Cleavage of laminin by thrombin and plasmin: Alpha thrombin selectively cleaves the beta chain of laminin

Highly purified -thrombin, plasmin and urokinase were incubated with laminin, type IV collagen and type V collagen. At 25°C (1:100 enzyme to substrate ratio on a weight/weight basis) -thrombin selectively degraded the β chain of native laminin, whereas plasmin degraded both the and β chains. The specific limited cleavage fragments of laminin produced by -thrombin and plasmin retained the ability to mediate binding of epithelial cells to type IV collagen. These serine proteases failed to degrade native type IV or type V collagen under identical experimental conditions. At 35°C type V collagen, but not type IV collagen, was partially cleaved by both -thrombin and plasmin. Urokinase failed to degrade any of the substrates. It is concluded that thrombin and plasmin could play a physiological role in the degradation of laminin during tissue remodeling or repair. Specific laminin cleavage products produced by these enzymes could be useful in structural, immunological, and biological studies.     

细胞表面半乳糖基转移酶介导外胎盘锥扩展和次生滋养层巨细胞迁移的证据

细胞表面半乳糖基转移酶（ＧａｌＴａｓｅ）是层粘连蛋白（ＬＮ）非整合素类受体，能与其上寡糖链的Ｎ－乙酰葡糖胺（ＧｌｃＮＡｃ）残基识别和结合。作者从妊娠第８．５天雌鼠子宫蜕膜中剥离胚胎，显微分离外胎盘锥（ＥＰＣ）后进行体外培养。用（１）ＬＮ预先半乳糖基化；（２）外源ＧｌｃＮＡｃ底物竞争；（３）ＧａｌＴａｓｅ抗体阻断；（４）α乳清蛋白处理等方法干扰滋养层细胞与ＬＮ的相互作用，均对ＥＰＣ粘附无影响，但抑制ＥＰＣ扩展和次生滋养层巨细胞（ＳＴＧＣｓ）迁移。证明ＧａｌＴａｓｅ虽不参与ＥＰＣ起初的粘附，但通过与ＬＮ寡糖链上ＧｌｃＮＡｃ的结合介导ＥＰＣ扩展和ＳＴＧＣｓ迁移。     

131I-层粘连九肽的合成及在荷乳腺癌裸鼠中的显像和分布

目的用１３１Ｉ层粘连九肽进行荷乳腺癌裸鼠的肿瘤受体显像及其生物分布研究。方法以固相法合成层粘连九肽，经ＨＰＬＣ纯化，用氯胺Ｔ法进行１３１Ｉ标记。标记物以ＳｅｐｈａｄｅｘＧ１０纯化。尾静脉注入荷乳腺癌裸鼠体内，分别在１、２、３、４、５、６小时进行肿瘤显像，划出感兴趣区，测其Ｔ／Ｂ值。静脉给药后分别在３、６小时观察生物分布，计算其每克组织摄取注射剂量百分数（％ＩＤ／ｇ）和肿瘤与非肿瘤的放射性比值（Ｔ／ＮＴ值）。结果化学合成的层粘连九肽经ＨＰＬＣ、ＦＡＢＭＳ纯化鉴定，Ｍ＋１＝９６６３。尾静脉注射后４～５小时肿瘤部位有明显浓聚，Ｔ／Ｂ值达４６２。肿瘤／肌肉的Ｔ／ＮＴ值在６小时为３４５。结论１３１Ｉ层粘连九肽有希望用于诊断人体乳腺癌     

Immunoprofile of reactive salivary myoepithelial cells in intraductal areas of carcinoma ex-pleomorphic adenoma

The myoepithelial cell (MC) is a component of various secretory glands, including salivary glands. Besides its function, a tumor suppressor and a tumor facilitating functions have been attributed to this cell. We investigated the immunoprofile of benign MC in intraductal areas of carcinoma ex-pleomorphic adenoma (CXPA), comparing them with the MC in duct-like areas of pleomorphic adenoma, origin of the malignant tumor. Antibodies against myoepithelial markers—CK14, -SMA, calponin, P63, CD10, and D2-40—plus laminin and maspin was applied in four selected cases of intracapsular and minimal invasive CXPA with only luminal differentiation presenting areas of intraductal carcinoma. The immunohistochemical reactions of all the antibodies showed stronger staining in benign MC surrounding the malignant epithelial cells than in benign MC in duct-like areas of pleomorphic adenoma, thus revealing that in the malignization process the benign MC become differentiated and produce important proteins related to the tumor suppressor function.     

Oxygen reduces accumulation of type IV collagen in endothelial cell subcellular matrix via oxidative stress

Anchorage-dependent cells in culture attach initially to proteins adsorbed to the culture substrate from the medium, and produce and deposit a subcellular matrix during the course of the cultivation. The aim of this study was to determine whether the concentration of O(2) in the culture atmosphere affects the accumulation of type IV collagen and laminin under human endothelial-cell monolayers. Enzyme-linked immunoassays on decellularized polystyrene substrates showed less type IV collagen, but not less laminin, under cells incubated in the standard atmosphere (5% CO(2) in air, i.e., approximately 20% O(2)) compared to an atmosphere of 5% O(2) and 5% CO(2) in N(2). Type IV collagen accumulation was inhibited via oxidative stress, because the inhibitory effect of 20% O(2) was antagonized by antioxidant ascorbic acid, and mimicked by prooxidant pyrogallol and exogenous H(2)O(2). Measurements of endogenous H(2)O(2) accumulation demonstrated that endothelial cells partially adapt to the high O(2) concentration. These results may have implications in endothelium modeling in vitro and in engineering of endothelial cell sheets and endothelialized vascular grafts.     

Identification of laminin-binding motifs of Yersinia pestis plasminogen activator by phage display

Yersinia pestis plasminogen activator (Pla), a surface virulence factor contributes to the highly invasive nature of the pathogen by binding various tissue matrix components. In this study we characterised the laminin-binding site(s) of Pla via phage display and alanine-scanning mutagenesis. Previously we isolated 18 different heptamer peptide sequences from a phage display library with biopanning on laminin, and have shown that two of them with sequences of WSLLTPA or YPYIPTL completely inhibited laminin binding of a Pla-expressing recombinant Escherichia coli strain. These phages themselves strongly bound laminin in an ELISA assay utilising horseradish peroxidase-labelled anti-M 13 antibody. In the present study, with the application of synthetic peptides, a 55% and a 33% inhibition was demonstrated using WSLLTPA and YPYIPTL, respectively. Peptide pattern alignment showed two homologous regions for WSLLTPA and one for YPYIPTL inside the Pla sequence. Amino acids were changed for alanine in one of the WSLLTPA regions and in the YPYIPTL region in order to prove the role of the LTP/PTL motifs in laminin binding. Of the four constructed mutants, the triple mutant L65A-T66A-L67A in the WSLLTPA region and the double mutant G178A-L179A in the YPYIPTL region decreased the laminin binding capacity of the Pla-expressing recombinant E. coli by about 50%.     

波形蛋白与层粘连蛋白在人胚早期神经管中的表达

目的观察层粘连蛋白（LN）和波形蛋白（Vim）在人胚早期神经管发育中的表达变化,探讨人胚神经管发育早期细胞微环境的特点。方法收集早期人胚23天和45天标本8例,分别用LN和Vim抗体对人胚标本的组织切片行免疫组织化学ABC法染色。图像分析不同观测指标阳性细胞的积分光密度值,结果用t检验进行统计分析。结果人胚发育早期,LN和Vim在神经管的神经上皮层、中间层和边缘层的神经细胞胞中表达。在神经管发育到23天时,LN的积分光密度值（1258．17±635）比Vim的积分光密度值（2611．34±502）低（P〈0．05）。结论在人胚早期,神经管的细胞基质成分以Vim为主,LN为辅,可能对神经干细胞的发育有重要作用。     

血清肝纤维化指标在各型肝病诊断中的应用

探讨联合检测层粘连蛋白（LN）、IV型胶原（C IV）、血清透明质酸（HA）和Ⅲ型前胶原（PCⅢ）在肝纤维化诊断中的应用价值。采用放射免疫分析法检测143例肝病患者的LN、C IV、HA、PCⅢ,同时与41名健康体检者进行比较。结果显示：各组肝病患者血清肝纤维化指标均高于正常对照组（P〈0.01）,且与慢性肝炎病情严重程度密切相关,在慢性肝炎重度组和肝硬化组中表达最高,以HA、PCⅢ升高最为显著。结论：血清LN、C IV、HA、PCⅢ联合检测可明显提高肝纤维化诊断的准确性和可靠性,对诊断肝纤维化有较高的临床价值,但不能用于慢性肝炎重度和肝硬化的鉴别诊断。     

二乙基亚硝胺诱发大鼠肝癌过程中癌前病变阶段差异表达蛋白质的筛选

目的 通过比较二乙基亚硝胺(DEN)诱发大鼠肝细胞癌发生过程中不同阶段的蛋白质表达谱,筛选在大鼠肝癌前病变阶段起重要作用的蛋白质分子. 方法大鼠分为DEN组和正常对照组,诱癌过程中定期处死动物并取其肝组织;以γ-谷氨酰转肽酶染色阳性的肝细胞增生灶为标志,识别和获取肝癌前病变的组织标本.用双向电泳及基质辅助激光解吸飞行时间串联质谱法对大鼠正常肝组织、癌前病变组织和肝癌组织的全蛋白质组表达谱进行比较和鉴定.用Western blot和RT-PCR方法对部分差异表达蛋白质(如层黏连蛋白受体1和鲱精胺酶)进行表达水平的验证.结果 筛选出表达水平差异≥2倍的蛋白质共82种,其中在癌前病变阶段即发生明显改变的差异表达蛋白质有47种,包括在正常对照组、癌前病变组、肝癌组呈现依次上调的层黏连蛋白受体1等8种蛋白质和在上述组织中呈现依次下调的鲱精胺酶等22种蛋白质.Western blot和RT-PCR方法对层黏连蛋白受体1和鲱精胺酶表达水平的验证结果与双向电泳的结果相似.结论 大鼠肝癌形成过程中不同阶段的蛋白质表达谱有所不同,对癌前病变的差异表达蛋白质如层黏连蛋白受体1和鲱精胺酶等进行深入探讨,有望为人类肝癌的早期诊断和治疗提供线索.     

Serum laminin assayed by Slot-Blot-ELISA in patients with combined viral hepatitis C and schistosomiasis

OBJECTIVES: Hepatic schistosomiasis and chronic hepatitis C virus (HCV) are the most prevalent agents causing hepatic fibrosis in humans. Laminin (LA) has been related to liver fibrosis and subsequent development of portal hypertension in chronic liver disease. There are no available data describing the pattern of laminin in combined HCV and schistosoma-infected patients, thus the rationale of this study was to assess the serum LA as an index of liver fibrosis in patients with schistosomiasis and/or chronic viral hepatitis C and to evaluate a developed Slot-Blot Enzyme-Linked Immunosorbant Assay (Slot-Blot-ELISA) as a method of estimation. DESIGNS AND METHODS: This study included four groups: group I included 34 patients with schistosomiasis, group II included 58 patients infected with HCV, group III included 68 patients with combined chronic viral hepatitis C and schistosomiasis and group IV included 50 healthy individuals who served as a control group. Serum LA was measured in the different groups quantitatively by ELISA and semi-quantitatively by Slot-Blot-ELISA. RESULTS: Significantly higher serum LA concentrations measured by ELISA were found in patients with combined chronic viral hepatitis C and schistosomiasis than in patients with either chronic HCV (P = 0.005) or schistosomiasis (P < 0.001) alone. Serum LA was significantly higher in the patient groups than the control group (P < 0.001). Serum LA concentration was positively correlated with fibrosis grading scores. Semi-quantitative results of serum LA using the developed SB-ELISA were found to have approximately the same power of ELISA results in different groups. The overall sensitivity, specificity, positive predictive value, negative predictive value and efficiency of ELISA for estimation of serum LA were 85.6%, 84.0%, 94.5%, 64.6% and 90%, respectively and for SB-ELISA were 87.5%, 82.0%, 94%, 67.2% and 88%, respectively. CONCLUSIONS: Serum LA was significantly increased in patients coinfected with HCV and Schistosoma mansoni. The newly developed Slot-Blot-ELISA is a simple, rapid and highly sensitive assay for detection of LA in hepatic fibrosis. Moreover, all steps were performed at room temperature without the need to use expensive equipment, and this may enhance the application of this assay in screening programs. Further study is warranted for confirmation of SB-ELISA reproducibility in a large population.