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1.
We had developed a conditional Laminin α 1 knockout-mouse model (Lama1cko) bypassing embryonic lethality of Lama1 deficient mice to study the role of this crucial laminin chain during late developmental phases and organogenesis. Here, we report a strong defect in the organization of the adult cerebellum of Lama1cko mice. Our study of the postnatal cerebellum of Lama1cko animals revealed a disrupted basement membrane correlated to an unexpected excessive proliferation of granule cell precursors in the external granular layer (EGL). This was counteracted by a massive cell death occurring between the postnatal day 7 (P7) and day 20 (P20) resulting in a net balance of less cells and a smaller cerebellum. Our data show that the absence of Lama1 has an impact on the Bergmann glia scaffold that aberrantly develops. This phenotype is presumably responsible for the observed misplacing of granule cells that may explain the overall perturbation of the layering of the cerebellum and an aberrant folia formation.  相似文献   

2.
We had developed a conditional Laminin α1 knockout-mouse model (Lama1cko) bypassing embryonic lethality of Lama1 deficient mice to study the role of this crucial laminin chain during late developmental phases and organogenesis. Here, we report a strong defect in the organization of the adult cerebellum of Lama1cko mice. Our study of the postnatal cerebellum of Lama1cko animals revealed a disrupted basement membrane correlated with an unexpected excessive proliferation of granule cell precursors in the external granular layer (EGL). This was counteracted by a massive cell death occurring between the postnatal day 7 (P7) and day 20 (P20) resulting in a net balance of less cells and a smaller cerebellum. Our data show that the absence of Lama1 has an impact on the Bergmann glia scaffold that aberrantly develops. This phenotype is presumably responsible for the observed misplacing of granule cells that may explain the overall perturbation of the layering of the cerebellum and an aberrant folia formation.Key words: cerebellum, development, laminin, cell migration, laminin-111  相似文献   

3.
Cell adhesive peptides have been widely applied for therapeutic drugs, drug delivery systems, and biomaterials. Previously, we identified various cell adhesive sequences in the G domains of four laminin α chains (α2-α5) by the systematic soluble peptide screening. We also identified five cell-binding sequences in the laminin α1 chain G domain using synthetic peptide-polystyrene beads. Here, we re-screened cell adhesive peptides in the laminin α1 chain G domain by the systematic soluble peptides screening. The 110 soluble peptides were evaluated for their cell adhesive activities using human fibrosarcoma HT1080 cells and human dermal fibroblasts. Fourteen peptides were newly identified as a cell adhesive. Additionally, four peptides (AG22: SSFHFDGSGYAM, AG42: TFDLLRNSYGVRK, AG76: HQNQMDYATLQLQ, AG86: LGGLPSHYRARNI) promoted integrin-mediated cell adhesion. Further, neurite outgrowth activity with rat pheochromocytoma PC12 cells was evaluated and two peptides (AG20: SIGLWNYIEREGK, AG26: SPNGLLFYLASNG) were newly identified for neurite outgrowth activity. These results suggested that the systematic soluble peptides screening approach is an accurate and powerful strategy for finding biologically active sequences. The active sequences newly identified here could be involved in the biological functions of this domain. The active peptides are useful for evaluating molecular mechanisms of laminin-receptor interactions and for developing cell adhesive biomaterials.  相似文献   

4.
Laminins, a multifunctional protein family of extracellular matrix, interact with various types of integrin. Here, integrin-mediated cell adhesive peptides have been systematically screened in the laminin α4 and α5 chain G domain peptide library consisting of 211 peptides by both the peptide-coated plastic plates and peptide-conjugated Sepharose bead assays using human dermal fibroblasts. Thirteen peptides promoted cell spreading and the activity was specifically inhibited by EDTA. Cell attachment to 11 peptides was inhibited by anti-integrin β1 antibody. Additionally, cell attachment to the A5G81 (AGQWHRVSVRWG) and A5G84 (TWSQKALHHRVP) peptides was specifically inhibited by anti-integrin α3 and α6 antibodies. These results suggest that the A5G81 and A5G84 peptides promote integrin α3β1- and α6β1-mediated cell attachment. Further, most of the integrin-mediated cell adhesive peptides are located in the loop regions in the G domains, suggesting that structure is important for the integrin specific recognition. Integrin binding peptides are useful for understanding laminin functions and have a potential to use for biomaterials and drug development.  相似文献   

5.
The biological activities of the laminin α2 chain LG4–5 module result from interactions with cell surface receptors, such as heparan sulfate proteoglycans and α-dystroglycan. In this study, heparin and α-dystroglycan binding sequences were identified using 42 overlapping synthetic peptides from the LG4–5 module and using recombinant LG4–5 protein (rec-α2LG4–5). Physiological activities of the active peptides were also examined in explants of submandibular glands. Heparin binding screens showed that the A2G78 peptide (GLLFYMARINHA) bound to heparin and prevented its binding to rec-α2LG4–5. Furthermore, alanine substitution of the arginine residue in the A2G78 site on rec-α2LG4–5 decreased heparin binding activity. When α-dystroglycan binding of the peptides was screened, two peptides, A2G78 and A2G80 (VQLRNGFPYFSY), bound α-dystroglycan. A2G78 and A2G80 also inhibited α-dystroglycan binding of rec-α2LG4–5. A2G78 and A2G80 specifically inhibited end bud formation of submandibular glands in culture. These results suggest that the A2G78 and A2G80 sites play functional roles as heparan sulfate- and α-dystroglycan-binding sites in the module. These peptides are useful for elucidating molecular mechanisms of heparan sulfate- and/or α-dystroglycan-mediated biological functions of the laminin α2 chain.  相似文献   

6.
The polymerization of laminin into a cell-associated network--a key step in basement membrane assembly--is mediated by the laminin amino-terminal (LN) domains at the tips of the three short arms of the laminin αβγ-heterotrimer. The crystal structure of a laminin α5LN-LE1-2 fragment shows that the LN domain is a β-jelly roll with several elaborate insertions that is attached like a flower head to the stalk-like laminin-type epidermal growth factor-like tandem. A surface loop that is strictly conserved in the LN domains of all α-short arms is required for stable ternary association with the β- and γ-short arms in the laminin network.  相似文献   

7.
The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.  相似文献   

8.
The N-terminal domains VI plus V (62 kDa) and V alone (43 kDa) of the laminin α1 chain were obtained as recombinant products and shown to be folded into a native form by electron microscopy and immunological assays. Domain VI alone, which corresponds to an LN module, did not represent an autonomously folding unit in mammalian cells, however. Fragment α1VI/V, but not fragment α1V, bound to purified α1β1 and α2β1 integrins, to heparin, and to heparan sulfate-substituted domains I and V of perlecan. This localized the binding activities to the LN module, which contains two basic sequences suitable for heparin interactions.  相似文献   

9.
10.
The basement membrane (BM) proteins laminins, which consist of alpha, beta and gamma chains, play critical roles in the maintenance of tissue structures. One of laminin alpha chains, alpha3 has two isoforms, the truncated form alpha3A and the full-sized form alpha3B. In contrast to alpha3A laminins, little is known about alpha3B laminins. To show the histological distribution of the laminin alpha3B chain, we prepared alpha3B-specific monoclonal antibodies. Immunohistochemical analysis showed that the alpha3B chain was colocalized with the alpha3A, beta3 and gamma2 chains in the epithelial BMs of the skin, esophagus, breast and lung, suggesting the presence of laminin-3B32 (laminin-5B) and laminin-3A32 (laminin-5A). In the lung alveoli, laminin-3B32 was dominant over laminin-3A32, but vice versa in other epithelial BMs. In contrast, the BMs of blood vessels including capillaries were strongly positive for alpha3B, but almost or completely negative for alpha3A, beta3 and gamma2. alpha3B was colocalized with beta1 and gamma1 in these BMs. The alpha3B chain was scarcely detected in the vessels of malignant skin cancers, though the gamma2 and beta3 chains were highly expressed in the cancer cells. These results strongly suggest that the laminin alpha3B chain is widely expressed in vascular BMs of normal tissues, probably as laminin-3B11/3B21 (laminin-6B/7B).  相似文献   

11.
Melanoma cells express and interact with laminins (LMs) and other basement membrane components during invasion and metastasis. In the present study we have investigated the production and migration-promoting activity of laminin isoforms in melanoma. Immunohistochemistry of melanoma specimens and immunoprecipitation/western blotting of melanoma cell lines indicated expression of laminin-111/121, laminin-211, laminin-411/421, and laminin-511/521. Laminin-332 was not detected. In functional assays, laminin-111, laminin-332, and laminin-511, but not laminin-211 and laminin-411, strongly promoted haptotactic cell migration either constitutively or following stimulation with insulin-like growth factors. Both placenta and recombinant laminin-511 preparations were highly active, and the isolated recombinant IVa domain of LMα5 also promoted cell migration. Function-blocking antibodies in cell migration assays revealed α6β1 integrin as the major receptor for laminin-111, and both α3β1 and α6β1 integrins for laminin-332 and laminin-511. In contrast, isolated LMα5 IVa domain-promoted melanoma cell migration was largely mediated via αVβ3 integrin and inhibited by RGD peptides. Given the ubiquitous expression of α5 laminins in melanoma cells and in melanoma-target tissues/anatomical structures, as well as the strong migration-promoting activity of these laminin isoforms, the α5 laminins emerge as putative primary extracellular matrix mediators of melanoma invasion and metastasis via α3β1 and other integrin receptors.  相似文献   

12.
The molecular weights calculated from the amino acid sequences of the A and B chains of the lens protein -crystallin differ only slightly (19830 and 20070, respectively). SDS gel electrophoresis of these chains and comparison with marker proteins yield apparent molecular weights of 19500 for A and 22500 for B. The discrepancy between the value of 22500 and the real molecular weight of 20070 for B vanishes by the combined use of SDS and 6 M urea in the polyacrylamide gels.  相似文献   

13.
14.
《FEBS letters》1997,412(1):211-216
Assembly of Drosophila laminin α, β and γ chains was analyzed by immunoprecipitation of the lysate from metabolically radiolabeled Kc 167 cells with chain-specific antibodies followed by two dimensional electrophoresis in which non-reducing and reducing SDS gel electrophoresis are combined. Precipitation of monomeric β (or γ) with anti-γ (or -β) antibody revealed that β and γ form stable dimer before they are disulfide-bonded to each other. In contrast, α associates with neither monomeric β, monomeric γ nor βγ dimer without disulfide-bonding but only with disulfide-bonded βγ dimer to form αβγ trimers. These results thus demonstrated that the interchain disulfide-boding between β and γ is essential for α to form αβγ trimer. We also found that the αβγ trimer can be secreted with α chain either disulfide-bonded or not bonded to the disulfide-bonded βγ dimer.  相似文献   

15.
Laminins are the major glycoproteins present in all basement membranes. Previously, we showed that perlecan is present during human development. Although an overview of mRNA-expression of the laminin β1 and β2 chains in various developing fetal organs is already available, a systematic localization of the laminin β1 and β2 chains on the protein level during embryonic and fetal human development is missing. Therefore, we studied the immunohistochemical expression and tissue distribution of the laminin β1 and β2 chains in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin β1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin β2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development.  相似文献   

16.
17.
Zhang Z  Zhang P  Hu H 《PloS one》2011,6(4):e19080
Mutations in genes encoding glycosyltransferases (and presumed glycosyltransferases) that affect glycosylation and extracellular matrix binding activity of α-dystroglycan (α-DG) cause congenital muscular dystrophies (CMDs) with central nervous system manifestations. Among the identified genes, LARGE is of particular interest because its overexpression rescues glycosylation defects of α-DG in mutations of not only LARGE but also other CMD-causing genes and restores laminin binding activity of α-DG. It is not known whether LARGE protein glycosylates other proteins in addition to α-DG. In this study, we overexpressed LARGE in DG-deficient cells and analyzed glycosylated proteins by Western blot analysis. Surprisingly, overexpression of LARGE in α-DG-deficient cells led to glycosylation dependent IIH6C4 and VIA4-1 immunoreactivity, despite the prevailing view that these antibodies only recognize glycosylated α-DG. Furthermore, the hyperglycosylated proteins in LARGE-overexpressing cells demonstrated the functional capacity to bind the extracellular matrix molecule laminin and promote laminin assembly at the cell surface, an effect that was blocked by IIH6C4 antibodies. These results indicate that overexpression of LARGE catalyzes the glycosylation of at least one other glycoprotein in addition to α-DG, and that this glycosylation(s) promotes laminin binding activity.  相似文献   

18.
《FEBS letters》1996,396(1):37-42
The laminin α1 chain carboxyl-terminal globular domain (G domain) contains multiple biological activities. Recently, we identified five cell binding sequences from the G domain by screening with overlapping 12-mer peptides encompassing the entire domain. The structures of these five sequences in the α1 chain are conserved in the corresponding regions of the different laminin α chains. Here we characterize the adhesion activities of the corresponding peptide segments from both the mouse laminin α2 chain and Drosophila laminin α chain using peptide-coated plastic plates and peptide-conjugated Sepharose beads. Using several cell lines, the laminin α2 chain peptides showed cell attachment and/or spreading activities with cell type specificities. Cell spreading on MG-10 was inhibited by integrin antibodies. Four of the Drosophila laminin peptides showed cell attachment activities. These results suggest that biologically active regions in the G domain are conserved in the laminin α1 and α2 chains, and that these regions in laminin play an important role in cell surface receptor interactions.  相似文献   

19.
The dramatic increase in bacterial resistance over the past three decades has greatly reduced the effectiveness of nearly all clinical antibiotics, bringing infectious disease to the forefront as a dire threat to global health. To combat these infections, adjuvant therapies have emerged as a way to reactivate known antibiotics against resistant pathogens. Herein, we report the evaluation of simplified α-pyrone adjuvants capable of potentiating penicillin G against Pseudomonas aeruginosa, a Gram-negative pathogen whose multidrug-resistant strains have been labeled by the Centers for Disease Control and Prevention as a serious threat to public health.  相似文献   

20.
As part of our ongoing programme for isolation of bioactive molecules from the flora of the Indo-Burma biodiversity belt, an unusual long chain alkylated α-methylene-γ-butyrolactone was isolated from the juice of ripe fruit of Artabotrys odoratissimus R.Br. Its structure was determined as 3-methylene-4-pentadecyldihydrofuran-2-one by spectroscopic methods. It was found to have good antifungal activity against Alternaria tenuissima Kunze Ex Pers. isolated from solasodine producing plant Solanum khasianum Clarke. Minimum Inhibitory Concentration (MIC) and IC50 for 3-methylene-4-pentadecyldihydrofuran-2-one were found as 300 and 51.37 μg/ml, respectively. The standard captan was found to have an MIC and IC50 of 200 and 35.52 μg/ml, respectively.  相似文献   

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