Cellular proliferation and differentiation induced by single-layer molybdenum disulfide and mediation mechanisms of proteins via the Akt-mTOR-p70S6K signaling pathway

Single-layer molybdenum disulfide (SLMoS₂) is a novel kind of 2D nanosheet that has attracted great attention regarding its use in biosensors, drug delivery, tissue engineering, and therapy. However, our results demonstrated that SLMoS₂ accelerated proliferation and promoted myogenic differentiation and epithelial–mesenchymal transition (EMT) in human embryonic lung fibroblasts (HELFs). The abnormal proliferation and differentiation of HELFs contribute to idiopathic pulmonary fibrosis. Specifically, SLMoS₂ significantly stimulated the expression of myofibroblast- and mesenchymal-associated genes and proteins. The Akt-mTOR-p70S6K signaling pathway plays a critical role in the acceleration of proliferation and promotion of myogenic differentiation and EMT in HELFs induced by SLMoS₂. After cell uptake, SLMoS₂ was primarily located in the cytoplasm and the perinuclear region and activated Akt-dependent signaling due to the generation of reactive oxygen species (ROS). Moreover, bovine serum albumin (BSA) binding markedly inhibited the cellular uptake of SLMoS₂ and the production of intracellular ROS due to an increased thickness and reduced adhesion of HELFs. BSA binding also mitigated the SLMoS₂-activated phosphorylation of Akt-dependent signaling pathways. This study is the first to illustrate the induction of cellular proliferation and differentiation by SLMoS₂ and the related mediation by proteins through Akt-mTOR-p70S6K signaling pathway.     

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.     

The Role of Diallyl Sulfides and Dipropyl Sulfides in the In Vitro Antimicrobial Activity of the Essential Oil of Garlic,Allium sativum L., and Leek,Allium porrum L.

The in vitro antibacterial activity of essential oils (EOs) obtained from fresh bulbs of garlic, Allium sativum L., and leek, Allium porrum L. ( Alliaceae), was studied. A. sativum (garlic) EO showed a good antimicrobial activity against Staphylococcus aureus (inhibition zone 14.8 mm), Pseudomonas aeruginosa (inhibition zone 21.1 mm), and Escherichia coli (inhibition zone 11.0 mm), whereas the EO of A. porrum (leek) had no antimicrobial activity. The main constituents of the garlic EO were diallyl monosulfide, diallyl disulfide (DADS), diallyl trisulfide, and diallyl tetrasulfide. The EO of A. porrum was characterized by the presence of dipropyl disulfide (DPDS), dipropyl trisulfide, and dipropyl tetrasulfide. The antimicrobial activities of the DADS and DPDS were also studied. The results obtained suggest that the presence of the allyl group is fundamental for the antimicrobial activity of these sulfide derivatives when they are present in Allium or in other species (DADS inhibition zone on S. aureus 15.9 mm, P. aeruginosa 21.9 mm, E. coli 11.4 mm). Copyright © 2012 John Wiley & Sons, Ltd.     

职业接触二硫化碳生物接触限值的探讨

目的 探讨我国职业接触二硫化碳(CS2)的生物接触限值.方法 用高效液相色谱法测定工人班末尿中2-硫代噻唑烷-4-羧酸(TTCA)含量,用气相色谱法测定接触CS2工人作业场所空气中CS2浓度,探讨二者的相关关系,比较生物接触限值和PC-TWA判定结果.结果 CS2作业工人班末尿中TTCA含量与其接触的工作场所空气中CS2浓度呈正相关,回归方程式Y=0.265X-0.165 (r=0.91,P＜0.01).基于本次研究的回归方程,根据GBZ 2.1-2007《工作场所有害因素职业接触限值第1部分:化学有害因素》规定的CS2的PC-TWA 5 mg/m3推算,CS2接触工人班末尿中TTCA浓度生物限值为1.2 mgTTCA/g Cr.结论 建议我国CS2生物接触限值修订为1.2 mg TTCA/g Cr.     

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.     

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.     

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.     

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     

饮酒对尿2 -硫代噻唑烷-4-羧酸排泄的影响

目的观察饮酒对二硫化碳(CS2)接触者及非接触者尿2-硫代噻唑烷4羧酸(TTCA)排泄的影响。方法(1)男性非接触CS2志愿者10人,一次饮用38。白酒150ml或250ml,高效液相色谱法观察其尿TTCA排泄动态;(2)CS2作业男工152人,非接触者60人,分别收集班末尿和晨尿进行TTCA测定并进行问卷调查;同时进行个体空气采样和CS2浓度气相色谱法测定。结果非接触者一次饮白酒150ml后3h尿TTCA水平达峰值,12h后降至饮前水平(餐前0．5h,饮酒后1、3、12h中位数分别为0．045、0．068、0．099、0．046mg/gCr,n=10);TTCA水平随饮白酒剂量的增加而增高,饮0、150、250ml白酒者TTCA水平(中位数)分别为0．036、0．064、0．609mg/gCr(n=5,饮后3h)。CS2浓度为≤10．0、10．1～50．0、>50．0mg/m3时,CS2接触者TTCA有随CS2浓度增高而上升的趋势;对照组中饮白酒和啤酒者TTCA水平似高于不饮者,而接触组TTCA水平则随饮酒指数的增加而呈降低趋势。结论大量饮酒可影响尿TTCA水平,在进行CS2生物监测时,应避免在大量饮酒后12h内采集尿样,以避免饮酒对监测结果的干扰作用。     

Short Toxin-like Proteins Attack the Defense Line of Innate Immunity

ClanTox (classifier of animal toxins) was developed for identifying toxin-like candidates from complete proteomes. Searching mammalian proteomes for short toxin-like proteins (coined TOLIPs) revealed a number of overlooked secreted short proteins with an abundance of cysteines throughout their sequences. We applied bioinformatics and data-mining methods to infer the function of several top predicted candidates. We focused on cysteine-rich peptides that adopt the fold of the three-finger proteins (TFPs). We identified a cluster of duplicated genes that share a structural similarity with elapid neurotoxins, such as α-bungarotoxin. In the murine proteome, there are about 60 such proteins that belong to the Ly6/uPAR family. These proteins are secreted or anchored to the cell membrane. Ly6/uPAR proteins are associated with a rich repertoire of functions, including binding to receptors and adhesion. Ly6/uPAR proteins modulate cell signaling in the context of brain functions and cells of the innate immune system. We postulate that TOLIPs, as modulators of cell signaling, may be associated with pathologies and cellular imbalance. We show that proteins of the Ly6/uPAR family are associated with cancer diagnosis and malfunction of the immune system.