Basement membranes in fetal,adult normal,hyperplastic and neoplastic human prostate

Summary The distribution of the various basement membrane (BM) components (type IV collagen, laminin and heparan sulphate proteoglycan) was studied in fetal, adult normal, hyperplastic and neoplastic prostates in formalin- and ethanol-fixed paraffin-embedded specimens. Stromal, epithelial and neoplastic BMs expressed differential susceptibility to pepsin treatment, suggesting conformational differences in the expression of epitopes on BM proteins in distinct anatomical structures and various lesions of the human prostate. In fetal prostate the acinar BM was regular and continuous in contrast to normal adult prostate and various hyperplastic conditions where the acinar BM was locally thickened or unreactive to the anti-BM antibodies. The localization pattern of BM components in grade I and grade II phases of prostatic cancer did not differ essentially from those found in various hyperplastic lesions. Regardless of the histopathological grade of malignancy, prostatic carcinoma cells were surrounded by distinct pericellular and periacinar membranes which were present even at points of contact with the stroma. This suggests that stroma invasion is invariably associated with neoplastic BM formations. Immunohistochemical evidence of the stromal or epithelial origin of neoplastic BMs could not be found. However, the consistent extracellular distribution of neoplastic BM components in contact with the stroma indicates that the elaboration of BM material requires a stromal influence.     

Multi-Molecular Gradients of Permissive and Inhibitory Cues Direct Neurite Outgrowth

Correct development of neuronal tracts requires the coordination of multiple permissive and inhibitory signals. By generating an in vitro microenvironment using soft lithography and microfluidic techniques, multiple guidance cues can be presented in a spatially defined way. Here we evaluated how neurites of dorsal root ganglia neurons responded to permissive and inhibitory cues presented by substrate-bound molecular gradients. Linear gradients containing inhibitory chondroitin sulfate proteoglycan (CSPG) and/or permissive laminin-1 (LN) were generated as single-cue gradients, parallel double-cue gradients, and opposing double-cue gradients with varying slopes. Neurite growth was analyzed using circular statistical methods, and for all gradients examined, neurons extended neurites toward regions of lower CSPG and higher LN concentrations. Single-cue gradients elicited similarly directed neurite growth responses at the higher concentrations tested for both LN and CSPG, and both gradient slope and fractional concentration change affected neurite growth. When the two contrasting molecular cues were presented together, neurites responded differently depending on the directions of the gradients. Neurite growth on LN-CSPG double gradients of opposite direction was strongly directed, while neurite growth on LN-CSPG double gradients of parallel direction was uniform. These results represent an important step toward understanding how neurite growth is guided by complex microenvironments containing multiple molecular cues.     

Ewing's sarcoma lines synthesize laminin and fibronectin

Summary Immunoelectron microscopy was employed to detect laminin and fibronectin cell surface expression on five Ewing's sarcoma lines plus a normal fibroblast line as control. Monospecific antibodies to both glycoproteins were detected on tumour cell and fibroblast layers with colloidal gold - protein A conjugates. All five tumour lines were positive for fibronectin and/or laminin, whereas the fibroblast line expressed fibronectin only, as expected. Fibronectin displayed a dense granular pattern, typically in the cell-cell and cell-matrix adhesion areas; laminin displayed a punctate pattern.³H-leucine metabolical labelling was also used to demonstrate laminin and fibronectin synthesis. The labelled proteins released in the culture media were separated by molecular weight on SDS-PAGE and identified by immunoprecipitation with the monospecific antibodies. The results substantiated the immunoelectron microscopy data. These findings indicate that Ewing's sarcoma lines produce a complex extracellular matrix including fibronectin and laminin, in addition to the collagens described by other workers. Histogenetic classification of this tumour in terms of extracellular matrix proteins synthesis is thus more difficult than has been supposed. The same complexity must also be borne in mind when using the matrix components as an aid to Ewing's sarcoma differentiation from other childhood tumours.This work was supported by Italian CNR Grant PFO 85002253.44     

Severe congenital muscular dystrophy in a Mexican family with a new nonsense mutation (R2578X) in the laminin α-2 gene

 The congenital muscular dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders. Approximately one half of cases diagnosed with classic CMD show primary deficiency of the laminin α2 chain of merosin. Complete absence of this protein is usually associated with a severe phenotype characterized by drastic muscle weakness and characteristic changes in white matter in cerebral magnetic resonance imaging (MRI). Here we report an 8-month-old Mexican female infant, from a consanguineous family, with classical CMD. Serum creatine kinase was elevated, muscle biopsy showed dystrophic changes, and there were abnormalities in brain MRI. Immunofluorescence analysis demonstrated the complete absence of laminin α2. In contrast, expression of α-, β-, γ-, and δ-sarcoglycans and dystrophin, all components of the dystrophin–glycoprotein complex, appeared normal. A homozygous C  T substitution at position 7781 that generated a stop codon in the G domain of the protein was identified by mutation analysis of the laminin α2 gene (LAMA2). Sequence analysis on available DNA samples of the family showed that parents and other relatives were carriers of the mutation. Received: August 22, 2002 / Accepted: November 11, 2002 Acknowledgments The authors wish to thank Dr. Pascale Guicheney (INSERMU523, Institute de Myologie), who kindly provided us with the primers' sequences and PCR conditions to amplify exons of LAMA2. This work was supported by CONACYT (Mexico) grant 34603-M, and Fondo de Fomento para la Investigacion-IMSS (Mexico) grant FP-0038/764. All DNA sequencing was carried out at the Centro de Instrumentos del Instituto Mexicano del Seguro Social (IMSS), Mexico City. The first two authors contributed equally to this work. Correspondence to:R.M. Coral-Vazquez     

Asbestos induced fibrosis in the omentum of rats

Summary Fibrosis and neoplasia are evoked by asbestos fibres. Different experimental models have been used to elucidate the cellular mechanism of their pathogenesis but there is no report available dealing with the role of structural glycoproteins and collagens in the development of the fibrosis. The omentums of 20 female SPF-Sprague-Dawley rats were investigated 1, 2 and 6 months after intraperitoneal injection of 15 mg UICC reference samples of crocidolite, by light- and immunofluorescence microscopy. Using monospecific antibodies, laminin, collagens types I and III and fibronectin were localized. After 6 months typical asbestos bodies were detected. By indirect marking of the basal lamina with anti-laminin-antibodies the marked degree of vessel proliferation occurring during the development of granuloma became visible. The deposition of connective tissue which was already established after 4 weeks was mainly due to collagen type III. After 4 and 8 weeks an accumulation of fibronectin associated with larger asbestos fibres was observed. The results suggest that fibrogenesis is promoted by the opsonic activity of fibronectin for long asbestos fibres. The fibrosis may derive from activated resident fibroblasts.     

Appearance and distribution of laminin a chain isoforms and integrin α2, α3, α6, β1, and β4 subunits in the developing human small intestinal mucosa

Background: Laminin, a major component of basement membranes, is well known in its classical heterotrimeric form (B1-A-B2) to regulate diverse biological functions, including cell polarization and differentiation. However, the role of merosin, a laminin-like molecule in which an M chain is substituted for its homologous A chain, remains largely unknown. Methods: In the present study, we analyzed by indirect immunofluorescence the expression and distribution of these four laminin chains as well as the integrins α2β1, α3β1,α6β1, and α6β4, four potential recptors, at the epithelial-mesenchymal interface of the developing human small intestine, with a panel of specific monoclonal antibodies. Results: Beginning at 7 weeks of gestation and throughout mucosal organogenesis, the B1 and B2 chains were uniformly detected at the epithelial basement membrane. The A chain also was detected beginning at 7 weeks, and its distribution at the basement membrane remained uniform throughout villus (9+ weeks) and crypt (16+ weeks) formation. In contrast, M chain expression was not observed until 16 weeks; between 16 and 20 weeks, it was exclusively associated with the base of epithelial cells that comprised the forming crypts. Integrins α6β1 and α6β4, as determined by their subunit immunolocalization, appeared to be expressed by all enterocytes from 7 to 20 weeks. In contrast, the expression of the α2β1 and α3β1 integrins was found time- and site-restricted. The α2 subunit was predominantly detected in the epithelial cells of the intervillous area and its derivative, the crypt, whereas the α3 subunit was strongly expressed by all epithelial cells except those located at the bottom of 19–20-week-old crypts. Conclusions: Taken together, these observations demonstrate that both compositional changes in the basement membrane and differential expression of receptors occur during human intestinal organogenesis, suggesting that epithelial cell-matrix interactions play a role during development. © 1995 Wiley-Liss, Inc.     

病毒性肝炎患者血清层粘连蛋白检测及其诊断价值的探讨

应用放射免疫方法测定了１６６例病毒性肝炎患者血清层粘连蛋白（ＬＮ）水平。结果从急性肝炎→慢性肝炎→肝硬化，血清ＬＮ逐步升高，慢性活动性肝炎及肝硬化患者分别为１４４．８４±３８．９０及１７１．１３±４０．４１μｇ／Ｌ，均较正常对照组１１６．６４±１９．８５μｇ／Ｌ显著升高。根据ＲＯＣ曲线分析，以１６０μｇ／Ｌ为临界值诊断肝硬化，灵敏度６９．９％，特异性８４．３％，准确性８１．１％。且血清ＬＮ水平与透     

层黏连蛋白促进人晶状体上皮细胞蛋白激酶B的活性

目的:探讨层黏连蛋白(Ln)对传代培养的人晶状体上皮细胞(hLECs)的蛋白激酶B(PKB)活性的影响,以及Ln激活PKB的信号分子途径。方法:选取第5代hLECs,分为对照组和处理组,在处理组的培养液中加入层黏连蛋白,终浓度为5mg/L。分别在处理后0,10,20,40,60min取出细胞,[γ-32P]-ATP掺入法测定胞膜和胞质PKB比活性。以PI3K的特异性抑制剂Wortmannin(终浓度100nmol/L)预处理1h,再加入层黏连蛋白,0,10,20,40,60min测定胞膜和胞质PKB比活性。结果:hLECs胞膜和胞质PKB比活性在层黏连蛋白作用40min时达到最高值,分别为空白对照的2.72和2.00倍。层黏连蛋白处理的各组胞膜和胞质PKB比活性显著高于对照组(P<0.05)。经Wortmannin预处理后,再加入层黏连蛋白,胞膜和胞质PKB比活性与未经预处理的各时间组相比均显著下降(P<0.05)。结论:层黏连蛋白通过PI3K途径激活人晶状体上皮细胞的PKB活性,比活性随时间变化而发生改变。     

肺癌患者化疗前后血清LN、CoIV水平变化及其临床意义

目的　探讨血清LN、CoIV在肺癌患者化疗前后水平变化及其临床价值。方法　应用放射免疫分析法 (RIA)检测 30例肺癌患者化疗前后血清中LN、CoIV水平 ,以 2 0例健康献血员及体检正常者为对照。结果　肺癌组层粘蛋白 (LN)、Ⅳ型胶原 (CoIV)明显高于对照组 ,化疗后有效者 (完全和部分缓解 )血清中LN、CoIV水平明显低于化疗前。结论　动态检测血清LN、CoIV水平对评价疗效有一定价值。     

Thy-1 is a neuronal and glial surface antigen which interacts with matrix proteins and plasminogen activator

Summary Thy-1 antigen is expressed at high levels in the thymus and in adult brain of rodents however its function remains undetermined. We report that immobilised Thy-1 binds laminin, fibronectin and the less active precursor form of the tissue type plasminogen activator (t-PA) yet it does not bind urokinase. The incorporation of serine protease inhibitors within the experimental procedures suggested that Thy-1 bound to the lysine-containing, protein-binding domain of t-PA thus leaving the active site available to interact with other proteins. By using an immunocytochemical approach designed to maximally preserve Thy-1 antigenicity, we were able to demonstrate that in the adult rat peripheral nervous system (PNS) Thy-1 was seen to co-localise with laminin on the Schwann cell membranes and accumulated at the nodes of Ranvier within sciatic nerve. The only neuronal structures to express Thy-1 within the PNS were the unmyelinated nerve fibres. In the adult rat central nervous system (CNS), the most distinct and novel association of Thy-1 was its presence along the myelin forming glial cells and their fibres.