Effect of nickel chloride and cadmium acetate on the development of preimplantation mouse embryos in vitro

Preimplantation mouse embryos were used to investigate the toxic effect of nickel chloride and cadmium acetate on early embryo development in vitro.Embryos at the 2- and 4–8 cell stage were cultured in approximately 0.05 ml of mouse embryo culture medium (No. 16), overlaid with paraffin oil and incubated in a humidified atmosphere of 5% CO₂ in air for 48 h. NiCl₂ · 6H₂O was added to the culture medium at concentrations of 10–1000 μM, Cd(CH₃COO)₂ · 2H₂O at concentrations of 10–50 μM. Morphological criteria were used to check embryonic development.Ten micromolars of nickel chloride affected adversely the development of Day 2 embryos (2-cell stage), whereas 300 μM was needed to affect Day 3 embryos (8-cell stage). Toxic effect of cadmium acetate on Day 2 embryos was observed at a concentration of 10 μM.     

柞蚕雄蛾提取液对荷瘤大鼠放疗后免疫功能的影响

柞蚕雄蛾提取液是纯中药制剂,具有多种保健功能,可明显增强小鼠细胞免疫功能和体液疫功能[1,2].     

Stimulation of the secretion of plasminogen activator from activated murine macrophages by microtubule disrupting agents and deuterium oxide

Murine peritoneal macrophages activated in vivo with thioglycollate broth secrete plasminogen activator and other neutral proteinases. The secretion of plasminogen activator by these cells is potentiated by continuous culture with the microtubule destabilizing drugs colchicine, demecolchicine, vinblastine and nocodazole but not by the colchicine analogues trimethylcolchicinic acid and colchicoside which do not destabilize microtubules. Pulse treatment with colchicine for 2 hr or with nocodazole for 16 hr also stimulated plasminogen activator secretion. Deuterium oxide, which stabilizes microtubules, also stimulated secretion of plasminogen activator and failed to antagonize the effects of destabilizing agents on enzyme secretion. It is concluded that secretion of plasminogen activator is not dependent on an intact microtubular system.     

苗药了哥王不同部位提取物的肾毒性研究

目的研究苗药了哥王不同部位提取物对健康大鼠的肾毒性作用,为了哥王的毒性作用机制及临床用药提供参考。方法以70%乙醇为溶剂,采用渗漉法提取,得了哥王乙醇总提取物;将上述提取物用水分散后,依次用石油醚、乙酸乙酯、正丁醇萃取得相应部位提取物,剩余为水部位提取物。将SD大鼠随机分为乙醇总提取物组、石油醚部位组、乙酸乙酯部位组、正丁醇部位组、水部位组和空白组,每组12只(雌雄各半)。给药组大鼠灌胃相应剂量的药液(乙醇总提取物317.520 mg/kg、石油醚部位7.875 mg/kg、乙酸乙酯部位78.435 mg/kg、正丁醇部位53.865 mg/kg、水部位76.545 mg/kg),每天1次,连续给药2周,再停药恢复2周;空白组大鼠灌胃等体积1.0%羧甲基纤维素钠溶液。实验期间观察大鼠的一般情况,取尿液(第14、28天)、血清和双肾组织(第15、29天),计算其肾脏指数,检测血清和尿液中的肾功能指标水平并观察肾组织的病理形态学变化。结果给药期间,与空白组比较,乙醇总提取物组、乙酸乙酯部位组大鼠出现精神萎靡、活动量和饮食量减少、大便稀薄、体质量下降等中毒行为活动特征;石油醚部位组、正丁醇部位组和水部位组大鼠精神状态较空白组稍差,活动量和饮食量稍减少,大便稀薄,体质量增长缓慢;但各组大鼠肛温无较大变化。给药2周后,乙醇总提取物组大鼠的肾脏指数,乙醇总提取物组、乙酸乙酯部位组大鼠血清中N-乙酰氨基葡萄糖苷酶(NAG)、尿素氮(BUN)、肌酐(Cr)水平,正丁醇部位组大鼠血清中NAG水平,水部位组大鼠血清中Cr水平,以及乙醇总提取物组、石油醚部位组大鼠尿液中NAG水平,乙酸乙酯部位组大鼠尿液中NAG、尿蛋白水平均显著升高(P<0.05或P<0.01);在病理形态学观察中,乙醇总提取物组、石油醚部位组和乙酸乙酯部位组大鼠有不同程度的肾小管结构不清、细胞肿胀、少数细胞坏死,伴有肾小球固缩、肾小管硬化、炎症细胞浸润。停药后,大鼠以上症状逐渐好转,精神状态有明显改善,活动量和饮食量增加,大便趋于正常。停药恢复2周后,各给药组大鼠血清和尿液中上述指标水平均恢复至与空白组接近(P>0.05);各给药组大鼠的肾小球结构逐渐恢复清晰,乙醇总提取物组、石油醚部位组和乙酸乙酯部位组大鼠少见细胞肿胀和炎症细胞浸润。结论苗药了哥王乙醇总提取物、石油醚部位和乙酸乙酯部位均具有一定的肾毒性,且具有一定的可逆性。其毒性成分可能为脂溶性成分。     

Absorption Characteristics of Dextrans with Different Molecular Weights from the Liver Surface Membrane in Rats: Implications for Targeting to the Liver

Abstract

We examined the importance of molecular weight on the absorption from the liver surface in rats using fluorescein isothiocyanate-dextrans (FDs) with molecular weights of 4,400 (FD-4), 9,300 (FD-10), 40,500 (FD-40) or 69,000 (FD-70). After application of FDs (5 mg) to the rat liver surface employing a cylindrical glass cell (i.d. 9 mm), each FD appeared gradually in the plasma, and the in vivo behavior was explained by two-compartment model with first-order absorption. The absorption ratios of FDs from the rat liver surface at 6 h, calculated from the amount recovered from the glass cell, decreased with an increase in the molecular weight (44.5% for FD-4, 29.3% for FD-10, 5.1% for FD-40 and 2.2% for FD-70). A linear relationship was observed between the absorption rate constant and the reciprocal value with square root of molecular weight of the model compounds. The limit of absorption from the rat liver surface was extrapolated to be at a molecular weight of 70,000. Furthermore, absorbed FDs were accumulated in the liver, as high liver/plasma concentration ratio as compared with that of i.v. administration.

We clarified the molecular weight dependence of drug absorption from the liver surface in rats. Moreover, the liver surface application appeared to be a promising route with enhancing the efficacy of drug targeting to the liver.     

Inhibition of the release of prostaglandins,leukotrienes and lysosomal acid hydrolases from macrophages by selective inhibitors of lecithin biosynthesis

The release of the inflammatory mediators, prostaglandins (PGs), leukotrienes (LT) and lysosomal acid hydrolases (LAH), by macrophages is stimulated by endocytic stimuli such as zymosan. This process can be interfered with by specific inhibitors of phosphatidylcholine (PC) biosynthesis. The diphenylsulfone dapsone and three analogs selectively inhibited [¹⁴C]choline incorporation into PC but had varied effects on inhibition of mediator release by macrophages. Dapsone inhibited the release of PGs, LT and LAH, whereas the three closely related structural analogs inhibited LT and LAH release only, with little or no effect on PG production.     

Donepezil down-regulates propionylation, 2-hydroxyisobutyrylation,butyrylation, succinylation,and crotonylation in the brain of bilateral common carotid artery occlusion-induced vascular dementia rats

Vascular dementia (VaD), caused by stroke or small vessel disease, is the second-most common type of dementia after Alzheimer's disease (AD). Donepezil is an acetylcholinesterase inhibitor that is currently used in patients with mild to moderate AD, and has recently been shown to improve cognitive performance in patients with VaD. In this study, we evaluated the effects of donepezil on VaD, and investigated the underlying molecular mechanisms of action. VaD was established by ligation of the bilateral common carotid artery occlusion (BCCAO). Executive function was tested by the Morris water maze (MWM) test and the attentional set shifting task (ASST). Our results showed that donepezil improved executive dysfunction and cognitive flexibility in BCCAO rats. In addition, we showed that donepezil treatment decreased the level of Aβ1-42 in BCCAO rats by enzyme-linked immunosorbent assay. Post-translational modifications (PTMs) are known to be critical mechanisms in the regulation of various cellular processes. Furthermore, PTMs have been linked to the central nervous system, which highlights the importance of PTMs in neurodegenerative diseases. In this study, we used western blot analysis to identify several novel PTMs in the hippocampus of BCCAO rats that were treated with or without donepezil. The data revealed that lysine propionylation, 2-hydroxyisobutyrylation, butyrylation, succinylation, and crotonylation were elevated in the hippocampus of BCCAO rats when compared to sham rats. This increase was abolished by donepezil treatment. Taken together, we speculate that donepezil treatment improves cognitive function in our animal model of VaD, possibly by reducing aberrant acyl-PTMs.     

马甲子叶提取物的抗肿瘤活性研究

目的 研究马甲子叶提取物的体内外抗肿瘤活性.方法 采用系统溶剂萃取法萃取马甲子叶醇提物,采用MTT法分别检测马甲子叶水提物、醇提物、石油醚部位、乙酸乙酯部位和正丁醇部位对人肝癌HepG-2、人胃癌MGC-803、人宫颈癌Hela细胞的体外抗肿瘤活性;考察马甲子叶乙酸乙酯部位对S180荷瘤小鼠的体内抗肿瘤活性.结果 马甲子乙酸乙酯部位对3种肿瘤细胞均有明显抑制作用,呈一定量效关系,IC50分别为23.68、24.91、75.32 μg·mL-;乙醇提取物和石油醚部位对3种肿瘤细胞均有一定抑制作用;正丁醇部位和水提物的抑制作用不明显.乙酸乙酯部位对S180荷瘤小鼠的体内抗肿瘤作用明显,中剂量的抑瘤效果与环磷酰胺的相当,抑瘤率达49.38％.结论 马甲子叶乙酸乙酯部位的抗肿瘤活性最强.     

Novel γ-secretase modulators for the treatment of Alzheimer's disease: a review focusing on patents from 2010 to 2012

Introduction: γ-Secretase is the enzyme responsible for the final step of amyloid precursor protein proteolysis to generate Aβ peptides including Aβ42 which is believed to be a toxic species involved in Alzheimer's disease (AD) progression. γ-Secretase modulators (GSMs) have been shown to selectively lower Aβ42 production without affecting total Aβ levels or the formation of γ-secretase substrate intracellular domains such as APP intracellular domain and Notch intracellular domain. Therefore, GSMs have emerged as an important therapeutic strategy for the treatment of AD.

Areas covered: The literature covering novel GSMs will be reviewed focusing on patents from 2010 to 2012.

Expert opinion: During the last review period (2008 – 2010) considerable progress was made developing GSMs with improved potency for lowering Aβ42 levels, but most of the compounds resided in unfavorable central nervous system (CNS) drug space. In this review period (2010 – 2012), there is a higher percentage of potent GSM chemical matter that resides in favorable CNS drug space. It is anticipated that clinical candidates will emerge out of this cohort that will be able to test the GSM mechanism of action in the clinic.     

Mutagenicity of extracts from Ceylon cinnamon in the rec assay

The extraction of about 1.9 kg of Ceylon cinnamon (Cinnamomum zeylanicum Nees) with 10 litres each of petroleum ether, chloroform and ethanol in a Soxhlet apparatus produced extracts weighing 76, 28 and 270 g respectively for the three solvents. In the preliminary test the ethanol extract showed no mutagenic activity. However, both the petroleum ether and the chloroform extracts showed mutagenicity when tested in the rec assay using Bacillus subtilis strains H17 (rec+) and M45 (rec-). When these extracts were studied quantitatively by the liquid and spore rec-assay methods, the minimum inhibitory concentrations of the extracts against strain H17 were higher than those against strain M45. However, in the presence of the liver S-9 mix, the minimum inhibitory concentrations of the petroleum ether and chloroform extracts against both strains of B. subtilis were equal, indicating that the mutagenicity of the extracts had been inactivated.     

人脐带沃顿胶间充质干细胞体外分离条件的优化及传代效应的流式细胞学检测

目的观察不同胶原酶消化方法对人脐带沃顿胶间充质干细胞(mesenchymal stem cell,MSC)分离和培养结果的影响,并鉴定其分化潜能;探讨传代效应对其免疫表型的影响。方法将制备好的脐带标本分别加入Ⅰ型、Ⅱ型及Ⅳ型胶原酶,持续消化4~18 h,筛网过滤,离心收集细胞,用DMEM/F12培养基重悬细胞,调整细胞密度4.8×103~1×104/cm2,接种培养,比较不同消化法分离人脐带沃顿胶MSC的效果。Von kossa钙结节染色、四环素荧光标记鉴定人脐带沃顿胶MSC向成骨方向分化的能力,RT-PCR鉴定其向心肌细胞分化的能力。应用流式细胞仪检测连续传代后MSC的免疫表型变化。结果Ⅰ型胶原酶消化法能够从人脐带沃顿胶获取了数量较多、活力较高的MSC,而且细胞出现伸展的时间及原代培养时间均短于Ⅱ型胶原酶及Ⅳ型胶原酶消化法。表面标记分析显示:随着传代次数的增加,阳性标记CD29、CD44、CD73、CD90、CD105的表达百分率没有变化,而阴性标记CD31、CD34和HLA-DR的表达率增加明显。体外诱导实验表明:来源于人脐带沃顿胶的MSC具有体外成骨和成心肌样细胞分化的能力。结论Ⅰ型胶原酶消化法简单易行,对细胞损伤小,能稳定、高效地从人脐带沃顿胶中分离出MSC。     

“Disease” of the Nation,Family and Individual: Three Moral Discourses of Alcohol Problems in Finnish Women's Magazines from the 1960s to the 2000s

Women's magazines can be seen as a genre that form feminized public spaces where everyday life contradictions of women's life are negotiated. The study examines the ways in which Finnish women's magazines have dealt with alcohol problems. The data covers six primary sampling years: 1968, 1976, 1984, 1992, 2000 and 2008. The data is analyzed by drawing on the concept of ‘moral regulation’. The analysis shows that a family-centered framing dominated the constructions of alcohol problem: fathers’ and husbands’ alcoholism appeared as a main object of regulation in all decades under study, while mothers’ and wives’ alcoholism was much less prevalent.     

Generation of T cell responses targeting the reactive metabolite of halothane in mice

Immune-mediated adverse drug reactions (IADRs) represent a significant problem in clinical practice and drug development. Studies of the underlying mechanisms of IADRs have been hampered by the lack of animal models. Halothane causes severe allergic hepatitis with clinical features consistent with an IADR. Our ultimate goal is to develop a mouse model of halothane hepatitis. Evidence suggests that adaptive immune responses targeting liver protein adducts of the reactive metabolite (trifluoroacetyl (TFA)) play an important role in the pathogenesis. The present study demonstrated that the combination of an anti-CD40 antibody (Ab) and a Toll-like receptor (TLR) agonist served as a potent adjuvant in generating TFA-specific T cell responses in mice. Both CD4⁺ and CD8⁺ subsets of T cells were activated and the TFA-specific responses were detected not only in the spleen but also in the liver of mice immunized with mouse serum albumin adducts of TFA (TFA-MSA) plus the combined CD40/TLR agonist. Whereas all three TLR agonists examined were effective in eliciting TFA-specific immune responses in BALB/cByJ mice, only polyI:C was effective in DBA/1 mice and none of the TLR agonists could aid the generation of TFA-specific T cells in C57BL/6J mice. This result, combined with our previous finding that BALB/cByJ mice were the most susceptible to halothane-induced acute liver injury, provides the basis for employing this strain in future studies. Collectively, our data demonstrated the successful completion of a crucial first step in the development of a murine model of halothane hepatitis.     

Interpretation of cell toxicity data for the estimation of potential irritation

Three cytotoxicity assays were evaluated using 57 chemicals of various classes (inorganic and organic metal salts, solvents, detergents, reagents, drugs) which have widely different mechanisms of cytotoxicity. Baby hamster kidney fibroblasts (BHK-21/C13) and early (Keller) and late (MRC-5) passage human fibroblasts were used to measure cell detachment, cloning efficiency, and growth inhibition under subconfluent culture conditions. For the majority of chemicals, for which comparisons were made, the ranking order was roughly the same in all three tests and with all three cell types. However, for some chemicals specific growth effects could either be detected or excluded because the relationship between the data from the detachment assay and that from one of the growth assays was characteristically altered. The ranking order resulting from our in vitro data correlated better with threshold limit values for human workroom air (TLV/TWA) than with LD50 values (rat, oral). Correlations with data from Draize skin and eye irritation tests were not determined since the available in vivo values were derived using various different scoring systems. However, when our in vitro data were used to divide the chemicals into three crude classes, (i) non-irritant, (ii) mild to moderate irritant, or (iii) strong irritant or corrosive, and the results were compared with the known irritation potential for skin and mucous membranes derived from human exposure data, the in vitro data were more than 80% predictive of the in vivo classifications.     

1H n.m.r. parameters of the N-terminal 19-residue S-peptide of ribonuclease in aqueous solution

The ¹H n.m.r. chemical shifts and the spin-spin coupling constants of the N-terminal 19-residue S-peptide of ribonuclease A have been measured in a 10mM solution in D₂O, _pD 3.0, 27d?, at 300 MHz. The titration parameters for end groups Lys-1 and Ala-19 and side chains Lys-1, Glu-2, Lys-7, Glu-9, Arg-10, His-12 and Asp-14 have been determined at 90 MHz. An assignment of observed signals to individual residue protons based upon characteristic shifts, spectral analysis, double resonance, titration shifts and comparison with the spectrum of C peptide (N-terminal 13 residue) is proposed. Differences in the observed chemical shifts, _pK_a‘s and titration shifts with reference to those proposed as “random coil” parameters are not large enough to assume the existence of a significant population of secondary structure in the conditions studied. The Hα chemical shifts differences can be accounted for by the Phe-8 phenyl ring current for an extended peptide backbone conformation and appropriate values for the torsion angles X₁ Phe-8 and X₂ Phe-8.     

Long-term toxicity studies in dogs support the safety of the special extract ERr 731 from the roots of Rheum rhaponticum.

The special extract ERr 731 from the roots of rhapontic rhubarb has been regularly prescribed for women with menopausal symptoms since 1993. As its constituents belong to the class of natural hydroxystilbenes, concerns have been raised about possible health risks similar to those known for the estrogenic diethylstilbestrol. To demonstrate the safety of the medical use of ERr 731, the extract was tested in long-term toxicity studies in dogs. In two independent studies, male and female beagle dogs were treated with 100, 300 and 1000 mg ERr 731/kg body weight (bw)/day and observed for 4 and 13 weeks followed by recovery periods. A histopathological examination of a full set of organs of all animals was examined. In both studies, all animals survived the scheduled treatment and recovery periods. The administration of ERr 731 resulted in increased incidences of feces with white particles due to an incomplete absorption of the extract. In the 13-week study, a slight decrease in glucose levels was recorded in both sexes at 1000 mg/kg bw/day. All other clinical changes were marginal and not related to ERr 731. Importantly, there was no increase in weight of organs of the genital tract due to ERr 731 intake. Based on these results, the no-observed-adverse-effect-level is 1000 mg/kg bw/day. No pathological findings where detected following ERr 731 treatment demonstrating that the toxicological risk for women taking ERr 731 regularly is extremely low.     

Depletion of melamine and cyanuric acid in serum from catfish Ictalurus punctatus and rainbow trout Onchorhynchus mykiss

Melamine and its triazine analogs, such as cyanuric acid, have been used to artificially inflate protein content both in animal feed ingredients, as well as in milk products produced for human consumption. We report here a LC–MS/MS method to quantify and confirm melamine and cyanuric acid in serum from channel catfish and rainbow trout with a limit of quantification of 0.8 μg/mL. The method was applied to serum samples from a residue depletion study in which fish were given a single oral dose of 20 mg/kg body weight melamine, cyanuric acid, or both compounds together. Samples were taken at 1, 3, 7, 14, and 28 days (an additional 42 day was added for trout). When given alone or in combination with cyanuric acid, melamine residues were highest on day 1 in both catfish and trout. Cyanuric acid was only quantifiable at day 1 in trout when given alone, and not at all in catfish. The serum half life of melamine in catfish was 1.50–1.62 days and 3.09–3.67 days in trout. This work highlights the differences of depletion kinetics in fish, which can be measured in days, as compared to the depletion in mammals, measured in hours.     

Estrogen-like effects of diet-derived cadmium differ from those of orally administered CdCl(2) in the ERE-luc estrogen reporter mouse model

Cadmium (Cd), an environmental and dietary contaminant, has been described to mimic the effects of 17β-estradiol (E₂) in selected model systems when studied as an inorganic salt. However, inorganic Cd salts do not represent the main form of Cd exposure in general human populations. The aims of this study were to compare the estrogen-like effects and the bioavailability of dietary Cd to inorganic CdCl₂. Adult ovariectomized ERE-luc reporter mice were administered two bread based diets containing different concentrations of Cd (17.57 and 49.22 μg/kg, corresponding to oral intakes of 1.8 and 5.1 μg/kg body weight (bw) per day, respectively), inorganic CdCl₂ (1 μg/kg bw per day by gavage) or E₂ (5 μg/kg bw per day pellet) for 21 days. The effects on estrogen signaling were investigated by studying the uterine weights, luciferase activation, and expression of endogenous estrogen target genes. The uterine weight was significantly increased by both CdCl₂ and E₂ but not by the Cd containing diets. All treatments modulated the expression of luciferase and the endogenous estrogen target genes; however, there was no consistent overlap between the responses triggered by the bread diets and the responses stimulated by CdCl₂ or E₂. Oral exposure to Cd was calculated and the concentrations in liver and kidneys quantified to estimate the amount of absorbed Cd retained in tissues. The results suggest significantly lower absorption and/or tissue retention of dietary Cd compared to CdCl₂ following oral exposure. Altogether, our results support previous reports on in vivo estrogenicity of CdCl₂ but do not suggest the same activity for diet bound Cd. This study calls for caution when extrapolating results from pure compound studies (e.g. estrogenicity of CdCl₂) to dietary exposure scenarios (e.g. estrogenicity of diet bound Cd). Further basic research is needed on the mechanisms of interaction between Cd and the estrogen signaling, biologically active species of Cd, and biomarkers of estrogen-like effects of Cd in vivo before human health risk assessment on the hormone disruptive effects of Cd can be carried out.