Laminin assembles into separate basement membrane and fibrillar matrices in Schwann cells.
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Laminins are important for Schwann cell basement membrane assembly and axonal function. In this study, we found that exogenous laminin-1, like neuromuscular laminins-2/4, formed two distinct extracellular matrices on Schwann cell surfaces, each facilitated by laminin polymerization. Assembly of one, a densely-distributed reticular matrix, was accompanied by a redistribution of cell-surface dystroglycan and cytoskeletal utrophin into matrix-receptor-cytoskeletal complexes. The other, a fibrillar matrix, accumulated in separate zones associated with pre-existing beta1-integrin arrays. The laminin-1 fragment E3 (LG-modules 4-5), which binds dystroglycan and heparin, inhibited reticular-matrix formation. By contrast, beta1-integrin blocking antibody (Ha2/5) prevented fibrillar assembly. Ultrastructural analysis revealed that laminin treatment induced the formation of a linear electron-dense extracellular matrix (lamina densa) separated from plasma membrane by a narrow lucent zone (lamina lucida). This structure was considerably reduced with non-polymerizing laminin, fully blocked by E3, and unaffected by Ha2/5. Although it formed in the absence of type IV collagen, it was nonetheless able to incorporate this collagen. Finally, cell competency to bind laminin and form a basement membrane was passage-dependent. We postulate that laminin induces the assembly of a basement membrane on competent cell surfaces probably mediated by anchorage through LG 4-5. Upon binding, laminin interacts with dystroglycan, mobilizes utrophin, and assembles a 'nascent' basement membrane, independent of integrin, that is completed by incorporation of type IV collagen. However, the fibrillar beta1-integrin dependent matrix is unlikely to be precursor to basement membrane. (+info)
The major structural subunits of Dr and F1845 fimbriae are adhesins.
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Fimbrial adhesins mediate the attachment of pathogenic Escherichia coli to various host tissues leading to the development of disease. The Dr hemagglutinin and F1845 fimbriae belong to the Dr family of adhesins, which is associated with urinary tract infections and diarrheal disease. These adhesins bind to the Dr(a) blood-group antigen present on decay-accelerating factor (DAF). The Dr hemagglutinin is unique in this family since it also binds to type IV collagen and its binding is inhibited by the presence of chloramphenicol. We have purified the major structural subunits of Dr and F1845 fimbriae, DraE and DaaE, as fusions to maltose-binding protein and to oligohistidine tags and examined their binding to erythrocytes, Chinese hamster ovary cell transfectants expressing DAF, and a DAF fusion protein. The DraE and DaaE fusion proteins bind to the DAF receptor in a specific manner resembling the distinct phenotypes of the corresponding Dr and F1845 fimbriae. In contrast to binding studies with the DAF receptor, the DraE fusion proteins did not bind to type IV collagen. (+info)
Expression of extracellular matrix proteins and vimentin in testes of azoospermic man: an immunohistochemical and morphometric study.
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AIM: To investigate the changes in the extracellular matrix protein expression and the morphology of seminiferous tubules in the testis of 88 azoospermic men. METHODS: The patients were of the following categories: (1) 22 cases of Sertoli-cell-only syndrome, (2) 20 cases of spermatogenic arrest, and (3) 46 cases with hypospermatogenesis. Testicular sections were immunohistochemically stained for fibronectin, vimentin, laminin and collagen type IV. The seminiferous tubular diameter and the connective matrix zone (CMZ, the acellular zone between the basement membrane [BM] and the peritubular cells) thickness were measured. Seminiferous tubules were typed according to the thickness of the connective matrix in the lamina propria. The predominant tubule type and the Johnsen and Silber scores were determined. RESULTS: The mean tubular diameter were 119 +/- 27, 117 +/- 20, and 140 +/- 38 microm for Groups 1, 2, and 3, respectively. Both the laminin and the type IV collagen were localized to the epithelial BM and peritubular cells. In most of the tubules, BM and peritubular cells were separated by a homogenous acellular layer, the CMZ, in which laminin, type IV collagen, fibronectin and vimentin were not present. It is perceived that the worse the testicular histology, the higher the thickness of the CMZ. CONCLUSION: In testis with no or low sperm production, the diameter of the seminiferous tubules is decreased, the thickness of the seminiferous tubular wall is increased and a CMZ is formed between the peritubular cells and the BM. The thickness of CMZ is increasing with the advancement of testiclar deterioration. The most important morphologic predictive factor for spermiogenesis is the predominant (+info)
Up-regulation of MMP-2 and MMP-9 leads to degradation of type IV collagen during skeletal muscle reperfusion injury; protection by the MMP inhibitor, doxycycline.
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OBJECTIVES: to determine the role of matrix metalloproteinases, MMP-2 and MMP-9, in reperfusion injury following skeletal muscle ischaemia and whether inhibition of MMPs by doxycycline protects against tissue damage. METHODS: rats were anaesthetised and a tourniquet applied to the proximal thigh to occlude blood flow. Four hours of ischaemia was followed by reperfusion for 0, 4, 24 or 72 h. Two further groups received doxycycline for 7 days prior to bilateral ischaemia and 24 h reperfusion. Skeletal muscle from both limbs, kidneys and lungs were harvested for zymography and immunohistochemical staining for type IV collagen. RESULTS: upregulation of MMP-2 and MMP-9 was detected by zymography in the ischaemic leg and lung but not in the kidney. Quantitative immunohistochemical analysis showed marked degradation of type IV collagen in reperfused muscle, lung and kidney. Doxycycline-treated rats showed significant preservation of type IV collagen in skeletal muscle and a trend towards preservation in kidney and lung. CONCLUSIONS: MMP-2 and MMP-9 are strongly upregulated in skeletal muscle ischaemia/reperfusion injury and are also upregulated in remote organs, leading to degradation of basement membranes. Inhibition of MMP activity may therefore be potentially therapeutically useful in reducing the severity of reperfusion injury. (+info)
Urinary type IV collagen: a specific indicator of incipient diabetic nephropathy.
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OBJECTIVE: To determine whether urinary type IV collagen can serve as an indicator specific for diabetic nephropathy. METHODS: Using a novel sandwich ABC-ELISA to measure type IV collagen directly, the 24-hour urinary type IV collagen excretion rate was determined in 120 diabetic patients and some groups of controls. Urinary albumin determinations were made with a RIA kit at the same time. A total of 13 diabetic patients with microalbuminuria underwent percutaneous renal biopsy for definitive diagnosis of diabetic nephropathy. Type IV collagen and TGF-beta 1 immunoreactivities were detected with ABC methods in renal biopsies. RESULTS: Urinary type IV collagen excretion was significantly increased in diabetic patients with microalbuminuria, especially those with albumin excretion above 200 mg/24 h. By comparison, collagen excretion was equivalent to that in healthy controls when measured in diabetics with normalbuminuria and in patients with primary glomerular disease, primary hypertension, or coronary heart disease. Urinary type IV collagen excretion in diabetics was negatively correlated with creatinine clearance. In renal biopsies from subjects with elevated collagen excretion, the glomeruli showed pathological changes typical of diabetic nephropathy. Also, excessive type IV collagen and TGF-beta 1 immunoreactivity were detected in the glomeruli, Bowman's capsule and interstitium. CONCLUSIONS: Excretion of type IV collagen, possibly reflecting increased production or decreased degradation of this protein, may be a clinically useful indicator of incipient diabetic nephropathy. (+info)
Regulation of fibronectin alternative splicing by a basement membrane-like extracellular matrix.
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Hepatocytes are the source of plasma fibronectin (FN) which lacks the alternatively spliced EDI segment, distinctive of oncofetal FN. When hepatic or other epithelial cells are cultured on plastic, EDI inclusion is triggered. Here we report that EDI inclusion is inhibited when hepatic cells are cultured on a basement membrane-like extracellular matrix (ECM), demonstrating a new role for the ECM in the control of gene expression. The effect is duplicated by collagen IV and laminin but not by collagen I; is not observed with another alternatively spliced FN exon (EDII); and correlates with a decrease in cell proliferation, consistently with high EDI inclusion levels observed in many physiological and pathological proliferative processes. (+info)
Antibody response against perlecan and collagen types IV and VI in chronic renal allograft rejection in the rat.
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Chronic rejection is the leading cause of late renal transplant failure. Various structural lesions are observed in grafts undergoing chronic rejection including glomerular basement membrane (GBM) duplications. The well-established Fisher (F344) to Lewis (LEW) rat renal transplant model for chronic rejection was used to assess the presence and role of the humoral immune response against graft antigens during chronic rejection. LEW recipients of F344 allografts develop transplant glomerulopathy and produce IgG1 antibodies directed against F344 GBM preparations that are detectable 3 weeks after transplantation. Glomerular IgG1 deposition was observed that in vitro co-localized with a rabbit anti-rat GBM antiserum in rejecting F344 grafts; elution experiments of isolated glomeruli yielded IgG1 antibodies reactive in vitro with F344 GBM, but not LEW GBM. Prevention of acute rejection by transient treatment of the recipients with cyclosporin A completely abrogated the production of anti-GBM antibodies. Using proteomic techniques we identified the antigens recognized by the LEW posttransplant sera as being the heparan sulfate proteoglycan perlecan and the alpha1 chain of collagen type VI in association with the alpha5 chain of collagen type IV. In conclusion, LEW recipients of F344 kidney grafts produce IgG1 antibodies against donor type perlecan and alpha1(VI)/alpha5(IV) collagen and develop transplant glomerulopathy. These data implicate an important role for the humoral immune response in the development of glomerulopathy during chronic rejection. (+info)
The LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes.
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Patients with nail-patella syndrome often suffer from a nephropathy, which ultimately results in chronic renal failure. The finding that this disease is caused by mutations in the transcription factor LMX1B, which in the kidney is expressed exclusively in podocytes, offers the opportunity for a better understanding of the renal pathogenesis. In our analysis of the nephropathy in nail-patella syndrome, we have made use of the Lmx1b knockout mouse. Transmission electron micrographs showed that glomerular development in general and the differentiation of podocytes in particular were severely impaired. The glomerular capillary network was poorly elaborated, fenestrae in the endothelial cells were largely missing, and the glomerular basement membrane was split. In addition podocytes retained a cuboidal shape and did not form foot processes and slit diaphragms. Expression of the alpha4 chain of collagen IV and of podocin was also severely reduced. Using gel shift assays, we demonstrated that LMX1B bound to two AT-rich sequences in the promoter region of NPHS2, the gene encoding podocin. Our results demonstrate that Lmx1b regulates important steps in glomerular development and establish a link between three hereditary kidney diseases: nail-patella syndrome (Lmx1b), steroid-resistant nephrotic syndrome (podocin), and Alport syndrome (collagen IV alpha4). (+info)