide connectivity of the resulting cyclic products was partly verified by mass spectrometry and sequence analysis of the fragments obtained after tryptic cleavage. In addition, the recently discovered antimicrobially active human peptide LEAP‐1/hepcidin, which contains four disulfide bonds, was successfully synthesized and subsequently oxidized. For Liver‐expressed anti microbial peptide (LEAP)‐1/hepcidin and hBD‐1, the identity of native and synthetic peptides was demonstrated by high‐pressure liquid chromatography and capillary electrophoretic analysis. The general synthetic procedure is suitable to rapidly perform the total chemical synthesis of novel fully bioactive defensins, which are expected to be identified soon, as well as of structurally modified analogs.     

降钙素基因相关肽的合成

降钙素基因相关肽是由３７个氨基酸残基组成，并有一对二硫键的生物活性多肽，本文采用固相合成方法，以Ｆｍｏｃ－氨基酸为原料，经ＨＢＴＵ－ＨＯＢＴ－ＮＭＭ综合合，三氟乙酸－本甲硫醚－三甲基溴硅烷脱保护，分别用铁氰化钾和二甲亚砚氧化形成二硫键，经ＰＲ－ＨＰＬＣ纯化，获得了ＲＰ－ＨＰＬＣ分析为单一峰的目的，目的肽的质谱和氨基酸组成分析结果均与理论值相符，同时具有较强物降血压生理活怀。     

鲑鱼降钙素合成中二硫键形成方法的探讨

在鲑鱼降钙素的合成中，采用空气氧化、二甲亚砜－三氟乙酸氧化、二四亚砜－水氧化和铁氰化钾氧化四种方法，对二硫键的形成方法进行了比较，实验结果表明用铁氰化钾法氧化时间短，氧化率高。     

Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nα‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side‐chain and backbone anchoring strategies,and compatible protection for N‐terminal cysteine*,†

Abstract: Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C‐terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N^α‐9‐fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N^α‐t‐butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side‐chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N‐terminal cysteine residue have been developed: (i) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐(N‐methyl‐N‐phenylcarbamoyl)sulfenylcysteine [Fmoc‐Cys(Snm)‐OH], allowing creation of an otherwise fully protected resin‐bound intermediate with N‐terminal free Cys; (ii) incorporation of N^α‐9‐fluorenylmethoxycarbonyl‐S‐triphenylmethylcysteine [Fmoc‐Cys(Trt)‐OH], generating a stable Fmoc‐Cys(H)‐peptide upon acidolytic cleavage; and (iii) incorporation of N^α‐t‐butyloxycarbonyl‐S‐fluorenylmethylcysteine [Boc‐Cys(Fm)‐OH], generating a stable H‐Cys(Fm)‐peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C‐termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on‐resin, as well as in solution after cleavage/purification.     

二烯丙基二硫上调p21、STAT1和CAMTA1诱导人白血病HL-60细胞分化

目的应用抑制性消减杂交(SSH)研究二烯丙基二硫(diallyl disulfide,DADS)诱导人白血病HL-60细胞分化的分子机制。方法在前期工作中,我们成功地构建了具有高消减效率的DADS诱导白血病分化的cDNA文库,本实验通过挑取文库中的30个克隆制备质粒并酶切分析、测序和同源性检索。结果18个克隆均具有100～600bp左右的插入片段,测序分析发现10个差异表达基因,分别与细胞周期、信号转导、代谢、RNA结合等有关。RT-PCR验证其中上调的3个基因:p21、STAT1和CAMTA1,结果与SSH相符。结论p21、STAT1和CAMTA1表达上调与DADS诱导白血病分化密切相关。     

Kinetics and intracellular location of intramolecular disulfide bond formation mediated by the cytoplasmic redox system encoded by vaccinia virus

Poxviruses encode a redox system for intramolecular disulfide bond formation in cytoplasmic domains of viral proteins. Our objectives were to determine the kinetics and intracellular location of disulfide bond formation. The vaccinia virus L1 myristoylated membrane protein, used as an example, has three intramolecular disulfide bonds. Reduced and disulfide-bonded forms of L1 were distinguished by electrophoretic mobility and reactivity with monoclonal and polyclonal antibodies. Because disulfide bonds formed during 5 min pulse labeling with radioactive amino acids, a protocol was devised in which dithiothreitol was present at this step. Disulfide bond formation was detected by 2 min after removal of reducing agent and was nearly complete in 10 min. When the penultimate glycine residue was mutated to prevent myristoylation, L1 was mistargeted to the endoplasmic reticulum and disulfide bond formation failed to occur. These data suggested that viral membrane association was required for oxidation of L1, providing specificity for the process.