615小鼠血红蛋白α链的氨基酸组成及个别肽段的氨基酸序列

用CM-Cellulose-23柱层析分离纯化了615小鼠珠蛋白α链,测定其N端氨基酸残基为缬氨酸.615小鼠珠蛋白α链含有141个氨基酸残基,其中19个亮氨酸残基,10个组氨酸残基,9个缬氨酸残基,上述氨基酸残基的数目与文献中其亲本C₅₇BL不同.用胰蛋白酶水解615小鼠珠蛋白α链,发现有不溶性的‘核心’和可溶性的酶解片段.其中一个酶解肽段从N端数第8位氨基酸残基发生了突变,由亲本的缬氨酸变为亮氨酸.     

镰刀形红细胞的形成

镰刀形红细胞贫血症是一种严重的溶血性疾病,其特点是在氧分压低的情况下,红细胞就由正常的双凹圆饼状变成镰刀形。产生镰刀形细胞贫血症的根本原因是基因突变,导致转录、翻译错误,使血红蛋白分子的β多肽链上,第六位的谷氨酸被缬氨酸代替,这一个氨基酸分子的改变是怎样引起红细胞形成镰刀状的呢? 从蛋白质分子的一级结构看,正常血红蛋白(HbA)与镰刀形红细胞血红蛋白(HbS)的差异,是位于β链N端第六位上的氨基酸残基不同,其氨基酸残基顺序如下: HbA:缬-组-亮-酷-脯-谷-谷-赖…… HbS:缬-组-亮-酪-脯-缬-谷-赖……谷氨酸的侧链基团带负电荷,缬氨酸的侧链基     

水基钻井液对诸氏鲻虾虎鱼抗氧化酶的影响

目的 研究我国南海区某海洋钻井水基钻井液对诸氏鲻虾虎鱼抗氧化酶的影响.方法在19.75～158 mg/L浓度下,对3月龄诸氏鲻虾虎鱼染毒,并用试剂盒法测定超氧化物歧化酶（SOD）和过氧化氢酶（CAT）的活性.结果 SOD对水基钻井液的毒性暴露不太敏感,在染毒第1天只有最低浓度组受到一定程度的抑制,染毒第2 天各浓度组酶活性均受到一定程度的诱导（12.4%～25.5%）,这种诱导作用在第4天即有所减弱,之后酶活性的变化变缓.CAT则对水基钻井液表现出较强的敏感性.染毒第1 天,各组酶活性均受到明显的诱导,并且各组酶活性表现出明显的浓度效应关系.最高浓度组酶活性最大诱导率达到97%（P＆gt;0.05）.随后虾虎鱼CAT酶活性逐渐下降,第7 天,各染毒组酶活性的抑制率达到58.4%～89.1%.结论 CAT在中毒反应中表现出了敏感性,具备了作为生物标志物的重要条件,有望在今后的实际应用中发挥作用.     

聚合牛血红蛋白抗原性的初步研究

对乙醇醛聚合的牛血红蛋白的抗原性进行了研究。将聚合的牛血红蛋白桉兔血量的10％和20％分别输入兔耳缘静脉,间隔7天后重复输液,共输注3次。ELISA检测未显示抗体产生。将聚合的牛血红蛋白、人血红蛋白和兔血红蛋白分别与免疫佐剂混合常规免疫家兔3次,并用上述抗原包被聚乙烯板,用ELISA方法检测抗体滴度,结果显示聚合牛血红蛋白和人血红蛋白加佐剂免疫家兔后均产生抗体,且有交叉反应,表明这两种血红蛋白有同源性,存在相似的抗厚决定簇。兔血红蛋白免疫家兔后无抗体产生,且无交叉反应,表明机体对自体蛋白有天然的耐受。     

溴氰菊酯对真鲷肝胰脏组织及细胞DNA的损伤

为研究溴氰菊酯亚急性染毒对真鲷肝胰脏的毒性作用,将真鲷分为5组进行不同剂量半静置染毒.染毒25 d后取真鲷肝胰脏进行组织切片显微观察,并采用彗星试验技术对其肝胰脏细胞进行DNA损伤分析.结果表明: 在0.025、0.125、0.250和0.375 μg·L^-1暴露浓度下,真鲷肝胰脏组织出现了不同程度的淤血、细胞核浓缩、细胞坏死等病理性损伤,且染毒浓度越高组织细胞损伤越显著.与空白对照组相比,各染毒浓度组肝胰脏细胞DNA均有不同程度损伤,彗星拖尾率、彗尾DNA相对含量、Olive距等指标均与对照有显著性差异.一元回归分析表明,染毒浓度与拖尾率、彗星尾长等参数呈极显著正相关; 染毒浓度与各指标均呈线性关系,回归方程拟合度(R)极高,范围为0.909～0.996.表明溴氰菊酯对真鲷肝胰脏组织和细胞DNA均可产生不同程度损伤,且损伤程度与染毒浓度之间具有高度线性相关关系.     

乙酰胆碱在钩吻素己致小鼠中毒死亡中的作用北大核心CSCD

本文旨在探讨全血乙酰胆碱浓度与钩吻素己致昆明小鼠中毒死亡的关系。给昆明小鼠腹腔注射生理盐水、钩吻素己或不同剂量的氯化乙酰胆碱溶液,并对注射钩吻素己和中剂量氯化乙酰胆碱的小鼠给予阿托品解救。中毒死亡小鼠死后即时取血;存活小鼠在注射药液后20 min处死取血。用试剂盒测定小鼠全血乙酰胆碱浓度与乙酰胆碱酯酶活性,计算各组死亡率并分析其死亡情况。结果显示,相对于生理盐水对照组,腹腔注射半数致死量(0.15 mg/kg)的钩吻素己后,小鼠全血乙酰胆碱酯酶活力降低,全血乙酰胆碱水平升高,但其中死亡小鼠的全血乙酰胆碱浓度显著低于中剂量氯化乙酰胆碱组死亡小鼠的水平,而与中剂量乙酰胆碱组存活小鼠的水平无差异。阿托品可有效解救氯化乙酰胆碱中毒小鼠,但对钩吻素己中毒小鼠的抢救效果不明显。以上结果提示,钩吻素己可引起小鼠全血胆碱酯酶活力下降,乙酰胆碱浓度升高,但体内乙酰胆碱堆积可能并不是钩吻素己致小鼠中毒死亡的主要或唯一原因。     

ω-芋螺毒素MVIIC的N及C端修饰对折叠及活性影响

 合成了 ω-芋螺毒素 MVIIC的三种 N及 C端修饰肽 ,应用高压液相色谱、CD及生物体内活性实验 ,研究了其 N及 C端修饰对折叠及活性的影响 .结果表明 :MVIIC N端用 Phe及 Ser修饰后降低其线性肽形成正确折叠的比例及结构的稳定性 ,对小鼠的脑室给药活性也相应降低 ;C端酰胺转为电负性羧基端后活性降低 ,CD谱存在显著差异 .     

肿瘤MUC1/Y的克隆及其胞外段在大肠杆菌中的可溶性表达、纯化和鉴定

为获得MUC1 Y全长cDNA及其胞外段蛋白 ,以用于进一步的生物学功能及肿瘤生物学治疗的研究 .利用RT PCR从HeLa细胞中扩增MUC1 Y全长cDNA ;PCR扩增其胞外段 ,克隆到原核表达载体pGEX 2T ,转化DH5a菌 ,诱导表达 ;亲和层析纯化 ;凝血酶酶切、GST活性及N端蛋白测序鉴定 ;免疫家兔制备多克隆抗体 .所得MUC1 YcDNA的开放读框为 759bp ,登录于GenBank(AF12 552 5) .其信号肽编码序列缺失 9bp ,第 3 3 1位发生G A转换 ,造成缬氨酸突变为蛋氨酸 .表达获得约 4 0kD融合蛋白GST Yex ,占菌体总蛋白 2 5%～ 3 0 % ,其中 70 %～ 80 %为可溶性 ,经亲和层析一步纯化 ,纯度 >90 % ,GST比活性为 0 2 1U μg .凝血酶酶切后的N端蛋白序列测定表明与已知序列完全一致 ,抗血清ELISA效价为 1∶2 50 0 0 0 .结果表明 ,克隆到发生碱基缺失和突变的MUC1 Y全长cDNA ,获得MUC1 Y胞外段蛋白及其多抗 ,可进一步用于相关研究 .     

腹腔注射法制备一氧化碳中毒迟发性脑病大鼠模型

目的：建立可靠的急性一氧化碳（CO）中毒迟发性脑病的动物模型。方法：雄性SD大鼠,分次腹腔注射CO染毒制备模型,动态监测尾血碳氧血红蛋白（HbCO）浓度;Morris水迷宫检测大鼠1-5w逃避潜伏期;尼氏染色及TUNEL原位末端凋亡染色检测大脑皮质及海马细胞损伤及凋亡。结果：染毒后,大鼠出现典型的CO重度中毒症状,体内血液HbCO浓度迅速升高,使用分次腹腔注射法,大鼠可维持长时间（〉12h）高HbCO状态（HbCO〉48%）;中毒组大鼠水迷宫检测认知功能较对照组下降,病理学检查显示大鼠出现脑细胞损伤、凋亡明显。结论：本研究建立了一种较为符合迟发性脑病临床特征的动物模型,具有简单、可靠、重复性好的特点,为深入研究急性CO中毒致迟发性脑损伤的机制提供可靠基础。     

乙酰胆碱在钩吻素己致小鼠中毒死亡中的作用

本文旨在探讨全血乙酰胆碱浓度与钩吻素己致昆明小鼠中毒死亡的关系。给昆明小鼠腹腔注射生理盐水、钩吻素己或不同剂量的氯化乙酰胆碱溶液,并对注射钩吻素己和中剂量氯化乙酰胆碱的小鼠给予阿托品解救。中毒死亡小鼠死后即时取血;存活小鼠在注射药液后20 min处死取血。用试剂盒测定小鼠全血乙酰胆碱浓度与乙酰胆碱酯酶活性,计算各组死亡率并分析其死亡情况。结果显示,相对于生理盐水对照组,腹腔注射半数致死量(0.15 mg/kg)的钩吻素己后,小鼠全血乙酰胆碱酯酶活力降低,全血乙酰胆碱水平升高,但其中死亡小鼠的全血乙酰胆碱浓度显著低于中剂量氯化乙酰胆碱组死亡小鼠的水平,而与中剂量乙酰胆碱组存活小鼠的水平无差异。阿托品可有效解救氯化乙酰胆碱中毒小鼠,但对钩吻素己中毒小鼠的抢救效果不明显。以上结果提示,钩吻素己可引起小鼠全血胆碱酯酶活力下降,乙酰胆碱浓度升高,但体内乙酰胆碱堆积可能并不是钩吻素己致小鼠中毒死亡的主要或唯一原因。     

L-Oxothiazolidine 4-carboxylate pretreatment of isolated human peripheral blood lymphocytes reduces sulfur mustard cytotoxicity

Sulfur mustard (HD, mustard gas) is a vesicant chemical warfare agent for which there is no specific medical countermeasure. A potential approach to combating the debilitating effects of this agent is the use of compounds that can react with this material before it interacts with critical macro-molecules. Glutathione (GSH), a tripeptide that exists in high concentrations in cells, reacts with HD and is involved in HD detoxification. Pretreatment of human peripheral blood lymphocytes (PBL) with 10 mmol/L L-oxothiazolidine-4-carboxylate (OTC), a "masked" cysteine precursor, increased intracellular glutathione levels 25-50% over control values. Pretreated PBL were harvested, washed, and exposed to 10, 50, or 100 µmol/L HD. Flow cytometry was used to measure cytotoxicity by propidium iodide uptake. Pretreatment of PBL with OTC led to small decreases in cytotoxicity after HD exposure. However, treatment of cells with OTC after HD exposure was not beneficial. Compounds that can modulate GSH levels within the cell may help to reduce the cytotoxicity of HD when used as a pretreatment.     

Sulfur mustard-increased proteolysis followingin vitro andin vivo exposures

The pathologic mechanisms underlying sulfur mustard (HD)-induced skin vesication are as yet undefined. Papirmeister et al. (1985) postulate enhanced proteolytic activity as a proximate cause of HD-induced cutaneous injury. Using a chromogenic peptide substrate assay, we previously reported that in vitro exposure of cell cultures to HD enhances proteolytic activity. We have continued our investigation of HD-increased proteolytic activity in vitro and have expanded our studies to include an in vivo animal model for HD exposure. In vitro exposure of human peripheral blood lymphocytes (PBL) to HD demonstrated that the increase in proteolytic activity is both time- and temperature-dependent. Using a panel of 10 protease substrates, we established that, the HD-increased proteolysis was markedly different from that generated by plasminogen activator. The hairless guinea pig is an animal model used for the study of HD-induced dermal pathology. When control and HD-exposed PBL and hairless guinea pig skin where examined, similarities in their protease substrate reactivities were observed. HD-exposed hairless guinea pig skin biopsies demonstrated increased proteolytic activity that was time-dependent. The HD-increased proteolytic response was similar in both in vitro and in vivo studies and may be useful for elucidating both the mechanism of HD-induced vesication and potential treatment compounds.Abbreviations CPSPA chromogenic peptide substrate protease assay - HD sulfur mustard - PBL human peripheral blood lymphocytes - pNA p-nitroaniline In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, revised 1985.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

The use of human epidermal keratinocytes in culture as a model for studying the biochemical mechanisms of sulfur mustard toxicity

Human epidermal keratinocytes in culture were studied to evaluate their usefulness in demonstrating toxic events following exposure to sulfur mustard. Exposure of keratinocytes to sulfur mustard over a concentration range of 1–1000 μM HD, reduced NAD+ levels from 96% to 32% of control levels. When keratinocytes were exposed to a concentration of 300 μM HD, NAD+ levels began to fall at 1 hour and reached a plateau of 47% of control levels at 4 hours. Niacinamide, an inhibitor of the enzyme poly(ADP-ribose) polymerase, partially protected mustard-exposed cells against NAD+ depletion. It also protected cellular viability as assessed by vital staining 24 hours after exposure. This protection was not seen in long-term (72 hr) cultures. These studies suggest that human epidermal keratinocytes in culture can serve as a usefulin vitro model for research into the biochemical mechanisms of sulfur mustard-induced cutaneous injury.     

Sulfur mustard exposure enhances Fc receptor expression on human epidermal keratinocytes in cell culture: Implications for toxicity and medical countermeasures

Sulfur mustard (HD) is a chemical warfare blister agent. The biochemical basis of HD-induced vesication is unknown, and no antidote currently exists. Basal epidermal cells are a major site of HD toxicity in vivo, with inflammation and HD-increased proteolytic activity implicated as factors that contribute to HD pathology. Fc receptors (FcR) bind to the Fc region of antibody to mediate many effector and regulatory functions that can influence inflammatory responses. FcR are found on all types of immune cells and are also expressed on the surface of human keratinocytes. Assay by fluorescent antibodies demonstrated significantly enhanced CD32 (FcRII) and CD16 (FcRIII) on human epidermal keratinocyte (HEK) cell cultures at 8 to 24 h after exposure to HD (50, 100 and 200 µmol/L). The enhanced CD32 was time- and concentration-dependent and agreed well with the time course of increased proteolysis and cutaneous pathology observed during HD vesication. HD-increased FcR on the surface of HEK might be a mechanism of vesication.     

Tissue distribution and urinary excretion of essential elements in rats orally exposed to aluminum chloride

The purpose of this study was to determine disorders in the metabolism of the essential elements (Ca, Fe, Cu, and Zn) in some tissues of rats, as well as to detect the dynamics of urinary excretion of these metals after oral administration of 20 mgAl/kg every day for 8 wk. The elements were determined in brain, kidneys, blood, and urine of the animals in 1st, 2nd, 3rd, 4th, and 8th wk after the exposure to AlCl₃. After the 1st wk of aluminium administration, we observed increase of Ca and a decrease of Fe in blood. In brain Ca, Fe, and Cu concentrations were significantly higher in Al-treated rats than in controls after 8-wk exposure. The concentration changes of the essential metals in the tissue were accompanied by increase of the Ca, Fe, and Zn urinary excretion. We assume that the increase in urinary excretion of Ca and the decrease of Fe in the blood may be sensitive indicators of oral aluminium administration.     

Using of metal-affinity chromatography for identification of alkylated rat globin

Rat globin peptides alkylated by sulfur mustard on amino-acid residues C-126, C-93 and E-27 with MH+ 1444.62 Da, 1561.66 Da, 1676.78 Da, respectively, were concentrated using metal-affinity chromatography on Cu2+. The peptides were received by trypic digestion after in vitro incubation of rat globin with 60 microM HD. Aklylated peptide with MH+ 1444.62 Da is the most sensitive biomarker, which can be concentrated from globin trypic digest, incubated with 3 microM sulfur mustard.     

Inhibition of sulfur mustard-increased protease activity by niacinamide,N-acetyl-L-cysteine or dexamethasone

The pathologic mechanisms underlying sulfur mustard-induced skin vesication remain undefined. Papirmeister et al. (1985) have postulated a biochemical mechanism for sulfur mustard-induced cutaneous injury involving DNA alkylation, metabolic disruption, and enhanced proteolysic activity. We have previously utilized a chromogenic peptide substrate assay to establish that human peripheral blood lymphocytes exposed to sulfur mustard exhibited enhanced proteolytic activity. In this study, compounds known to alter the biochemical events associated with sulfur mustard exposure or to reduce protease activity were tested for their ability to block the sulfur mustard-increased proteolysis. Treatment of cells with niacinamide, N-acetyl-L-cysteine, or dexamethasone resulted in a decrease of sulfur mustard-increased protease activity. Complete inhibition of sulfur mustard-increased proteolysis was achieved by using protease inhibitors (antipain, leupeptin, and 4-(2-aminoethyl)-benzenesulfonylfluoride). These data suggest that therapeutic intervention in the biochemical pathways that culminate in protease activation or direct inhibition of proteolysis might serve as an approach to the treatment of sulfur mustard-induced pathology.Abbreviations APMSF 4-(2-aminoethyl)-benzenesulfonylfluoride, HCI - CPSPA Chromogenic Peptide Substrate Protease Assay - EDTA ethylenediaminetetraacetic acid - HD sulfur mustard - PBL human peripheral blood lymphocytes - pNA p-nitroaniline     

Parameterizing Potential Exposure to Sulfur Mustard (HD) Using Mixed Model Regression

The possible threat posed by terrorists using chemical warfare agents (CWAs) against civilian targets is a major concern, reflecting the fact that CWAs are highly toxic to unprotected populations, with releases as vapors or aerosols likely to produce mass casualties on a highly localized basis within minutes or hours after an incident. A conceptual site model is developed and mixed model regression is used to estimate concentration values for the vesicant sulfur mustard (HD) based on the output from computational fluid dynamics (CFD) simulation following wind tunnel experimentation. The analysis provides a first-approximation of the spatial and temporal distribution of potential exposures within a set of 50 m × 50 m × 2 m grids across a 1000 m width by 300 m height by 2250 m length domain in a geographic information system (GIS) environment. The HD concentration values are calculated as log-averaged mean and the 95% confidence intervals for each grid at 1.9 d and 6.0 d after initial release. The technique offers a statistically valid means for rapidly generating unbiased first-approximations of concentration values subsequent to an initial release as an alternative to extensive monitoring or multiple runs of CFD models to parameterize potential exposure to HD spatially and temporally.