177,319 research outputs found
A synaptic nidogen: developmental regulation and role of nidogen-2 at the neuromuscular junction
No full textBackground: The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.Results: In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen- 2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.Conclusion: All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction
Structural-properties of Recombinant Domain Iii-3 of Perlecan Containing A Globular Domain Inserted Into An Epidermal-growth-factor-like Motif Rid C-4254-2008
No full textA fragment comprising approximately domain III-3 of the basement membrane heparan sulfate proteoglycan perlecan was prepared in recombinant form from kidney cell clones. This fragment was predicted to contain a cysteine-free globular domain inserted within an epidermal-growth-factor(EGF)-like motif (L4 module) and three additional EGF-like motifs (LE module) without large inserts. This prediction was confirmed by electron microscopy, which demonstrated a globule joined to a very short rod-like segment. The globule was selectively destroyed by pepsin, which also demonstrated that its insertion into an EGF-like motif did not prevent the typical disulfide connections known far such motifs. Yet the globule was more stable against neutral proteinases. The fragment showed a distinct content (55-60%) of alpha helical and beta structure and a partially reversible melting of the conformation in 6 M guanidine. Antibodies raised against recombinant domain III-3 demonstrated a complete cross-reaction with tissue-derived perlecan but not with laminin and a distinct basement membrane staining of tissue sections. Most of the epitopes were lost after reduction and alkylation. Together the data demonstrated a proper folding of recombinant domain III-3 similar to its structure in the native protein and provided the first structural evidence for a novel globular protein motif L4 based on an EGF-like scaffold
Binding of the basement membrane protein laminin to Escherichia coli
No full textHere, we report that strains of
uropathogenic E. coli bind to the basement membrane
protein laminin. Laminin, which is a glycoprotein
with Mr - 900 000 serves as a substrate
mediating the attachment and spreading of different
eucaryotic cellsbasement membrane
protein laminin
Evidence of nidogen-2 compensation for nidogen-1 deficiency in transgenic mice
No full textPrevious studies have shown that inhibition of nidogen-laminin binding interferes with basement membrane stabilization in various mouse organ cultures while no overt phenotype has been observed following inactivation of the nidogen-1 gene in mice. We have now used recombinant mouse nidogen-1 and nidogen-2 in order to evaluate a possible compensation between the two isoforms in the knock-out mice. Essentially, a comparable in vitro binding of nidogens-1 and -2 to the same laminin-1 chain structure and to several other basement membrane proteins has been revealed. Quantitative radioimmuno-assays have demonstrated high concentrations of nidogen-1 exceeding those of laminin gamma1 and nidogen-2 by factors of 5 and 20-50, respectively, in tissue extracts of wild-type mice. A three- to sevenfold increase in nidogen-2 was observed in heart and muscle of mice with nidogen-1 deficiency and confirmed by a similar increase in the intensity of immunogold staining of these tissues. However, a few of the tissues from mice with the gene knock-out still contained some nidogen-1-like immunoreactivity (1% of wild-type). Furthermore, both nidogen isoforms showed a similar distribution in various organs during embryonic development which, however, as shown previously, changed in some adult tissues. The data support the nidogen-2 compensation hypothesis to explain the limited phenotype observed following elimination of the nidogen-1 gene. (C) 2002 Elsevier Science B.V. and International Society of Matrix Biology. All rights reserved
Laminin gamma 3 chain binds to nidogen and is located in murine basement membranes
No full textRecently a novel laminin γ 3 chain was identified in mouse and human and shown to have the same modular structure as the laminin γ 1 chain. We expressed two fragments of the γ 3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3-5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The γ 3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the γ 2 VI/ V fragment. The γ 3 III3-5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the γ 1 III3-5 fragment. These data suggested that laminins containing the γ 3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against γ 3 VI/V and γ 3 III3-5 showed no cross-reaction with homologous fragments from the γ 1 and γ 2 chains of laminin and allowed the establishment of γ chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20-100-fold lower content of the γ 3 chain compared with the γ 1 chain in various tissue extracts of adult mice. The expression of γ 3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the γ 3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the γ 3 chain to basement membranes of these tissues
Binding of Streptococcus pyogenes to laminin
No full textSome strains of Streptococcus pyogenes isolated from infected human tissues were shown to bind laminin, a major component of basement membranes. Binding of 125I-laminin to bacteria was time dependent and functionally irreversible. Of several unlabeled proteins tested in competition experiments, laminin and fibrinogen inhibited binding of the radiolabeled protein. The inhibitory effect exerted by fibrinogen was apparently not caused by a binding to the laminin receptors. The number of receptors available for laminin on cells of the strain examined ranged from 0 to 10(3) depending on the media used to grow the bacteria and an apparent KD of 4 X 10(-8)M was calculated for the reaction. Bacterial cells incubated with proteolytic enzymes lose the ability to bind laminin, and a trypsin digest contained active receptors capable of competing with intact cells for 125I-laminin. Active receptors may be adsorbed on a column of laminin-Sepharose but not on Sepharose gels substituted with fibrinogen or fibronectin. After radiolabeling the proteins in the trypsin digest a laminin-binding 125I-labeled protein (Mr greater than 10(6] was isolated by affinity chromatography from a receptor positive strain. Similar components could not be isolated from a strain apparently lacking laminin receptors. Therefore, this protein was tentatively identified as a laminin receptor of streptococci
Structural and functional analysis of the recombinant G domain of the laminin alpha 4 chain and its proteolytic processing in tissues
No full textThe C-terminal G domains of laminin a chains have been implicated in various cellular and other interactions. The G domain of the alpha4 chain was now produced in transfected mammalian cells as two tandem arrays of LG modules, alpha 4LG1-3 and alpha 4LG4-5. The recombinant fragments Were shown to fold into globular structures and could be distinguished by specific antibodies. Both fragments were able to bind to heparin, sulfatides, and the microfibrillar fibulin-1 and fibulin-2. They were, however, poor substrates for cell adhesion and had only a low affinity;for the alpha -dystroglycan receptor when compared with: the G domains of the laminin alpha1 and alpha2 chains. Yet antibodies to alpha 4LG1-3 but not to alpha 4LG4-5 clearly inhibited alpha (6)beta (1) integrin-mediated cell adhesion to laminin-8, indicating the participation of alpha 4LG1-3 in a cell-adhesive structure of higher complexity. Proteolytic: processing within a link region between the alpha 4LG3 and alpha 4LG4 modules was shown to occur during recombinant production and in endothelial and Schwann cell culture. Cleavage could be attributed to three different peptide bonds and is accompanied by the release of the alpha 4LG4-5 segment. Immunohistology demonstrated abundant staining of alpha 4LG1-3 in vessel walls, adipose, and perineural tissue. No significant staining was found for alpha 4LG4-5 indicating their loss from tissues. Immunogold staining demonstrated an association of the alpha4 chain primarily with microfibrillar regions rather than with basement membranes, while laminin alpha2 chains appear primarily associated with various basement membranes
Ultrastructural colocalization of nidogen-1 and nidogen-2 with laminin-1 in murine kidney basement membranes
No full textNidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen type IV networks. Recently a new member of the nidogen family, nidogen-2, has been characterized. Preliminary immunohistochemical data indicated that nidogen-1 and nidogen-2 show a similar tissue distribution at the light microscopic level. We have now localized nidogen-1 and nidogen-2, as well as their corresponding mRNAs, at the light and electron microscopic levels in adult mouse kidney, by in situ hybridization and immunogold histochemistry, as well as carrying out double labeling with laminin-1. Both nidogen-1 and nidogen-2 mRNAs are found not only in mesenchymal cells of embryonic tissues, but also in all epithelial and endothelial cells in adult mouse kidney. Both nidogens are ubiquitous basement membrane components in the mouse kidney, being found in glomerular, tubular, and capillary compartments and Bowman's capsule. Further more, a substantial fraction of nidogen-1 and nidogen-2 colocalizes with laminin-1. The results indicate that nidogen-1 and nidogen-2 could well substitute for one another in some of their biological activities in kidney, for example, stabilizing basement membrane networks in vivo
Appropriate Similarity Measures for Author Cocitation Analysis
No full textWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Evidence for coiled-coil alpha-helical regions in the long arm of laminin.
No full textThree new laminin fragments, E8, E9 and 25K with mol. wt. 50 000-280 000, were prepared from a limited elastase digest of laminin and from tissue extracts. They were similar with respect to their rod-like structure, a high alpha-helix content, the assembly from two chain segments and immunological cross-reactivity. Two of the fragments (E8 and E9) possess in addition globular domains which lack alpha-helices. Chemical, immunological and physical data together with sequence analysis strongly indicate that the alpha-helical segments are assembled in coiled-coil structures which are located in the rod of the long arm of laminin. These data give new insights into the overall structure of the protein
- …
