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Sample records for 67-kda laminin receptor

  1. Laminin receptors for neurite formation

    SciTech Connect

    Kleinman, H.K.; Ogle, R.C.; Cannon, F.B.; Little, C.D.; Sweeney, T.M.; Luckenbill-Edds, L.

    1988-02-01

    Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. The authors report here that /sup 125/I-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein was found in a variety of epithelial cell lines and in brain, whereas the 180-kDa protein was neural specific. Antibodies prepared against the 110-kDa and 180-kDa proteins inhibited neurite outgrowth induced by the neurite-promoting domain of laminin, whereas antibodies to the 67-kDa laminin receptor had no effect on neurite outgrowth. They conclude that neuronal cells have multiple cell-surface laminin receptors and that the 110-kDa and 180-kDa proteins are involved in neurite formation.

  2. Presence of Laminin Receptors in Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Lopes, J. D.; Dos Reis, M.; Brentani, R. R.

    1985-07-01

    A characteristic feature of infection by Staphylococcus aureus is bloodstream invasion and widespread metastatic abscess formation. The ability to extravasate, which entails crossing the vascular basement membrane, appears to be critical for the organism's pathogenicity. Extravasation by normal and neoplastic mammalian cells has been correlated with the presence of specific cell surface receptors for the basement membrane glycoprotein laminin. Similar laminin receptors were found in Staphylococcus aureus but not in Staphylococcus epidermidis, a noninvasive pathogen. There were about 100 binding sites per cell, with an apparent binding affinity of 2.9 nanomolar. The molecular weight of the receptor was 50,000 and pI was 4.2. Eukaryotic laminin receptors were visualized by means of the binding of S. aureus in the presence of laminin. Prokaryotic and eukaryotic invasive cells might utilize similar, if not identical, mechanisms for invasion.

  3. The Human Laminin Receptor is a Member of the Integrin Family of Cell Adhesion Receptors

    NASA Astrophysics Data System (ADS)

    Gehlsen, Kurt R.; Dillner, Lena; Engvall, Eva; Ruoslahti, Erkki

    1988-09-01

    A receptor for the adhesive basement membrane protein, laminin, was isolated from human glioblastoma cells by affinity chromatography on laminin. This receptor has a heterodimeric structure similar to that of receptors for other extracellular matrix proteins such as fibronectin and vitronectin. Incorporation of the laminin receptor into liposomal membranes makes it possible for liposomes to attach to surfaces coated with laminin. The receptor liposomes also attached to some extent to surfaces coated with fibronectin, but not with other matrix proteins. These properties identify the laminin receptor as a member of the integrin family of cell adhesion receptors.

  4. Crystal Structure of the Human Laminin Receptor Precursor

    SciTech Connect

    Jamieson,K.; Wu, J.; Hubbard, S.; Meruelo, D.

    2008-01-01

    The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR.

  5. Expression of 32/67-kDa laminin receptor in laminin adhesion-selected human colon cancer cell lines.

    PubMed Central

    Kim, W. H.; Lee, B. L.; Jun, S. H.; Song, S. Y.; Kleinman, H. K.

    1998-01-01

    Laminin promotes the malignant phenotype, and the expression of certain laminin receptors is increased in malignancy. Previously, we demonstrated that a laminin-adhesive subclone of a human colon cancer cell line showed increased tumorigenicity in nude mice and increased affinity of the beta1 integrin for laminin relative to the laminin-non-adhesive subclone. The total amount of either beta1 integrin protein or mRNA did not increase. As levels of the 32/67-kDa laminin receptor (67LR) correlate with malignancy, we examined 67LR expression in the laminin adhesion-selected human colon cancer cells. The laminin-adhesive subclone, which was more tumorigenic in both heterotopic and orthotopic locations than in a laminin-non-adhesive subclone, showed cell-surface membrane staining of 67LR, whereas the laminin-non-adhesive subclone showed cytoplasmic staining of 67LR. No difference in either the amount of 67LR mRNA or the amount of protein was observed in the parental cells than in the laminin-adhesive and non-adhesive subclones. When assayed on a laminin affinity column, more 67LR molecules bound to the column with cell extracts from the laminin-adhesive subclone than was observed with the non-adhesive subclone. These findings suggest that the increased tumorigenicity of laminin adhesion-selected tumour cells might be due to an alteration in the distribution and/or adhesiveness of multiple receptors including 67LR and beta1 integrin. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:9459140

  6. The human integrin VLA-2 is a collagen receptor on some cells and a collagen/laminin receptor on others

    SciTech Connect

    Elices, M.J.; Hemler, M.E. )

    1989-12-01

    The integrin heterodimer VLA-2, previously known as a collagen receptor, is now shown also to be a laminin receptor. Adhesion of the human melanoma cell line LOX to laminin was inhibited by anti-VLA {alpha}{sup 2} antibodies. Because VLA-2-mediated LOX cell attachment to laminin was not inhibited by digestion with collagenase, collagen contamination of laminin was not a factor. In addition, VLA-2 from LOX cells bound to immobilized laminin, and binding was disrupted by EDTA but not by Arg-Gly-Asp (RGD) peptides. VLA-3 also bound to laminin-Sepharose, although less avidly than VLA-2. Thus, at least four separate members of the integrin {beta}{sub 1} subfamily serve as laminin receptors - i.e., VLA-2 and VLA-3 (this study) together with VLA-1 and VLA-6 (other reports). Whereas LOX and other cell lines used VLA-2 as both a laminin and collagen receptor, fibroblast VLA-2 mediated collagen but not laminin binding. Likewise, VLA-2 from platelets did not interact with laminin. Despite this functional discordancy, VLA-2 from laminin-binding and nonbinding sources was indistinguishable by all immunochemical and biochemical criteria examined. Thus, functional differences in VLA-2 may be due to cell type-specific modulation.

  7. Comprehensive Proteomic Analysis of Non-Integrin Laminin Receptor Interacting Proteins

    PubMed Central

    Venticinque, Lisa; Meruelo, Daniel

    2012-01-01

    Human non-integrin laminin receptor is a multifunctional protein acting as an integral component of the ribosome and a cell surface receptor for laminin-1. Laminin receptor is overexpressed in several human cancers and is also the cell surface receptor for several viruses and pathogenic prion protein, making it a pathologically significant protein. This study focused on the proteomic characterization of laminin receptor interacting proteins from mus musculus. The use of affinity chromatography with immobilized recombinant laminin receptor coupled with mass spectrometry analysis identified 45 proteins with high confidence. Following validation through co-immunoprecipitation, the proteins were classified based on predicted function into ribosomal, RNA processing, signal transduction/ metabolism, protein processing, cytoskeleton/ cell anchorage, DNA/ chromatin and unknown functions. A significant portion of the identified proteins is related to functions or localizations previously described for laminin receptor. This work represents a comprehensive proteomic approach to studying laminin receptor, and provides an essential stepping-stone to a better mechanistic understanding of this protein’s diverse functions. PMID:22909348

  8. Looking into laminin receptor: critical discussion regarding the non-integrin 37/67-kDa laminin receptor/RPSA protein.

    PubMed

    DiGiacomo, Vincent; Meruelo, Daniel

    2016-05-01

    The 37/67-kDa laminin receptor (LAMR/RPSA) was originally identified as a 67-kDa binding protein for laminin, an extracellular matrix glycoprotein that provides cellular adhesion to the basement membrane. LAMR has evolutionary origins, however, as a 37-kDa RPS2 family ribosomal component. Expressed in all domains of life, RPS2 proteins have been shown to have remarkably diverse physiological roles that vary across species. Contributing to laminin binding, ribosome biogenesis, cytoskeletal organization, and nuclear functions, this protein governs critical cellular processes including growth, survival, migration, protein synthesis, development, and differentiation. Unsurprisingly given its purview, LAMR has been associated with metastatic cancer, neurodegenerative disease and developmental abnormalities. Functioning in a receptor capacity, this protein also confers susceptibility to bacterial and viral infection. LAMR is clearly a molecule of consequence in human disease, directly mediating pathological events that make it a prime target for therapeutic interventions. Despite decades of research, there are still a large number of open questions regarding the cellular biology of LAMR, the nature of its ability to bind laminin, the function of its intrinsically disordered C-terminal region and its conversion from 37 to 67 kDa. This review attempts to convey an in-depth description of the complexity surrounding this multifaceted protein across functional, structural and pathological aspects.

  9. The Laminin Receptor Is a Cellular Attachment Receptor for Classical Swine Fever Virus

    PubMed Central

    Chen, Jianing; He, Wen-Rui; Shen, Liang; Dong, Hong; Yu, Jiahui; Wang, Xiao; Yu, Shaoxiong; Li, Yongfeng; Li, Su; Luo, Yuzi; Sun, Yuan

    2015-01-01

    ABSTRACT Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious, economically important viral disease in many countries. The Erns and E2 envelope glycoproteins are responsible for the binding to and entry into the host cell by CSFV. To date, only one cellular receptor, heparan sulfate (HS), has been identified as being involved in CSFV attachment. HS is also present on the surface of various cells that are nonpermissive to CSFV. Hence, there must be another receptor(s) that has been unidentified to date. In this study, we used a set of small interfering RNAs (siRNAs) against a number of porcine cell membrane protein genes to screen cellular proteins involved in CSFV infection. This approach resulted in the identification of several proteins, and of these, the laminin receptor (LamR) has been demonstrated to be a cellular receptor for several viruses. Confocal analysis showed that LamR is colocalized with CSFV virions on the membrane, and a coimmunoprecipitation assay indicated that LamR interacts with the CSFV Erns protein. In inhibition assays, anti-LamR antibodies, soluble laminin, or LamR protein significantly inhibited CSFV infection in a dose-dependent manner. Transduction of PK-15 cells with a recombinant lentivirus expressing LamR yielded higher viral titers. Moreover, an attachment assay demonstrated that LamR functions during virus attachment. We also demonstrate that LamR acts as an alternative attachment receptor, especially in SK6 cells. These results indicate that LamR is a cellular attachment receptor for CSFV. IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only heparan sulfate (HS) has been identified to be an attachment receptor for CSFV. Here, using RNA interference screening with small interfering RNAs (siRNAs) against a number of porcine membrane

  10. In situ hybridization reveals temporal and spatial changes in cellular expression of mRNA for a laminin receptor, laminin, and basement membrane (type IV) collagen in the developing kidney

    PubMed Central

    1989-01-01

    The appearance of extracellular matrix molecules and their receptors represent key events in the differentiation of cells of the kidney. Steady-state mRNA levels for a laminin receptor, the laminin B1, B2, and A chains, and the alpha 1-chain of collagen IV (alpha 1[IV]), were examined in mouse kidneys at 16 d gestation and birth, when cell differentiation is active, and 1-3 wk after birth when this activity has subsided. Northern analysis revealed that mRNA expression of laminin receptor precedes the alpha 1(IV) and laminin B chains whereas laminin A chain mRNA expression was very low. In situ hybridization reflected this pattern and revealed the cells responsible for expression. At 16 d gestation, laminin receptor mRNA was elevated in cells of newly forming glomeruli and proximal and distal tubules of the nephrogenic zone located in the kidney cortex. These cells also expressed mRNA for alpha 1(IV) and laminin chains. At birth, mRNA expression of receptor and all chains remained high in glomeruli but was reduced in proximal and distal tubules. At 1 wk after birth, expression was located in the medulla over collecting ducts and loops of Henle. Little expression was detectable by 3 wk. These results suggest that cellular expression of steady-state mRNA for laminin receptor, laminin, and collagen IV is temporally linked, with laminin receptor expression proceeding first and thereafter subsiding. PMID:2527859

  11. Drosophila PS1 integrin is a laminin receptor and differs in ligand specificity from PS2.

    PubMed Central

    Gotwals, P J; Fessler, L I; Wehrli, M; Hynes, R O

    1994-01-01

    We have expressed Drosophila position-specific (PS) integrins on the surfaces of Schneider S2 cells and tested for adhesion and spreading on various matrix molecules. We report that PS1 integrin is a laminin receptor and that PS1 and PS2 integrins promote cell spreading on two different Drosophila extracellular matrix molecules, laminin and tiggrin, respectively. The differing ligand specificities of these two integrins, combined with data on the in vivo expression patterns of the integrins and their ligands, lead to a model for the structure of integrin-dependent attachments in the pupal wings and embryonic muscles of Drosophila. Images PMID:7972082

  12. 67-Kilodalton laminin receptor expression correlates with worse prognostic indicators in non-small cell lung carcinomas.

    PubMed

    Fontanini, G; Vignati, S; Chiné, S; Lucchi, M; Mussi, A; Angeletti, C A; Ménard, S; Castronovo, V; Bevilacqua, G

    1997-02-01

    Tumor samples obtained from 72 patients resected for non-small cell lung cancer were stained immunohistochemically using an immunoperoxidase method and the MLuC5 monoclonal antibody specific for the 67-kDa laminin receptor. Sixty-one of 72 patients (84.7%) displayed a MLuC5-positive reaction, which was usually localized in both the inner surface of the plasmatic membranes and the cytoplasm of neoplastic cells. When we compared the laminin receptor expression with clinicopathological and biological parameters such as histotype, grading, T status, N status, ploidy, proliferative activity, vessel invasion, and p53 protein accumulation, the following results were observed: (a) the mean expression of the receptor was higher in the group of patients with metastatic nodal involvement than in those with uninvolved lymph nodes (P = 0.02); (b) a high Ki-67 score (>13% of positive cells) was observed in tumors with a higher mean value of laminin receptor (P = 0.004); (c) the tumors harboring neoplastic emboli in their vessels showed a higher laminin receptor immunoreactivity (P = 0.02); and (d) a borderline association was found between the high mean value of laminin receptor immunopositivity and p53 accumulation in neoplastic cell nuclei (P = 0.05). Our observations indicate that detection of high tissue levels of 67-kDa laminin receptor is associated with an invasive phenotype in non-small cell lung cancer and may provide further information in the biological characterization of this type of cancer.

  13. Immunodetection of the metastasis-associated laminin receptor in human breast cancer cells obtained by fine-needle aspiration biopsy.

    PubMed Central

    Castronovo, V.; Colin, C.; Claysmith, A. P.; Chen, P. H.; Lifrange, E.; Lambotte, R.; Krutzsch, H.; Liotta, L. A.; Sobel, M. E.

    1990-01-01

    Fine-needle aspiration biopsy of the breast is a very useful technique for the evaluation of a suspect lesion before surgical removal. Increased expression of the 67-kd laminin receptor has been associated with the metastatic phenotype of cancer cells, particularly in colon and breast cancers. In this study, the expression of laminin receptor was evaluated using the immunoperoxidase technique in 81 breast aspirates (26 benign and 55 neoplastic lesions). Cells obtained from benign samples exhibited a low level of laminin receptor antigen detected by affinity-purified antibody raised against a cDNA-derived laminin receptor peptide. In contrast, 71% of smears obtained from malignant breast lesions contained cells that were strongly stained by the antibody. Heterogeneous expression of the laminin receptor was noted in both breast aspirates and fixed tissue specimens. These data suggest that the immunodetection of laminin receptor in cells obtained by fine-needle aspiration of breast lesions could be a valuable adjunct in the prognostic evaluation of breast lesions. Images Figure 3 Figure 4 Figure 5 Figure 7 PMID:2148054

  14. Laminin Receptor in Shrimp Is a Cellular Attachment Receptor for White Spot Syndrome Virus

    PubMed Central

    Liu, Wang-Jing; Li, Yi-Chieh; Kou, Guang-Hsiung; Lo, Chu-Fang

    2016-01-01

    White spot syndrome virus (WSSV, genus Whispovirus, family Nimaviridae) is causing huge economic losses in global shrimp farming, but there is no effective control. Shrimp cell laminin receptor (Lamr) may have a role in WSSV infection. The objective was to characterize interactions between Penaeus monodon Lamr (PmLamr) and WSSV structural proteins. In this study, PmLamr interacted with nine WSSV structural proteins (based on yeast two-hybrid screening), of which one (VP31) was characterized. Protein pull-down assay confirmed the interaction between PmLamr and VP31; the latter was an envelope protein exposed outside the WSSV virion (based on membrane topology assays). Furthermore, similar to mammalian Lamr, there were two major protein bands in shrimp cells. Cellular localization assay demonstrated VP31 co-localized with PmLamr on transfected cells. Enzyme-link immunosorbent assay (ELISA) and competitive ELISA demonstrated binding of VP31 on PmLamr was dose-dependent; however, addition of WSSV virion competed for binding affinity. Furthermore, based on an in vivo neutralization assay, both VP31 and PmLamr delayed mortality in shrimp challenged with WSSV. We concluded Lamr was an important receptor for WSSV infection and the viral envelope protein VP31 may have a role in host cell recognition and binding. These data contributed to elucidating pathogenesis of WSSV infection and may help in controlling this disease. PMID:27257954

  15. Proteolytic fragments of laminin promote excitotoxic neurodegeneration by up-regulation of the KA1 subunit of the kainate receptor.

    PubMed

    Chen, Zu-Lin; Yu, Huaxu; Yu, Wei-Ming; Pawlak, Robert; Strickland, Sidney

    2008-12-29

    Degradation of the extracellular matrix (ECM) protein laminin contributes to excitotoxic cell death in the hippocampus, but the mechanism of this effect is unknown. To study this process, we disrupted laminin gamma1 (lamgamma1) expression in the hippocampus. Lamgamma1 knockout (KO) and control mice had similar basal expression of kainate (KA) receptors, but the lamgamma1 KO mice were resistant to KA-induced neuronal death. After KA injection, KA1 subunit levels increased in control mice but were unchanged in lamgamma1 KO mice. KA1 levels in tissue plasminogen activator (tPA)-KO mice were also unchanged after KA, indicating that both tPA and laminin were necessary for KA1 up-regulation after KA injection. Infusion of plasmin-digested laminin-1 into the hippocampus of lamgamma1 or tPA KO mice restored KA1 up-regulation and KA-induced neuronal degeneration. Interfering with KA1 function with a specific anti-KA1 antibody protected against KA-induced neuronal death both in vitro and in vivo. These results demonstrate a novel pathway for neurodegeneration involving proteolysis of the ECM and KA1 KA receptor subunit up-regulation.

  16. Shrimp laminin receptor binds with capsid proteins of two additional shrimp RNA viruses YHV and IMNV.

    PubMed

    Busayarat, Nattaphon; Senapin, Saengchan; Tonganunt, Moltira; Phiwsaiya, Kornsunee; Meemetta, Watcharachai; Unajak, Sasimanas; Jitrapakdee, Sarawut; Lo, Chu-Fang; Phongdara, Amornrat

    2011-07-01

    Laminin receptor (Lamr) in shrimp was previously proposed to be a potential receptor protein for Taura syndrome virus (TSV) based on yeast two-hybrid assays. Since shrimp Lamr bound to the VP1 capsid protein of TSV, we were interested to know whether capsid/envelope proteins from other shrimp viruses would also bind to Lamr. Thus, capsid/envelope encoding genes from 5 additional shrimp viruses were examined. These were Penaeus stylirostris densovirus (PstDNV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), Macrobrachium rosenbergii nodavirus (MrNV), and yellow head virus (YHV). Protein interaction analysis using yeast two-hybrid assay revealed that Lamr specifically interacted with capsid/envelope proteins of RNA viruses IMNV and YHV but not MrNV and not with the capsid/envelope proteins of DNA viruses PstDNV and WSSV. In vitro pull-down assay also confirmed the interaction between Lamr and YHV gp116 envelope protein, and injection of recombinant Lamr (rLamr) protein produced in yeast cells protected shrimp against YHV in laboratory challenge tests. PMID:21414409

  17. The Transition of the 37-Kda Laminin Receptor (Rpsa) to Higher Molecular Weight Species: Sumoylation or Artifact?

    PubMed

    Digiacomo, Vincent; Gando, Ivan A; Venticinque, Lisa; Hurtado, Alicia; Meruelo, Daniel

    2015-12-01

    The 37-kDa laminin receptor (37LRP or RPSA) is a remarkable, multifaceted protein that functions in processes ranging from matrix adhesion to ribosome biogenesis. Its ability to engage extracellular laminin is further thought to contribute to cellular migration and invasion. Most commonly associated with metastatic cancer, RPSA is also increasingly found to be important in other pathologies, including microbial infection, neurodegenerative disease and developmental malformations. Importantly, it is thought to have higher molecular weight forms, including a 67-kDa species (67LR), the expression of which is linked to strong laminin binding and metastatic behavior. The composition of these larger forms has remained elusive and controversial. Homo- and heterodimerization have been proposed as events capable of building the larger species from the monomeric 37-kDa precursor, but solid evidence is lacking. Here, we present data suggesting that higher molecular weight species require SUMOylation to form. We also comment on the difficulty of isolating larger RPSA species for unambiguous identification and demonstrate that cell lines stably expressing tagged RPSA for long periods of time fail to produce tagged higher molecular weight RPSA. It is possible that higher molecular weight species like 67LR are not derived from RPSA.

  18. Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis

    PubMed Central

    Alqahtani, Fulwah; Mahdavi, Jafar; Wheldon, Lee M.; Vassey, Matthew; Pirinccioglu, Necmettin; Royer, Pierre-Joseph; Qarani, Suzan M.; Morroll, Shaun; Stoof, Jeroen; Holliday, Nicholas D.; Teo, Siew Y.; Oldfield, Neil J.; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

    2014-01-01

    The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C173) of Gal-3 or lysine (K166) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial–host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization. PMID:25274119

  19. Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis.

    PubMed

    Alqahtani, Fulwah; Mahdavi, Jafar; Wheldon, Lee M; Vassey, Matthew; Pirinccioglu, Necmettin; Royer, Pierre-Joseph; Qarani, Suzan M; Morroll, Shaun; Stoof, Jeroen; Holliday, Nicholas D; Teo, Siew Y; Oldfield, Neil J; Wooldridge, Karl G; Ala'Aldeen, Dlawer A A

    2014-10-01

    The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C(173)) of Gal-3 or lysine (K(166)) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial-host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization.

  20. Interactions of laminin β3 fragment with β1-integrin receptor

    PubMed Central

    Lie, Pearl PY; Mok, Ka-wai; Cheng, Yan-ho; Wong, Elissa WP; Mannu, Jayakanthan; Mathur, Premendu P; Yan, Helen H N; Mruk, Dolores D

    2011-01-01

    Recent studies have demonstrated the presence of a functional axis that coordinates the events of spermiation and blood-testis barrier (BTB) restructuring which take place simultaneously at the opposite ends of the seminiferous epithelium at stage VIII of the epithelial cycle of spermatogenesis in the rat testis. In short, the disruption of the apical ectoplasmic specialization (apical ES) at the Sertoli cell-elongated spermatid interface, which facilitates the release of sperm at spermiation near the tubule lumen, is coordinated with restructuring at the BTB to accommodate the transit of preleptotene spermatocytes across the immunological barrier near the basement membrane. These two events are likely coordinated by a functional axis involving hemidesmosome at the Sertoli cell-basement membrane interface, and it was designated the apical ES-BTB-hemidesmosome axis. It was demonstrated that fragments of laminin chains (e.g., laminin β3 or γ3 chains) derived from the α6β1-integrin-laminin333 protein complex at the apical ES, which were likely generated via the action of MMP-2 (matrix metalloprotease-2, MMP2) prior to spermiation, acted as biologically active peptides to perturb the BTB permeability function by accelerating protein endocytosis (e.g., occludin) at the site, thereby destabilizing the BTB integrity to facilitate the transit of preleptotene spermatocytes. These laminin fragments also perturbed hemidesmosome function via their action on β1-integrin, a component of hemidesmosome in the testis, which in turn, sent a signal to further destabilize the BTB function. As such, the events of spermiation and BTB restructuring are coordinated via this functional axis. Recent studies using animal models treated with toxicants, such as mono-(2-ethylhexyl) phthalate (MEHP), or adjudin, a male contraceptive under investigation, have also supported the presence of this functional axis in the mouse. In this short review, we critically evaluate the role of this local

  1. Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic PSP94-derived peptide PCK3145 cell surface binding

    SciTech Connect

    Annabi, Borhane; Currie, Jean-Christophe; Bouzeghrane, Mounia; Dulude, Helene; Daigneault, Luc; Garde, Seema; Rabbani, Shafaat A.; Panchal, Chandra; Wu, Jinzi J.; Beliveau, Richard . E-mail: oncomol@nobel.si.uqam.ca

    2006-07-21

    Purpose: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. Results: [{sup 14}C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145 binding but unexpectedly not to its uptake. Conclusions: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.

  2. Evidence for the presence of a high-affinity laminin receptor-like molecule on the surface of Candida albicans yeast cells.

    PubMed Central

    López-Ribot, J L; Casanova, M; Monteagudo, C; Sepúlveda, P; Martínez, J P

    1994-01-01

    Two polypeptides of 37 and 67 kDa that bind laminin were detected in cell wall extracts of Candida albicans blastoconidia. The 37-kDa species, found only in yeast cell wall extracts, cross-reacted with a rabbit polyclonal antibody (PAb 4160) directed towards the carboxyl-terminal laminin-binding domain present in the human 67-kDa high-affinity laminin receptor (67LR) and its 37-kDa precursor (37LRP), whereas another antibody (PAb 4056), directed against internal domains of 67LR and 37LRP, recognized a 37-kDa species in wall extracts from both blastoconidia and germinated blastoconidia. Indirect immunofluorescence with PAb 4160 showed a patchy binding pattern only on yeast cells that represented about 10% of the entire blastoconidia population. Images PMID:8300236

  3. Serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor.

    PubMed

    Thepparit, Chutima; Smith, Duncan R

    2004-11-01

    Dengue virus, the causative agent of dengue fever, dengue shock syndrome, and dengue hemorrhagic fever, infects susceptible cells by initially binding to a receptor(s) located on the host cell surface. Evidence to date suggests that receptor usage may be cell and serotype specific, and this study sought to identify dengue virus serotype 1 binding proteins on the surface of liver cells, a known target organ. By using a virus overlay protein binding assay (VOPBA), in both nondenaturing and denaturing gel systems, a putative dengue virus serotype 1 binding protein of approximately 37 kDa expressed on the surface of liver (HepG2) cells was identified. Mass spectrometry analysis identified a candidate protein, the 37/67-kDa high-affinity laminin receptor. Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.

  4. INS-1 cell glucose-stimulated insulin secretion is reduced by the downregulation of the 67 kDa laminin receptor.

    PubMed

    Sabra, Georges; Dubiel, Evan A; Kuehn, Carina; Khalfaoui, Taoufik; Beaulieu, Jean-François; Vermette, Patrick

    2015-12-01

    Understanding β cell-extracellular matrix (ECM) interactions can advance our knowledge of the mechanisms that control glucose homeostasis and improve culture methods used in islet transplantation for the treatment of diabetes. Laminin is the main constituent of the basement membrane and is involved in pancreatic β cell survival and function, even enhancing glucose-stimulated insulin secretion. Most of the studies on cell responses towards laminin have focused on integrin-mediated interactions, while much less attention has been paid on non-integrin receptors, such as the 67 kDa laminin receptor (67LR). The specificity of the receptor-ligand interaction through the adhesion of INS-1 cells (a rat insulinoma cell line) to CDPGYIGSR-, GRGDSPC- or CDPGYIGSR + GRGDSPC-covered surfaces was evaluated. Also, the effects of the 67LR knocking down over glucose-stimulated insulin secretion were investigated. Culture of the INS-1 cells on the bioactive surfaces was improved compared to the low-fouling carboxymethyl dextran (CMD) surfaces, while downregulation of the 67LR resulted in reduced cell adhesion to surfaces bearing the CDPGYIGSR peptide. Glucose-stimulated insulin secretion was hindered by downregulation of the 67LR, regardless of the biological motif available on the biomimetic surfaces on which the cells were cultured. This finding illustrates the importance of the 67LR in glucose-stimulated insulin secretion and points to a possible role of the 67LR in the mechanisms of insulin secretion.

  5. Laminin receptor mediates anti-inflammatory and anti-thrombogenic effects of pigment epithelium-derived factor in myeloma cells.

    PubMed

    Matsui, Takanori; Higashimoto, Yuichiro; Yamagishi, Sho-ichi

    2014-01-17

    Pigment epithelium-derived factor (PEDF) has anti-inflammatory and anti-thrombogenic properties both in cell culture and animal models. Although adipose triglyceride lipase (ATGL) and laminin receptor (LR) are two putative receptors for PEDF, which receptor mainly mediates the beneficial effects of PEDF is largely unknown. In this study, we addressed the issue. siRNA raised against LR (siLR) and siATGL transfection dramatically decreased LR and ATGL levels in human cultured myeloma cells, respectively. Ten nM PEDF significantly reduced vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels in siCon- or siATGL-transfected myeloma cells, whereas PEDF increased rather than decreased these gene expressions in siLR-transfected cells. Neutralizing antibody directed against LR (LR-Ab) or LR antagonist actually bound to LR and reduced mRNA levels of VEGF, MCP-1, ICAM-1 and PAI-1 in myeloma cells. Further, pre-treatment of LR-Ab or LR antagonist suppressed the binding of PEDF to LR and resultantly blocked the effects of PEDF in myeloma cells. In addition, high concentration of LR agonist mimicked the actions of PEDF on these gene expressions in myeloma cells. This study indicates that PEDF causes anti-angiogenic, anti-inflammatory and anti-thrombogenic reactions in myeloma cells through the interaction with LR. Target domain of LR agonist and antagonist might be involved in the PEDF-signaling to gene suppression in myeloma cells.

  6. Laminin receptor specific therapeutic gold nanoparticles (198AuNP-EGCg) show efficacy in treating prostate cancer

    SciTech Connect

    Shukla, R.; Chanda, N.; Zambre, A.; Upendran, A.; Katti, K.; Kulkarni, R. R.; Nune, S. K.; Casteel, S. W.; Smith, C. J.; Vimal, J.; Boote, E.; Robertson, J. D.; Kan, P.; Engelbrecht, H.; Watkinson, L. D.; Carmack, T. L.; Lever, J. R.; Cutler, C. S.; Caldwell, C.; Kannan, R.; Katti, K. V.

    2012-07-16

    Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechingallate( EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), will circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein provide unequivocal validation of our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from Au-198 isotope; the range of 198Au β-particle ( ~ 11 mm in tissue or ~1100 cell diameters) is sufficiently long to provide cross-fire effects of radiation dose delivered to cells within the prostate gland and short enough to minimize radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible 198AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed ~72% retention of 198AuNP-EGCg in tumors 24 h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28 days demonstrating significant inhibition of tumor growth compared to controls. This innovative “green nanotechnological“approach serves as a basis for designing target specific antineoplastic agents. This novel intratumorally injectable 198AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors.

  7. Laminin Receptor-Avid Nanotherapeutic EGCg-AuNPs as a Potential Alternative Therapeutic Approach to Prevent Restenosis

    PubMed Central

    Khoobchandani, Menka; Katti, Kavita; Maxwell, Adam; Fay, William P.; Katti, Kattesh V.

    2016-01-01

    In our efforts to develop new approaches to treat and prevent human vascular diseases, we report herein our results on the proliferation and migration of human smooth muscles cells (SMCs) and endothelial cells (ECs) using epigallocatechin-3-gallate conjugated gold nanoparticles (EGCg-AuNPs) as possible alternatives to drug coated stents. Detailed in vitro stability studies of EGCg-AuNPs in various biological fluids, affinity and selectivity towards SMCs and ECs have been investigated. The EGCg-AuNPs showed selective inhibitory efficacy toward the migration of SMCs. However, the endothelial cells remained unaffected under similar experimental conditions. The cellular internalization studies have indicated that EGCg-AuNPs internalize into the SMCs and ECs within short periods of time through laminin receptor mediated endocytosis mode. Favorable toxicity profiles and selective affinity toward SMCs and ECs suggest that EGCg-AuNPs may provide attractive alternatives to drug coated stents and therefore offer new therapeutic approaches in treating cardiovascular diseases. PMID:26938531

  8. Laminin receptor is an interacting partner for viral outer capsid protein VP5 in grass carp reovirus infection.

    PubMed

    Wang, Hao; Yu, Fei; Li, Jiale; Lu, Liqun

    2016-03-01

    Grass carp reovirus (GCRV) is responsible for viral hemorrhagic disease in cultured grass carp Ctenopharyngon idellus. Through yeast two-hybrid screen, laminin receptor (LamR) was identified as a potential interacting partner for the outer capsid protein VP5 of GCRV. We cloned and sequenced the gene encoding grass carp LamR. Viral attachment assay demonstrated the involvement of membrane-associated LamR in GCRV infection. Solid-phase overlay assays demonstrated that GCRV interacted with GST-tagged LamR in vitro. In contrast to VP7, GST-tagged VP5 was shown to associate with LamR in both pull-down and solid-phase blot overlay assays. With the reduction of LamR expression in CIK cells achieved by RNAi, remarkably reduced infection efficiency of GCRV was observed. CIK cells pretreated with polyclonal antibody against LamR resulted in dose-dependent inhibition of GCRV infection. These results collectively indicated that grass carp LamR was involved in GCRV infection by interacting with viral outer capsid protein VP5.

  9. Photodynamic treatment of epithelial tissue derived from patients with endometrial cancer: a contribution to the role of laminin and epidermal growth factor receptor in photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Ziolkowski, Piotr P.; Symonowicz, Krzysztof; Osiecka, Beata J.; Rabczynski, Jerzy; Gerber, Jerzy

    1999-07-01

    Photodynamic therapy (PDT) was used to treat endometrial G1 cancer tissue derived from patients who had undergone a total hysterectomy and bilateral salpingo-oophorectomy. After surgical treatment the cancerous tissue was kept in a medium containing Dulbecco solution, fetal calf serum, and antibiotics. The tissue was then exposed to hematoporphyrin derivative (0.1 mg/l) and 24 h later exposed to light (total light dose--18 J/sq cm). Necrosis depth was evaluated 24 h later using a light microscope. In order to assess the possible role of the basal membrane component laminin, as well as epidermal growth factor receptor susceptibility to PDT, immunohistochemical studies were carried out. Additionally, nucleolar organizer regions evaluation was performed. Our experiment confirmed that PDT results in the necrosis in the treated endometrial cancer, while not affecting the laminin in the cancerous tissue. In contrast, PDT strongly affects the epidermal growth factor receptor and nucleolar organizer regions in cancer cells. We suggest that laminin may contribute to the prevention of cancer dissemination in the cases where PDT has to be repeated, and that after PDT the cells become less susceptible to a mitogen, like, e.g., epidermal growth factor.

  10. YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells.

    PubMed

    Poitelon, Yannick; Lopez-Anido, Camila; Catignas, Kathleen; Berti, Caterina; Palmisano, Marilena; Williamson, Courtney; Ameroso, Dominique; Abiko, Kansho; Hwang, Yoonchan; Gregorieff, Alex; Wrana, Jeffrey L; Asmani, Mohammadnabi; Zhao, Ruogang; Sim, Fraser James; Wrabetz, Lawrence; Svaren, John; Feltri, Maria Laura

    2016-07-01

    Myelination is essential for nervous system function. Schwann cells interact with neurons and the basal lamina to myelinate axons using known receptors, signals and transcription factors. In contrast, the transcriptional control of axonal sorting and the role of mechanotransduction in myelination are largely unknown. Yap and Taz are effectors of the Hippo pathway that integrate chemical and mechanical signals in cells. We describe a previously unknown role for the Hippo pathway in myelination. Using conditional mutagenesis in mice, we show that Taz is required in Schwann cells for radial sorting and myelination and that Yap is redundant with Taz. Yap and Taz are activated in Schwann cells by mechanical stimuli and regulate Schwann cell proliferation and transcription of basal lamina receptor genes, both necessary for radial sorting of axons and subsequent myelination. These data link transcriptional effectors of the Hippo pathway and of mechanotransduction to myelin formation in Schwann cells.

  11. Multiple Functions of the 37/67-kd Laminin Receptor Make It a Suitable Target for Novel Cancer Gene Therapy

    PubMed Central

    Scheiman, Jonathan; Tseng, Jen-Chieh; Zheng, Yun; Meruelo, Daniel

    2009-01-01

    The 37/67-kd laminin receptor, LAMR, is a multifunctional protein that associates with the 40S ribosomal subunit and also localizes to the cell membrane to interact with the extracellular matrix. LAMR is overexpressed in many types of cancer, playing important roles in tumor-cell migration and invasion. Here, we show that LAMR is also vital for tumor-cell proliferation, survival, and protein translation. Small-interfering RNA (siRNA)–mediated reduction in expression of LAMR leads to G1 phase cell-cycle arrest in vitro by altering cyclins A2/B1, cyclin-dependent kinases (CDKs) 1/2, Survivin, and p21 expression levels. In vivo, reduction in LAMR expression results in inhibition of HT1080 cells to develop tumors. We also found that LAMR's ribosomal functions are critical for translation as reduction in LAMR expression leads to a dramatic decrease in newly synthesized proteins. Further, cells with lower expression of LAMR have fewer 40S subunits and 80S monosomes, causing an increase in free 60S ribosomal subunits. These results indicate that LAMR is able to regulate tumor development in many ways; further enhancing its potential as a target for gene therapy. To test this, we developed a novel Sindbis/Lenti pseudotype vector carrying short-hairpin RNA (shRNA) designed against lamr. This pseudotype vector effectively reduces LAMR expression and specifically targets tumors in vivo. Treatment of tumor-bearing severe combine immunodeficient (SCID) mice with this pseudotype vector significantly inhibits tumor growth. Thus, we show that LAMR can be used as a target in novel therapy for tumor reduction and elimination. PMID:19724263

  12. A simplified laminin nomenclature.

    PubMed

    Aumailley, Monique; Bruckner-Tuderman, Leena; Carter, William G; Deutzmann, Rainer; Edgar, David; Ekblom, Peter; Engel, Jürgen; Engvall, Eva; Hohenester, Erhard; Jones, Jonathan C R; Kleinman, Hynda K; Marinkovich, M Peter; Martin, George R; Mayer, Ulrike; Meneguzzi, Guerrino; Miner, Jeffrey H; Miyazaki, Kaoru; Patarroyo, Manuel; Paulsson, Mats; Quaranta, Vito; Sanes, Joshua R; Sasaki, Takako; Sekiguchi, Kiyotoshi; Sorokin, Lydia M; Talts, Jan F; Tryggvason, Karl; Uitto, Jouni; Virtanen, Ismo; von der Mark, Klaus; Wewer, Ulla M; Yamada, Yoshihiko; Yurchenco, Peter D

    2005-08-01

    A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered. PMID:15979864

  13. Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes

    PubMed Central

    1989-01-01

    We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co- localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes. PMID:2480353

  14. Laminin-111 Inhibits Bovine Fertilization but Improves Embryonic Development in vitro, and Receptor Integrin-β1 is Involved in Sperm-Oocyte Binding.

    PubMed

    Lin, F; Huang, C-J; Liu, C-S; Guo, L-L; Liu, G; Liu, H-J

    2016-10-01

    This study detected the distribution of laminin during embryonic formation by immunofluorescence. To determine the possible function of laminin on developmental ability of in vitro fertilized embryos, the presumptive zygotes were divided and transferred to CR1aa medium supplemented with different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml) of laminin. To explore the association with sperm-oocyte fusion, oocytes and/or sperm were pre-incubated with laminin or anti-β1 antibody before insemination. Laminin was absent in mature oocytes and could be detected first at the 8-cell stage and then displayed an increasing tendency. Adding 10 μg/ml laminin to the culture medium improved embryonic development including cleavage rate, blastocyst rate, total cell numbers in the blastocyst and cell numbers in the inner cell mass. Laminin inhibited sperm-oocyte fusion when incubated with oocytes and/or sperm before in vitro fertilization, and only integrin-β1 of sperm was involved in sperm-oocyte binding. Inhibition may be caused by blocking β1, but why laminin inhibits fertilization is still unknown. The results suggest that laminin plays an important role during embryonic formation and has a negative function in sperm-oocyte fusion, but improves embryonic development. However, only integrin-β1 is involved in sperm-oocyte binding. PMID:27491353

  15. Extraribosomal Functions Associated with the C Terminus of the 37/67 kDa Laminin Receptor are Required for Maintaining Cell Viability

    SciTech Connect

    J Scheiman; K Jamieson; J Ziello; J Tseng; D Meruelo

    2011-12-31

    The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G{sub 1} phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization.

  16. Polyphenols from green tea prevent antineuritogenic action of Nogo-A via 67-kDa laminin receptor and hydrogen peroxide.

    PubMed

    Gundimeda, Usha; McNeill, Thomas H; Barseghian, Barsegh A; Tzeng, William S; Rayudu, David V; Cadenas, Enrique; Gopalakrishna, Rayudu

    2015-01-01

    Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5 μM) or more effectively through a steady-state generation (1-2 μM), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.>

  17. Polyphenols from green tea prevent antineuritogenic action of Nogo-A via 67-kDa laminin receptor and hydrogen peroxide.

    PubMed

    Gundimeda, Usha; McNeill, Thomas H; Barseghian, Barsegh A; Tzeng, William S; Rayudu, David V; Cadenas, Enrique; Gopalakrishna, Rayudu

    2015-01-01

    Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5 μM) or more effectively through a steady-state generation (1-2 μM), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.> PMID

  18. Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

    SciTech Connect

    Byun, Eui-Baek; Choi, Han-Gyu; Sung, Nak-Yun; Byun, Eui-Hong

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited MAPKs activation and NF-{kappa}B p65 translocation via 67LR. Black-Right-Pointing-Pointer EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-{alpha}, interleukin [IL]-1{beta}, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor {kappa}B (NF-{kappa}B) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

  19. The helper-component protease transmission factor of tobacco etch potyvirus binds specifically to an aphid ribosomal protein homologous to the laminin receptor precursor.

    PubMed

    Fernández-Calvino, Lourdes; Goytia, Elisa; López-Abella, Dionisio; Giner, Ana; Urizarna, María; Vilaplana, Lluisa; López-Moya, Juan José

    2010-11-01

    Potyviruses are plant pathogens transmitted by aphids in a non-persistent manner. During transmission, the virus-encoded factor helper-component protease (HCPro) is presumed to act as a molecular bridge, mediating the reversible retention of virions to uncharacterized binding sites in the vector mouthparts. Whilst the predicted interaction between HCPro and the coat protein (CP) of virions has been confirmed experimentally, the characterization of putative HCPro-specific receptors in aphids has remained elusive, with the exception of a report that described binding of HCPro of zucchini yellow mosaic virus to several cuticle proteins. To identify other aphid components that could play a role during transmission, this study used purified HCPro of tobacco etch virus (TEV) in far-Western blotting assays as bait to select interactors among proteins extracted from aphid heads. With this approach, new HCPro-interacting proteins were found, and several were identified after mass spectrometry analysis and searches in databases dedicated to aphid sequences. Among these interactors, a ribosomal protein S2 (RPS2) was chosen for further investigation due to its homology with the laminin receptor precursor, known to act as the receptor of several viruses. The specific interaction between RPS2 and TEV HCPro was confirmed after cloning and heterologous expression of the corresponding Myzus persicae gene. The possible involvement of RPS2 in the transmission process was further suggested by testing a variant of HCPro that was non-functional for transmission due to a mutation in the conserved KITC motif (EITC variant). This variant retained its ability to bind CP but failed to interact with RPS2.

  20. The helper-component protease transmission factor of tobacco etch potyvirus binds specifically to an aphid ribosomal protein homologous to the laminin receptor precursor.

    PubMed

    Fernández-Calvino, Lourdes; Goytia, Elisa; López-Abella, Dionisio; Giner, Ana; Urizarna, María; Vilaplana, Lluisa; López-Moya, Juan José

    2010-11-01

    Potyviruses are plant pathogens transmitted by aphids in a non-persistent manner. During transmission, the virus-encoded factor helper-component protease (HCPro) is presumed to act as a molecular bridge, mediating the reversible retention of virions to uncharacterized binding sites in the vector mouthparts. Whilst the predicted interaction between HCPro and the coat protein (CP) of virions has been confirmed experimentally, the characterization of putative HCPro-specific receptors in aphids has remained elusive, with the exception of a report that described binding of HCPro of zucchini yellow mosaic virus to several cuticle proteins. To identify other aphid components that could play a role during transmission, this study used purified HCPro of tobacco etch virus (TEV) in far-Western blotting assays as bait to select interactors among proteins extracted from aphid heads. With this approach, new HCPro-interacting proteins were found, and several were identified after mass spectrometry analysis and searches in databases dedicated to aphid sequences. Among these interactors, a ribosomal protein S2 (RPS2) was chosen for further investigation due to its homology with the laminin receptor precursor, known to act as the receptor of several viruses. The specific interaction between RPS2 and TEV HCPro was confirmed after cloning and heterologous expression of the corresponding Myzus persicae gene. The possible involvement of RPS2 in the transmission process was further suggested by testing a variant of HCPro that was non-functional for transmission due to a mutation in the conserved KITC motif (EITC variant). This variant retained its ability to bind CP but failed to interact with RPS2. PMID:20631085

  1. Expression and identification of a laminin-binding protein in Aspergillus fumigatus conidia.

    PubMed Central

    Tronchin, G; Esnault, K; Renier, G; Filmon, R; Chabasse, D; Bouchara, J P

    1997-01-01

    Adhesion of Aspergillus fumigatus, the causative agent of human aspergillosis, to the extracellular matrix protein laminin has been previously demonstrated. This study investigated the expression of laminin receptors during swelling of conidia, a step leading to germination and subsequent colonization of tissues. Scanning electron microscopy showed that the laminin binding sites were distributed over the external rodlet layer of resting conidia. During swelling, the characteristic rodlet layer progressively disintegrated and conidia surrounded by a smooth cell wall layer appeared. Flow cytometry using fluorescein isothiocyanate-conjugated laminin demonstrated that expression of laminin receptors at the surface of conidia was swelling dependent. Resting conidia expressed high levels of laminin receptors on their surface. A gradual decrease of laminin binding was then observed as swelling occurred, reaching a minimum for 4-h-swollen conidia. This correlated with a loss of adherence of swollen conidia to laminin immobilized on microtiter plates. Trypsin pretreatment of conidia reduced laminin binding. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting with laminin identified in a cell wall extract a major 72-kDa cell wall glycoprotein which binds laminin. Thus, one of the initial events in the host colonization may be the recognition of basement membrane laminin by this 72-kDa cell wall surface component. PMID:8975886

  2. Attenuated migration by green tea extract (-)-epigallocatechin gallate (EGCG): involvement of 67 kDa laminin receptor internalization in macrophagic cells.

    PubMed

    Ren, Xuezhi; Guo, Xingzhi; Chen, Li; Guo, Minxia; Peng, Ning; Li, Rui

    2014-08-01

    Excessive activation of the microglia in the brain is involved in the development of several neurodegenerative diseases. Previous studies have indicated that (-)-epigallocatechin gallate (EGCG), a major active constituent of green tea, exhibits potent suppressive effects on the activation of microglia. As the 67 kDa laminin receptor (67LR) is a key element in cellular activation and migration, we investigated the effect of EGCG on cell migration and 67LR in lipopolysaccharide (LPS)-activated macrophagic RAW264.7 cells. The presence of EGCG (1-25 μM) markedly attenuated LPS-induced cell migration in a dose-dependent manner. However, the total amount of 67LR protein in the RAW264.7 cells was unaffected by EGCG, as revealed by Western blot analysis. In addition, confocal immunofluorescence microscopy indicated that EGCG caused a marked membrane translocation of 67LR from the membrane surface towards the cytoplasm. Cell-surface biotinylation analysis confirmed that EGCG induced a significant internalization of 67LR by 24-68% in a dose-dependent manner. This study helps to explain the pharmacological action of EGCG on 67LR, suggesting its potential use in the treatment of diseases associated with macrophage/microglia activation, such as neurodegenerative diseases and cancer.

  3. Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity.

    PubMed

    Naidoo, Kerrilyn; Malindisa, Sibusiso T; Otgaar, Tyrone C; Bernert, Martin; Da Costa Dias, Bianca; Ferreira, Eloise; Reusch, Uwe; Knackmuss, Stefan; Little, Melvyn; Weiss, Stefan F T; Letsolo, Boitelo T

    2015-01-01

    Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85-90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity. PMID:26545108

  4. Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8

    PubMed Central

    1990-01-01

    The involvement of integrins in mediating interaction of cells to well- characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody- sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules

  5. Electric Field–directed Cell Motility Involves Up-regulated Expression and Asymmetric Redistribution of the Epidermal Growth Factor Receptors and Is Enhanced by Fibronectin and Laminin

    PubMed Central

    Zhao, Min; Dick, Andrew; Forrester, John V.; McCaig, Colin D.

    1999-01-01

    Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell–substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell–substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor β and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor β and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM. PMID:10198071

  6. Green tea epigallocatechin gallate inhibits insulin stimulation of adipocyte glucose uptake via the 67-kilodalton laminin receptor and AMP-activated protein kinase pathways.

    PubMed

    Hsieh, Chi-Fen; Tsuei, Yi-Wei; Liu, Chi-Wei; Kao, Chung-Cheng; Shih, Li-Jane; Ho, Low-Tone; Wu, Liang-Yi; Wu, Chi-Peng; Tsai, Pei-Hua; Chang, Hsin-Huei; Ku, Hui-Chen; Kao, Yung-Hsi

    2010-10-01

    Insulin and (-)-epigallocatechin gallate (EGCG) are reported to regulate obesity and fat accumulation, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated glucose uptake in 3T3-L1 and C3H10T1/2 adipocytes. EGCG inhibited insulin stimulation of adipocyte glucose uptake in a dose- and time-dependent manner. The concentration of EGCG that decreased insulin-stimulated glucose uptake by 50-60% was approximately 5-10 µM for a period of 2 h. At 10 µM, EGCG and gallic acid were more effective than (-)-epicatechin, (-)-epigallocatechin, and (-)-epicatechin 3-gallate. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and extended the findings for this study to clarify whether EGCG-induced changes in insulin-stimulated glucose uptake in adipocytes could be mediated through the 67LR. Pretreatment of adipocytes with a 67LR antibody, but not normal rabbit immunoglobulin, prevented the effects of EGCG on insulin-increased glucose uptake. This suggests that the 67LR mediates the effect of EGCG on insulin-stimulated glucose uptake in adipocytes. Moreover, pretreatment with an AMP-activated protein kinase (AMPK) inhibitor, such as compound C, but not with a glutathione (GSH) activator, such as N-acetyl-L-cysteine (NAC), blocked the antiinsulin effect of EGCG on adipocyte glucose uptake. These data suggest that EGCG exerts its anti-insulin action on adipocyte glucose uptake via the AMPK, but not the GSH, pathway. The results of this study possibly support that EGCG mediates fat content.

  7. Dystroglycan loss disrupts polarity and beta-casein induction inmammary epithelial cells by perturbing laminin anchoring

    SciTech Connect

    Weir, M. Lynn; Oppizzi, Maria Luisa; Henry, Michael D.; Onishi,Akiko; Campbell, Kevin P.; Bissell, Mina J.; Muschler, John L.

    2006-02-17

    Precise contact between epithelial cells and their underlying basement membrane is critical to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG expression in cultured mammary epithelial cells (MECs). Strikingly, DG loss disrupted laminin-111-induced polarity and {beta}-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. DG re-expression restored these deficiencies. Investigations of mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when the entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in MECs that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity, and {beta}-casein induction. The observed loss of laminin-111 assembly and signaling in DG-/-MECs provides insights into the signaling changes occurring in breast carcinomas and other cancers, where DG's laminin-binding function is frequently defective.

  8. Nucleus pulposus cell-matrix interactions with laminins.

    PubMed

    Gilchrist, C L; Francisco, A T; Plopper, G E; Chen, J; Setton, L A

    2011-01-01

    The cells of the nucleus pulposus (NP) region of the intervertebral disc play a critical role in this tissue's generation and maintenance, and alterations in NP cell viability, metabolism, and phenotype with aging may be key contributors to progressive disc degeneration. Relatively little is understood about the phenotype of NP cells, including their cell-matrix interactions which may modulate phenotype and survival. Our previous work has identified strong and region-specific expression of laminins and laminin cell-surface receptors in immature NP tissues, suggesting laminin cell-matrix interactions are uniquely important to the biology of NP cells. Whether these observed tissue-level laminin expression patterns reflect functional adhesion behaviors for these cells is not known. In this study, we examined NP cell-matrix interactions with specific matrix ligands, including various laminin isoforms, using quantitative assays of cell attachment, spreading, and adhesion strength. NP cells were found to attach in higher numbers and exhibited rapid cell spreading and higher resistance to detachment force on two laminin isoforms (LM-511,LM-332) identified to be uniquely expressed in the NP region, as compared to another laminin isoform (LM-111) and several other matrix ligands (collagen, fibronectin). Additionally, NP cells were found to attach in higher numbers to laminins as compared to cells isolated from the disc's annulus fibrosus region. These findings confirm that laminin and laminin receptor expression documented in NP tissues translates into unique functional NP cell adhesion behaviors that may be useful tools for in vitro cell culture and biomaterials that support NP cells.

  9. Angiotensin II type 1 receptor antagonists alleviate muscle pathology in the mouse model for laminin-α2-deficient congenital muscular dystrophy (MDC1A)

    PubMed Central

    2012-01-01

    Background Laminin-α2-deficient congenital muscular dystrophy (MDC1A) is a severe muscle-wasting disease for which no curative treatment is available. Antagonists of the angiotensin II receptor type 1 (AT1), including the anti-hypertensive drug losartan, have been shown to block also the profibrotic action of transforming growth factor (TGF)-β and thereby ameliorate disease progression in mouse models of Marfan syndrome. Because fibrosis and failure of muscle regeneration are the main reasons for the severe disease course of MDC1A, we tested whether L-158809, an analog derivative of losartan, could ameliorate the dystrophy in dyW/dyW mice, the best-characterized model of MDC1A. Methods L-158809 was given in food to dyW/dyW mice at the age of 3 weeks, and the mice were analyzed at the age of 6 to 7 weeks. We examined the effect of L-158809 on muscle histology and on muscle regeneration after injury as well as the locomotor activity and muscle strength of the mice. Results We found that TGF-β signaling in the muscles of the dyW/dyW mice was strongly increased, and that L-158809 treatment suppressed this signaling. Consequently, L-158809 reduced fibrosis and inflammation in skeletal muscle of dyW/dyW mice, and largely restored muscle regeneration after toxin-induced injury. Mice showed improvement in their locomotor activity and grip strength, and their body weight was significantly increased. Conclusion These data provide evidence that AT1 antagonists ameliorate several hallmarks of MDC1A in dyW/dyW mice, the best-characterized mouse model for this disease. Because AT1 antagonists are well tolerated in humans and widely used in clinical practice, these results suggest that losartan may offer a potential future treatment of patients with MDC1A. PMID:22943509

  10. Structure and function of laminin LG modules.

    PubMed

    Timpl, R; Tisi, D; Talts, J F; Andac, Z; Sasaki, T; Hohenester, E

    2000-08-01

    Laminin G domain-like (LG) modules of approximately 180-200 residues are found in a number of extracellular and receptor proteins and often are present in tandem arrays. LG modules are implicated in interactions with cellular receptors (integrins, alpha-dystroglycan), sulfated carbohydrates and other extracellular ligands. The recently determined crystal structures of LG modules of the laminin alpha2 chain reveal a compact beta sandwich fold and identify a novel calcium binding site. Binding epitopes for heparin, sulfatides and alpha-dystroglycan have been mapped by site-directed mutagenesis and show considerable overlap. The epitopes are located in surface loops around the calcium site, which in other proteins (agrin, neurexins) are modified by alternative splicing. Efficient ligand binding often requires LG modules to be present in tandem. The close proximity of the N- and C-termini in the LG module, as well as a unique link region between laminin LG3 and LG4, impose certain constraints on the arrangement of LG tandems. Further modifications may be introduced by proteolytic processing of laminin G domains, which is known to occur in the alpha2, alpha3 and alpha4 chains.

  11. Interaction of human laminin receptor with Sup35, the [PSI⁺] prion-forming protein from S. cerevisiae: a yeast model for studies of LamR interactions with amyloidogenic proteins.

    PubMed

    Pampeno, Christine; Derkatch, Irina L; Meruelo, Daniel

    2014-01-01

    The laminin receptor (LamR) is a cell surface receptor for extracellular matrix laminin, whereas the same protein within the cell interacts with ribosomes, nuclear proteins and cytoskeletal fibers. LamR has been shown to be a receptor for several bacteria and viruses. Furthermore, LamR interacts with both cellular and infectious forms of the prion protein, PrP(C) and PrP(Sc). Indeed, LamR is a receptor for PrP(C). Whether LamR interacts with PrP(Sc) exclusively in a capacity of the PrP receptor, or LamR specifically recognizes prion determinants of PrP(Sc), is unclear. In order to explore whether LamR has a propensity to interact with prions and amyloids, we examined LamR interaction with the yeast prion-forming protein, Sup35. Sup35 is a translation termination factor with no homology or functional relationship to PrP. Plasmids expressing LamR or LamR fused with the green fluorescent protein (GFP) were transformed into yeast strain variants differing by the presence or absence of the prion conformation of Sup35, respectively [PSI⁺] and [psi⁻]. Analyses by immunoprecipitation, centrifugal fractionation and fluorescent microscopy reveal interaction between LamR and Sup35 in [PSI⁺] strains. The presence of [PSI⁺] promotes LamR co-precipitation with Sup35 as well as LamR aggregation. In [PSI⁺] cells, LamR tagged with GFP or mCherry forms bright fluorescent aggregates that co-localize with visible [PSI⁺] foci. The yeast prion model will facilitate studying the interaction of LamR with amyloidogenic prions in a safe and easily manipulated system that may lead to a better understanding and treatment of amyloid diseases.

  12. Laminin alpha5 chain is required for intestinal smooth muscle development.

    PubMed

    Bolcato-Bellemin, Anne Laure; Lefebvre, Olivier; Arnold, Christiane; Sorokin, Lydia; Miner, Jeffrey H; Kedinger, Michèle; Simon-Assmann, Patricia

    2003-08-15

    Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent.

  13. Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5, and migrate on the newly deposited basement membrane.

    PubMed Central

    Tani, T.; Lumme, A.; Linnala, A.; Kivilaakso, E.; Kiviluoto, T.; Burgeson, R. E.; Kangas, L.; Leivo, I.; Virtanen, I.

    1997-01-01

    We studied the adhesion mechanism of pancreatic carcinoma using in vitro adhesion and migration assays of stable cell lines and tumors grown from these cell lines in nude mice. We also compared the results with the expression profiles of laminins and their receptors in pancreatic carcinomas to evaluate the relevance of these mechanisms in vivo. All of the cell lines preferably adhered to laminin-5, irrespective of their capability to synthesize laminin-5. Cell migration was studied in the presence of hepatocyte growth factor, as it increased the speed of migration manyfold. Herbimycin A treatment and antibodies against the beta 1 and alpha 3 integrin subunits and laminin alpha 3 chain almost entirely blocked cell migration of the BxPC-3 cell line, whereas migration was nearly unaffected by RGD peptide and only moderately inhibited by antibody against the alpha 6 integrin subunit. Indirect immunofluorescence microscopy of wounded BxPC-3 cells suggested a rapid endocytosis of alpha 3 integrin subunit in the cells at the margin of the wound and a rapid, polarized rearrangement of the alpha 6 beta 4 integrin. Especially HGF-treated cultures showed a prominent cytoplasmic reaction for laminin-5 at the margin of the wound. Xenografted cells formed tumors that produced and deposited the same laminin chains as the in vitro cultures. Frozen sections of human pancreatic carcinomas showed reactivity for laminin chains suggestive for expression of laminin-1 and laminin-5. Both xenografted tumors and human pancreatic carcinomas also showed stromal reactivity for laminin-5. Electron microscopy of the human tumors suggested that this was due to an abundant reduplication the basement-membrane-like material around the nests of malignant cells. Our results suggest that pancreatic carcinomas synthesize and deposit laminin-5 in the basement membrane in an abnormal manner. Invading cells adhere to this newly produced basement membrane and migrate on it by using the alpha 3 beta 1

  14. Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors

    PubMed Central

    1994-01-01

    Kalinin was purified from squamous cell carcinoma (SCC25) spent culture media using an immunoaffinity column prepared from the mAb BM165. The affinity-purified material was separated by SDS-PAGE into three bands of 165-155, 140, and 105 kD identical to those obtained from normal human keratinocyte cultures and previously identified as kalinin. Kalinin promoted adhesion of a large number of normal cells and established cell lines with an activity similar to other adhesion molecules such as the laminin-nidogen complex, fibronectin, or collagen IV. However, kalinin was a much better substrate than laminin-nidogen complex for adhesion of cells of epithelial origin including primary human keratinocytes. Adhesion to kalinin was followed by cell shape changes ranging from rounded to fully spread cells depending on the cell types. The adhesion-promoting activity of kalinin was conformation dependent and was abolished by heat denaturation. mAb BM165 prevented cell adhesion to kalinin but not to other extracellular matrix substrates. However, either complete or partial inhibition was observed with different cells suggesting the existence of at least two cell- binding sites on the kalinin molecule. Experiments inhibiting cell adhesion with function-blocking anti-integrin subunit antibodies indicated that both alpha 3 beta 1 and alpha 6 beta 1 integrins are involved in the cellular interactions with kalinin, while for cell adhesion to classical mouse Engelbreth-Holm-Swarm laminin only alpha 6 beta 1 integrins, and not alpha 3 beta 1, appeared to be functional. Altogether, these results suggest that kalinin may fulfill additional functions than laminin, particularly for epithelial cells. PMID:8138572

  15. The 37/67kDa laminin receptor (LR) inhibitor, NSC47924, affects 37/67kDa LR cell surface localization and interaction with the cellular prion protein

    PubMed Central

    Sarnataro, Daniela; Pepe, Anna; Altamura, Gennaro; De Simone, Imma; Pesapane, Ada; Nitsch, Lucio; Montuori, Nunzia; Lavecchia, Antonio; Zurzolo, Chiara

    2016-01-01

    The 37/67 kDa laminin receptor (LR) is a non-integrin protein, which binds both laminin-1 of the extracellular matrix and prion proteins, that hold a central role in prion diseases. The 37/67 kDa LR has been identified as interactor for the prion protein (PrPC) and to be required for pathological PrP (PrPSc) propagation in scrapie-infected neuronal cells, leading to the possibility that 37/67 kDa LR-PrPC interaction is related to the pathogenesis of prion diseases. A relationship between 37/67 kDa LR and PrPC in the presence of specific LR inhibitor compounds has not been investigated yet. We have characterized the trafficking of 37/67 kDa LR in both neuronal and non-neuronal cells, finding the receptor on the cell surface and nuclei, and identified the 67 kDa LR as the almost exclusive isoform interacting with PrPC. Here, we show that the treatment with the 37/67 kDa LR inhibitor, NSC47924, affects both the direct 37/67 kDa LR-PrPC interaction in vitro and the formation of the immunocomplex in live cells, inducing a progressive internalization of 37/67 kDa LR and stabilization of PrPC on the cell surface. These data reveal NSC47924 as a useful tool to regulate PrPC and 37/67 kDa LR trafficking and degradation, representing a novel small molecule to be tested against prion diseases. PMID:27071549

  16. [Expression of human LSB 32/67 KD gene in E. coli and analysis of its interactions with laminin].

    PubMed

    Siianova, E Iu

    1992-01-01

    Earlier we have cloned cDNA coding for a polypeptide that reacts with monoclonal antibodies specific for some cytoskeleton structures. This gene is homologous to the laminin receptor 67 KD. However, cDNA suffices only for a polypeptide of 32 Da, far smaller than the 67 rDa laminin receptor. We have constructed a vector that produces the fusion protein LBP-TrpE in the bacterial strain CAG-456. Our studies show that hybrid protein LBP-TrpE is able to interact with laminin. This result was confirmed by using following methods: immunoblotting, "ELISA" and affinity chromatography on laminin.

  17. Neuronal migration on laminin in vitro.

    PubMed

    Liang, S; Crutcher, K A

    1992-03-20

    Chick sympathetic (E-9) or telencephalic (E-7) neurons were cultured at low density on poly-DL-ornithine (PORN), poly-L-lysine (POLS), laminin or laminin-covered PORN or POLS and monitored with time-lapse videomicroscopy. Neurons migrated on laminin, or laminin-covered PORN or POLS, but not on PORN or POLS alone. Neuronal migration did not involve interactions with other cells indicating that neurons are capable of independent migration when exposed to a laminin substrate.

  18. Neuronal migration on laminin in vitro.

    PubMed

    Liang, S; Crutcher, K A

    1992-03-20

    Chick sympathetic (E-9) or telencephalic (E-7) neurons were cultured at low density on poly-DL-ornithine (PORN), poly-L-lysine (POLS), laminin or laminin-covered PORN or POLS and monitored with time-lapse videomicroscopy. Neurons migrated on laminin, or laminin-covered PORN or POLS, but not on PORN or POLS alone. Neuronal migration did not involve interactions with other cells indicating that neurons are capable of independent migration when exposed to a laminin substrate. PMID:1600626

  19. Aberrant Development of Thymocytes in Mice Lacking Laminin-2

    PubMed Central

    Magner, William J.; Chang, Andrew C.; Owens, Jennie; Hong, M-J. P.; Brooks, Andrew

    2000-01-01

    In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin- 2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4-CD8-) and SP (CD4+CD8-, CD4-CD8+) populations and a significant decrease in the DP (CD4+CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice. PMID:11097211

  20. Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity

    NASA Astrophysics Data System (ADS)

    Yao, Yao; Chen, Zu-Lin; Norris, Erin H.; Strickland, Sidney

    2014-03-01

    Blood brain barrier (BBB) breakdown is not only a consequence of but also contributes to many neurological disorders, including stroke and Alzheimer’s disease. How the basement membrane (BM) contributes to the normal functioning of the BBB remains elusive. Here we use conditional knockout mice and an acute adenovirus-mediated knockdown model to show that lack of astrocytic laminin, a brain-specific BM component, induces BBB breakdown. Using functional blocking antibody and RNAi, we further demonstrate that astrocytic laminin, by binding to integrin α2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, and thus maintains the integrity of BBB. Additionally, loss of astrocytic laminin decreases aquaporin-4 (AQP4) and tight junction protein expression. Altogether, we report a critical role for astrocytic laminin in BBB regulation and pericyte differentiation. These results indicate that astrocytic laminin maintains the integrity of BBB through, at least in part, regulation of pericyte differentiation.

  1. Alpha 1 beta 1 integrin on neural crest cells recognizes some laminin substrata in a Ca(2+)-independent manner

    PubMed Central

    1992-01-01

    Neural crest cells migrate along pathways containing laminin and other extracellular matrix molecules. In the present study, we functionally and biochemically identify an alpha 1 beta 1 integrin heterodimer which bears the HNK-1 epitope on neural crest cells. Using a quantitative cell adhesion assay, we find that this heterodimer mediates attachment to laminin substrata prepared in the presence of Ca2+. Interestingly, neural crest cells bind to laminin-Ca2+ substrata in the presence or absence of divalent cations in the cell attachment medium. In contrast, the attachment of neural crest cells to laminin substrata prepared in the presence of EDTA, heparin, Mg2+, or Mn2+ requires divalent cations. Interactions with these laminin substrata are mediated by a different integrin heterodimer, since antibodies against beta 1 but not alpha 1 integrins inhibit neural crest cell attachment. Thus, the type of laminin substratum appears to dictate the choice of laminin receptor used by neural crest cells. The laminin conformation is determined by the ratio of laminin to Ca2+, though incorporation of heparin during substratum polymerization alters the conformation even in the presence of Ca2+. Once polymerized, the substratum appears stable, not being altered by soaking in either EDTA or divalent cations. Our findings demonstrate: (a) that the alpha 1 beta 1 integrin can bind to some forms of laminin in the absence of soluble divalent cations; (b) that substratum preparation conditions alter the conformation of laminin such that plating laminin in the presence of Ca2+ and/or heparin modulates its configuration; and (c) that neural crest cells utilize different integrins to recognize different laminin conformations. PMID:1280273

  2. High Resolution Imaging Study of Interactions between the 37 kDa/67 kDa Laminin Receptor and APP, Beta-Secretase and Gamma-Secretase in Alzheimer's Disease

    PubMed Central

    Jovanovic, Katarina; Loos, Ben; Da Costa Dias, Bianca; Penny, Clement; Weiss, Stefan F. T.

    2014-01-01

    Alzheimer's disease (AD) is the most prevalent form of dementia affecting the elderly. Neurodegeneration is caused by the amyloid beta (Aβ) peptide which is generated from the sequential proteolytic cleavage of the Amyloid Precursor Protein (APP) by the β– and γ- secretases. Previous reports revealed that the 37 kDa/67 kDa laminin receptor (LRP/LR) is involved in APP processing, however, the exact mechanism by which this occurs remains largely unclear. This study sought to assess whether LRP/LR interacted with APP, β- or γ-secretase. Detailed confocal microscopy revealed that LRP/LR showed a strong co-localisation with APP, β- and γ-secretase, respectively, at various sub-cellular locations. Superresolution Structured Illumination Microscopy (SR-SIM) showed that interactions were unlikely between LRP/LR and APP and β-secretase, respectively, while there was strong co-localisation between LRP/LR and γ-secretase at this 80 nm resolution. FRET was further employed to assess the possibility of protein-protein interactions and only an interaction between LRP/LR and γ-secretase was found. FLAG co-immunoprecipitation confirmed these findings as LRP/LR co-immunoprecipitated with γ-secretase, but failed to do so with APP. These findings indicate that LRP/LR exerts its influence on Aβ shedding via a direct interaction with the γ-secretase and possibly an indirect interaction with the β-secretase. PMID:24972054

  3. Inhibition of laminin-5 production in breast epithelial cells by overexpression of p300.

    PubMed

    Miller, K A; Chung, J; Lo, D; Jones, J C; Thimmapaya, B; Weitzman, S A

    2000-03-17

    The transcriptional coactivator p300 is essential for normal embryonic development and cellular differentiation. We have been studying the role of p300 in the transcription of a variety of genes, and we became interested in the role of this coactivator in the transcription of genes important in breast epithelial cell biology. From MCF-10A cells (spontaneously immortalized, nontransformed human breast epithelial cells), we developed cell lines that stably overexpress p300. These p300-overexpressing cells displayed reduced adhesion to culture dishes and were found to secrete an extracellular matrix deficient in laminin-5. Laminin-5 is the major extracellular matrix component produced by breast epithelium. Immunofluorescence studies, as well as experiments using normal matrix, confirmed that the decreased adhesion of p300-overexpressing cells is due to laminin-5-deficient extracellular matrix and not due to loss of laminin-5 receptors. Northern blots revealed markedly decreased levels of expression of two of the genes (designated LAMA3 and LAMC2) encoding the alpha3 and gamma2 chains of the laminin-5 heterotrimer in the cells that overexpress p300, whereas LAMB3 mRNA, encoding the third or beta3 chain of laminin-5, was not markedly reduced. Transient transfection experiments with a vector containing a murine LAMA3 promoter demonstrate that overexpressing p300 down-regulates the LAMA3 promoter. In summary, overexpression of p300 leads to down-regulation of laminin-5 production in breast epithelial cells, resulting in decreased adhesion. PMID:10713141

  4. Laminin promotes metalloproteinase-mediated dystroglycan processing to regulate oligodendrocyte progenitor cell proliferation.

    PubMed

    Leiton, Cindy V; Aranmolate, Azeez; Eyermann, Christopher; Menezes, Michael J; Escobar-Hoyos, Luisa F; Husain, Solomon; Winder, Steve J; Colognato, Holly

    2015-11-01

    The cell surface receptor dystroglycan mediates interactions between oligodendroglia and laminin-211, an extracellular matrix protein that regulates timely oligodendroglial development. However, dystroglycan's precise role in oligodendroglial development and the potential mechanisms to regulate laminin-dystroglycan interactions remain unknown. Here we report that oligodendroglial dystroglycan is cleaved by metalloproteinases, thereby uncoupling oligodendroglia from laminin binding. Dystroglycan cleavage is selectively stimulated by oligodendrocyte progenitor cell attachment to laminin-211, but not laminin-111 or poly-D-lysine. In addition, dystroglycan cleavage occurs most prominently in oligodendrocyte progenitor cells, with limited dystroglycan cleavage observed in differentiating oligodendrocytes. When dystroglycan cleavage is blocked by metalloproteinase inhibitors, oligodendrocyte progenitor cell proliferation is substantially decreased. Conversely, expression of the intracellular portion of cleaved dystroglycan results in increased oligodendrocyte progenitor cell proliferation, suggesting that endogenous dystroglycan cleavage may promote oligodendrocyte progenitor cell cycle progression. Intriguingly, while matrix metalloproteinase-2 and/or -9 have been reported to be responsible for dystroglycan cleavage, we find that these two metalloproteinases are neither necessary nor sufficient for cleavage of oligodendroglial dystroglycan. In summary, laminin-211 stimulates metalloproteinase-mediated dystroglycan cleavage in oligodendrocyte progenitor cells (but not in differentiated oligodendrocytes), which in turn promotes oligodendrocyte progenitor cell proliferation. This novel regulation of oligodendroglial laminin-dystroglycan interactions may have important consequences for oligodendroglial differentiation, both during development and during disease when metalloproteinase levels become elevated.

  5. Variable region structure and staphylococcal protein A binding specificity of a mouse monoclonal IgM anti-laminin-receptor antibody.

    PubMed Central

    Feijó, G C; Sabbaga, J; Carneiro, C R; Brígido, M M

    1997-01-01

    Staphylococcal protein A is a cell wall-attached polypeptide that acts as a B-lymphocyte superantigen. This activation correlates with specific VH gene segment usage in the B-cell receptor. B-cell receptor assembled from members of the VH3 family in humans, or S107 family in mice, has an intrinsic affinity for protein A. Human VH3-derived antibodies bind to domain D of protein A. We have characterized a mouse IgM monoclonal antibody that binds protein A. The sequencing of the variable region suggests an almost germline-encoded VH derived from S107 family and a V kappa 8-derived VL. The binding specificity of the monoclonal antibody was tested with various recombinant constructions derived from protein A. We show that, unlike human VH3-derived antibody, mouse S107-derived immunoglobulin binds to the B domain of the bacterial superantigen. PMID:9301540

  6. Green tea polyphenols precondition against cell death induced by oxygen-glucose deprivation via stimulation of laminin receptor, generation of reactive oxygen species, and activation of protein kinase Cε.

    PubMed

    Gundimeda, Usha; McNeill, Thomas H; Elhiani, Albert A; Schiffman, Jason E; Hinton, David R; Gopalakrishna, Rayudu

    2012-10-01

    As the development of synthetic drugs for the prevention of stroke has proven challenging, utilization of natural products capable of preconditioning neuronal cells against ischemia-induced cell death would be a highly useful complementary approach. In this study using an oxygen-glucose deprivation and reoxygenation (OGD/R) model in PC12 cells, we show that 2-day pretreatment with green tea polyphenols (GTPP) and their active ingredient, epigallocatechin-3-gallate (EGCG), protects cells from subsequent OGD/R-induced cell death. A synergistic interaction was observed between GTPP constituents, with unfractionated GTPP more potently preconditioning cells than EGCG. GTPP-induced preconditioning required the 67-kDa laminin receptor (67LR), to which EGCG binds with high affinity. 67LR also mediated the generation of reactive oxygen species (ROS) via activation of NADPH oxidase. An exogenous ROS-generating system bypassed 67LR to induce preconditioning, suggesting that sublethal levels of ROS are indeed an important mediator in GTPP-induced preconditioning. This role for ROS was further supported by the fact that antioxidants blocked GTPP-induced preconditioning. Additionally, ROS induced an activation and translocation of protein kinase C (PKC), particularly PKCε from the cytosol to the membrane/mitochondria, which was also blocked by antioxidants. The crucial role of PKC in GTPP-induced preconditioning was supported by use of its specific inhibitors. Preconditioning was increased by conditional overexpression of PKCε and decreased by its knock-out with siRNA. Collectively, these results suggest that GTPP stimulates 67LR and thereby induces NADPH oxidase-dependent generation of ROS, which in turn induces activation of PKC, particularly prosurvival isoenzyme PKCε, resulting in preconditioning against cell death induced by OGD/R.

  7. Laminin 411 and 511 promote the cholangiocyte differentiation of human induced pluripotent stem cells.

    PubMed

    Takayama, Kazuo; Mitani, Seiji; Nagamoto, Yasuhito; Sakurai, Fuminori; Tachibana, Masashi; Taniguchi, Yukimasa; Sekiguchi, Kiyotoshi; Mizuguchi, Hiroyuki

    2016-05-20

    The drug discovery research for cholestatic liver diseases has been hampered by the lack of a well-established human cholangiocyte model. Functional cholangiocyte-like cells differentiated from human induced pluripotent stem (iPS) cells are expected to be a promising candidate for such research, but there remains no well-established method for differentiating cholangiocytes from human iPS cells. In this study, we searched for a suitable extracellular matrix to promote cholangiocyte differentiation from human iPS cells, and found that both laminin 411 and laminin 511 were suitable for this purpose. The gene expression levels of the cholangiocyte markers, aquaporin 1 (AQP1), SRY-box 9 (SOX9), cystic fibrosis transmembrane conductance regulator (CFTR), G protein-coupled bile acid receptor 1 (GPBAR1), Jagged 1 (JAG1), secretin receptor (SCTR), and γ-glutamyl transferase (GGT1) were increased by using laminin 411 or laminin 511 as a matrix. In addition, the percentage of AQP1-positive cells was increased from 61.8% to 92.5% by using laminin 411 or laminin 511. Furthermore, the diameter and number of cysts consisted of cholangiocyte-like cells were increased when using either matrix. We believe that the human iPS cell-derived cholangiocyte-like cells, which were generated by using our differentiation technology, would be useful for the drug discovery research of cholestatic liver diseases. PMID:27103433

  8. Loss of cell-surface laminin anchoring promotes tumor growth and is associated with poor clinical outcomes.

    PubMed

    Akhavan, Armin; Griffith, Obi L; Soroceanu, Liliana; Leonoudakis, Dmitri; Luciani-Torres, Maria Gloria; Daemen, Anneleen; Gray, Joe W; Muschler, John L

    2012-05-15

    Perturbations in the composition and assembly of extracellular matrices (ECM) contribute to progression of numerous diseases, including cancers. Anchoring of laminins at the cell surface enables assembly and signaling of many ECMs, but the possible contributions of altered laminin anchoring to cancer progression remain undetermined. In this study, we investigated the prominence and origins of defective laminin anchoring in cancer cells and its association with cancer subtypes and clinical outcomes. We found loss of laminin anchoring to be widespread in cancer cells. Perturbation of laminin anchoring originated from several distinct defects, which all led to dysfunctional glycosylation of the ECM receptor dystroglycan. In aggressive breast and brain cancers, defective laminin anchoring was often due to suppressed expression of the glycosyltransferase LARGE. Reduced expression of LARGE characterized a broad array of human tumors in which it was associated with aggressive cancer subtypes and poor clinical outcomes. Notably, this defect robustly predicted poor survival in patients with brain cancers. Restoring LARGE expression repaired anchoring of exogenous and endogenous laminin and modulated cell proliferation and tumor growth. Together, our findings suggest that defects in laminin anchoring occur commonly in cancer cells, are characteristic of aggressive cancer subtypes, and are important drivers of disease progression.

  9. Laminins: Roles and Utility in Wound Repair

    PubMed Central

    Iorio, Valentina; Troughton, Lee D.; Hamill, Kevin J.

    2015-01-01

    Significance: Laminins are complex extracellular macromolecules that are major players in the control of a variety of core cell processes, including regulating rates of cell proliferation, differentiation, adhesion, and migration. Laminins, and related extracellular matrix components, have essential roles in tissue homeostasis; however, during wound healing, the same proteins are critical players in re-epithelialization and angiogenesis. Understanding how these proteins influence cell behavior in these different conditions holds great potential in identifying new strategies to enhance normal wound closure or to treat chronic/nonhealing wounds. Recent Advances: Laminin-derived bioactive peptides and, more recently, laminin-peptide conjugated scaffolds, have been designed to improve tissue regeneration after injuries. These peptides have been shown to be effective in decreasing inflammation and granulation tissue, and in promoting re-epithelialization, angiogenesis, and cell migration. Critical Issues: Although there is now a wealth of knowledge concerning laminin form and function, there are still areas of some controversy. These include the relative contribution of two laminin-based adhesive devices (focal contacts and hemidesmosomes) to the re-epithelialization process, the impact and implications of laminin proteolytic processing, and the importance of laminin polymer formation on cell behavior. In addition, the roles in wound healing of the laminin-related proteins, netrins, and LaNts are still to be fully defined. Future Directions: The future of laminin-based therapeutics potentially lies in the bioengineering of specific substrates to support laminin deposition for ex vivo expansion of autologous cells for graft formation and transplantation. Significant recent advances suggest that this goal is within sight. PMID:25945287

  10. Laminin and Fibronectin in Cell Adhesion: Enhanced Adhesion of Cells from Regenerating Liver to Laminin

    NASA Astrophysics Data System (ADS)

    Carlsson, Roland; Engvall, Eva; Freeman, Aaron; Ruoslahti, Erkki

    1981-04-01

    Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing regeneration. After regeneration, the attachment to laminin returned to the low levels found in animals not subjected to partial hepatectomy but attachment to fibronectin remained high. Immunofluorescent staining of sections of normal liver with antilaminin revealed the presence of laminin in or adjacent to the walls of the bile ducts and blood vessels. After induction of regeneration by partial hepatectomy, increased amounts of laminin appeared in the sinusoidal areas. After carbon tetrachloride poisoning, staining for laminin was especially pronounced in the necrotic and postnecrotic areas around the central veins. This additional expression of laminin was transient. It reached a maximum around 5-6 days after the injury and then gradually disappeared. These findings show that laminin is an adhesive protein. The increase of laminin in regenerating liver and the adhesiveness of cells from such livers to laminin suggest a role for laminin in the maintenance of a proper tissue organization during liver regeneration.

  11. Human laminin B2 chain

    SciTech Connect

    Pikkarainen, T.; Kallunki, T.; Tryggvason, K.

    1988-05-15

    The complete amino acid sequence of the human laminin B2 chains has been determined by sequencing of cDNA clones. The six overlapping clones studied cover approximately 7.5 kilobases of which 5312 nucleotides were sequenced from the 5' end. The open reading frame codes for a 33-residue signal peptide and a 1576-residue B2 chain proper, which is 189 residues less than in the highly homologous B1 chain. Computer analysis revealed that the B2 chain consists of distinct domains that contain helical structures, cysteine-rich repeats, and globular regions, as does the B1 chain. However, domain ..cap alpha.. and domain ..beta.. of the B1 chain have no counterpart in B2, and the number of cysteine-rich repeats is 12, or 1 less than in the B1 chain. The degree of homology between the two chains is highest in the cysteine repeat-containing domains III and V where 40% of the residues match. However, in helical domains I/II only 16% of residues match. The results demonstrate that the B1 and B2 chains of laminin are highly homologous proteins that are probably the products of related genes.

  12. The adaptor protein LAD/TSAd mediates laminin-dependent T cell migration via association with the 67 kDa laminin binding protein

    PubMed Central

    Park, Eunkyung; Choi, Youngbong; Ahn, Eunseon; Park, Inyoung

    2009-01-01

    The adaptor protein, LAD/TSAd, plays essential roles in T cell activation. To further understand the functions of this protein, we performed yeast two-hybrid screening using TSAd as bait and identified 67 kDa laminin binding protein (LBP) as the interacting partner. Subsequently, TSAd-LBP interaction was confirmed in D1.1 T cell line. Upon costimulation by T cell receptor (TCR) plus laminin crosslinking or TCR plus integrin α6 crosslinking, LBP was coimmunoprecipitated with TSAd. Moreover, TCR plus laminin costimulation-dependent T cell migration was enhanced in D1.1 T cells overexpressing TSAd but was disrupted in D1.1 cells overexpressing dominant negative form of TSAd or TSAd shRNA. These data show that, upon TCR plus integrin costimulation, TSAd associates with LBP and mediates T lymphocyte migration. PMID:19561400

  13. Screening of integrin-binding peptides in a laminin peptide library derived from the mouse laminin β chain short arm regions.

    PubMed

    Katagiri, Fumihiko; Takagi, Masaharu; Nakamura, Minako; Tanaka, Yoichiro; Hozumi, Kentaro; Kikkawa, Yamato; Nomizu, Motoyoshi

    2014-05-15

    Laminins, major components of basement membrane, consist of three different subunits, α, β, and γ chains, and so far, five α, three β, and three γ chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin α chain sequences (α1-α5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the β1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin β2 and β3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin β1 chain, 255-266), B67 (IPYSMEYEILIRY, mouse laminin β1 chain, 604-616), B2-105 (APNFWNFTSGRG, mouse laminin β2 chain, 1081-1092), and B3-19 (GHLTGGKVQLNL, mouse laminin β3 chain, 182-193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2-105, and B3-19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with α3, α6, and β1 integrins, and B34 and B3-19 interact with β1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2-64 (LPRAMDYDLLLRW, mouse laminin β2 chain, 618-630), the Glu(8) residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide

  14. Distinct and overlapping ligand specificities of the alpha 3A beta 1 and alpha 6A beta 1 integrins: recognition of laminin isoforms.

    PubMed Central

    Delwel, G O; de Melker, A A; Hogervorst, F; Jaspars, L H; Fles, D L; Kuikman, I; Lindblom, A; Paulsson, M; Timpl, R; Sonnenberg, A

    1994-01-01

    The ligand specificity of the alpha 3A beta 1 integrin was analyzed using K562 cells transfected with full-length alpha 3A cDNA and was compared with that of alpha 6A beta 1 in similarly transfected K562 cells. Clones were obtained that showed comparable surface expression of either alpha 3A beta 1 or alpha 6A beta 1 integrins. Those expressing alpha 3A beta 1 attached to and spread on immunopurified human kalinin and cellular matrices containing human kalinin, which is a particular isoform of laminin. In addition, alpha 3A transfectants adhered to bovine kidney laminins possessing a novel A chain variant. Binding to kalinin was blocked by a monoclonal antibody against the A chain constituent of kalinin and adhesion to both kalinin and kidney laminins by anti-alpha 3 and beta 1 monoclonal antibodies. The alpha 3A transfected cells bound more strongly to kalinin and bovine kidney laminins after treatment with the beta 1 stimulatory antibody TS2/16. A distinctly weaker and activation-dependent adhesion of alpha 3A transfectants was observed on human placental laminins possessing the Am chain variant (merosin), and no adhesion occurred on bovine heart laminins and murine EHS tumor laminin. Further inactive substrates were fibronectin, nidogen, and collagen types IV and VI, indicating that the alpha 3A beta 1 integrin is a much less promiscuous receptor than thought before. By contrast, alpha 6A transfected cells adhered to all laminin isoforms when stimulated with TS2/16. Adhesion also occurred only on bovine kidney laminins in the absence of TS2/16. These results demonstrate that both alpha 3A beta 1 and alpha 6A beta 1 integrins are typical laminin receptors but that their affinity and activation dependence for binding to various laminin isoforms differ considerably. Images PMID:8019006

  15. Ultrastructural immunolocalization of laminin 332 (laminin 5) at dento-gingival interface in Macaca fuscata monkey.

    PubMed

    Sawada, Takashi; Yamazaki, Takaki; Shibayama, Kazuko; Yamaguchi, Yoko; Ohshima, Mitsuhiro

    2015-06-01

    Although laminin 332 (laminin 5), an extracellular matrix molecule involved in cell adhesion and migration, has been localized at the interface between the tooth enamel and junctional epithelium, its ultrastructural localization remains to be fully clarified. The purpose of the present study was to investigate the ultrastructural distribution of laminin 332 at the dento-gingival interface in Japanese monkey (Macaca fuscata) using pre- and post-embedding immunoelectron microscopy. Pre-embedding immunoelectron microscopy revealed a broad band of internal basal lamina together with supplementary lamina densa, and both showed immunolabeling for laminin 332. Immunoreaction products for laminin 332 were observed in the rough-surfaced endoplasmic reticulum of the junctional epithelial cells close to the tooth enamel. Post-embedding immunoelectron microscopy revealed an increase in the number of immunogold particles toward the coronal portion, resulting in a large accumulation of particles on the basal lamina, preferentially on the lamina densa. Concomitantly the dental cuticle at the dento-gingival interface was sporadically, but specifically, immunogold-labeled with anti-laminin 332 antibody. These data suggest that junctional epithelium actively produces laminin 332, and that the products accumulate at the dento-gingival interface during cell migration coronally towards the gingival sulcus.

  16. Deletion of integrin α7 subunit does not aggravate the phenotype of laminin α2 chain-deficient mice

    PubMed Central

    Gawlik, Kinga I.; Durbeej, Madeleine

    2015-01-01

    Laminin-211 is a major constituent of the skeletal muscle basement membrane, exerting its biological functions by binding to cell surface receptors integrin α7β1 and dystroglycan (the latter is part of the dystrophin-glycoprotein complex). The importance of these molecules for normal muscle function is underscored by the fact that their respective deficiency leads to different forms of muscular dystrophy with different severity in humans and animal models. We recently demonstrated that laminin α2 chain and members of the dystrophin-glycoprotein complex have overlapping but non-redundant roles despite being part of the same adhesion complex. To analyse whether laminin-211 and integrin α7 subunit have non-redundant functions we generated mice deficient in laminin α2 chain and integrin α7 subunit (dy3K/itga7). We show that lack of both molecules did not exacerbate the severe phenotype of laminin α2-chain deficient animals. They displayed the same weight, survival and dystrophic pattern of muscle biopsy, with similar degree of inflammation and fibrosis. These data suggest that laminin-211 and integrin α7β1 have intersecting roles in skeletal muscle. PMID:26355035

  17. Dystroglycan is involved in laminin-1-stimulated motility of Müller glial cells: combined velocity and directionality analysis.

    PubMed

    Méhes, Elöd; Czirók, András; Hegedüs, Balázs; Szabó, Bálint; Vicsek, Tamás; Satz, Jakob; Campbell, Kevin; Jancsik, Veronika

    2005-03-01

    We investigate the role of dystroglycan, a major laminin-1 receptor and central member of the dystrophin-glycoprotein complex, in the laminin-1 induced motility of cultured Muller glial cells. Binding of laminin-1 to dystroglycan was prevented by IIH6, a function-blocking monoclonal antibody against alpha-dystroglycan. As an alternative means of inhibition, we used heparin to mask the dystroglycan binding site of the laminin-1, known to overlap with heparin binding sites. Cell motility was characterized in a two-dimensional motility assay based on computer-controlled videomicroscopy and statistical analysis of cellular trajectories. We obtained data on both the cell velocity and the diffusion index, a measure of direction-changing frequency. Both means of inhibition of dystroglycan function led to a significant decrease in the ability of laminin-1 to stimulate cell migration. At the same time, dystroglycan function does not appear to be involved in laminin-1-dependent increase in process dynamism and direction-changing activity.

  18. Laminin-211 in skeletal muscle function

    PubMed Central

    Holmberg, Johan; Durbeej, Madeleine

    2013-01-01

    A chain is no stronger than its weakest link is an old idiom that holds true for muscle biology. As the name implies, skeletal muscle’s main function is to move the bones. However, for a muscle to transmit force and withstand the stress that contractions give rise to, it relies on a chain of proteins attaching the cytoskeleton of the muscle fiber to the surrounding extracellular matrix. The importance of this attachment is illustrated by a large number of muscular dystrophies caused by interruption of the cytoskeletal-extracellular matrix interaction. One of the major components of the extracellular matrix is laminin, a heterotrimeric glycoprotein and a major constituent of the basement membrane. It has become increasingly apparent that laminins are involved in a multitude of biological functions, including cell adhesion, differentiation, proliferation, migration and survival. This review will focus on the importance of laminin-211 for normal skeletal muscle function. PMID:23154401

  19. Laminin database: a tool to retrieve high-throughput and curated data for studies on laminins

    PubMed Central

    Golbert, Daiane C. F.; Linhares-Lacerda, Leandra; Almeida, Luiz G.; Correa-de-Santana, Eliane; de Oliveira, Alice R.; Mundstein, Alex S.; Savino, Wilson; de Vasconcelos, Ana T. R.

    2011-01-01

    The Laminin(LM)-database, hosted at http://www.lm.lncc.br, is the first database focusing a non-collagenous extracellular matrix protein family, the LMs. Part of the knowledge available in this website is automatically retrieved, whereas a significant amount of information is curated and annotated, thus placing LM-database beyond a simple repository of data. In its home page, an overview of the rationale for the database is seen and readers can access a tutorial to facilitate navigation in the website, which in turn is presented with tabs subdivided into LMs, receptors, extracellular binding and other related proteins. Each tab opens into a given LM or LM-related molecule, where the reader finds a series of further tabs for ‘protein’, ‘gene structure’, ‘gene expression’ and ‘tissue distribution’ and ‘therapy’. Data are separated as a function of species, comprising Homo sapiens, Mus musculus and Rattus novergicus. Furthermore, there is specific tab displaying the LM nomenclatures. In another tab, a direct link to PubMed, which can be then consulted in a specific way, in terms of the biological functions of each molecule, knockout animals and genetic diseases, immune response and lymphomas/leukemias. LM-database will hopefully be a relevant tool for retrieving information concerning LMs in health and disease, particularly regarding the hemopoietic system. PMID:21087995

  20. Relationship between laminin binding capacity and laminin expression on tumor cells sensitive or resistant to natural cell-mediated cytotoxicity

    SciTech Connect

    Laybourn, K.A.; Varani, J.; Fligiel, S.E.G.; Hiserodt, J.C.

    1986-03-01

    Previous studies have identified the presence of laminin binding sites on murine NK and NC sensitive tumor cells by /sup 125/I-laminin binding and laminin induced cell-cell aggregation. The finding that the addition of exogenous laminin inhibits NK/NC binding to sensitive tumor cells suggests laminin binding sites may serve as target antigens for NK cells. The present study extends earlier reports by analyzing a large panel of tumor cells for laminin binding capacity, laminin expression and sensitivity to NK/NC killing. The data indicate that all tumor cells which bind to NK/NC cells (8 lines tested) express laminin binding sites. All of these tumor cells were capable of competing for NK lysis of YAC-1 cells in cold target competition assays, and all bound enriched NK cells in direct single cell binding assays. In contrast, tumor cells expressing high levels of surface laminin (B16 melanomas, C57B1/6 fibrosarcomas, and RAS transfected 3T3 fibroblasts) but low levels of laminin binding capacity did not bind NK/NC cells and were resistant to lysis. These data support the hypothesis that expression of laminin/laminin binding sites may contribute to tumor cell sensitivity to NK/NC binding and/or killing.

  1. Linker molecules between laminins and dystroglycan ameliorate laminin-α2–deficient muscular dystrophy at all disease stages

    PubMed Central

    Meinen, Sarina; Barzaghi, Patrizia; Lin, Shuo; Lochmüller, Hanns; Ruegg, Markus A.

    2007-01-01

    Mutations in laminin-α2 cause a severe congenital muscular dystrophy, called MDC1A. The two main receptors that interact with laminin-α2 are dystroglycan and α7β1 integrin. We have previously shown in mouse models for MDC1A that muscle-specific overexpression of a miniaturized form of agrin (mini-agrin), which binds to dystroglycan but not to α7β1 integrin, substantially ameliorates the disease (Moll, J., P. Barzaghi, S. Lin, G. Bezakova, H. Lochmuller, E. Engvall, U. Muller, and M.A. Ruegg. 2001. Nature. 413:302–307; Bentzinger, C.F., P. Barzaghi, S. Lin, and M.A. Ruegg. 2005. Matrix Biol. 24:326–332.). Now we show that late-onset expression of mini-agrin still prolongs life span and improves overall health, although not to the same extent as early expression. Furthermore, a chimeric protein containing the dystroglycan-binding domain of perlecan has the same activities as mini-agrin in ameliorating the disease. Finally, expression of full-length agrin also slows down the disease. These experiments are conceptual proof that linking the basement membrane to dystroglycan by specifically designed molecules or by endogenous ligands, could be a means to counteract MDC1A at a progressed stage of the disease, and thus opens new possibilities for the development of treatment options for this muscular dystrophy. PMID:17389231

  2. A Fractal Nature for Polymerized Laminin

    PubMed Central

    Hochman-Mendez, Camila; Cantini, Marco; Moratal, David; Salmeron-Sanchez, Manuel; Coelho-Sampaio, Tatiana

    2014-01-01

    Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system. PMID:25296244

  3. The role of laminins in basement membrane function

    PubMed Central

    AUMAILLEY, MONIQUE; SMYTH, NEIL

    1998-01-01

    Laminins are a family of multifunctional macromolecules, ubiquitous in basement membranes, and represent the most abundant structural noncollagenous glycoproteins of these highly specialised extracellular matrices. Their discovery started with the difficult task of isolating molecules produced by cultivated cells or extracted from tissues. The development of molecular biology techniques has facilitated and accelerated the identification and the characterisation of new laminin variants making it feasible to identify full-length polypeptides which have not been purified. Further, genetically engineered laminin fragments can be generated for studies of their structure-function relationship, permitting the demonstration that laminins are involved in multiple interactions with themselves, with other components of the basal lamina, and with cells. It endows laminins with a central role in the formation, the architecture, and the stability of basement membranes. In addition, laminins may both separate and connect different tissues, i.e. the parenchymal and the interstitial connective tissues. Laminins also provide adjacent cells with a mechanical scaffold and biological information either directly by interacting with cell surface components, or indirectly by trapping growth factors. In doing so they trigger and control cellular functions. Recently, the structural and biological diversity of the laminins has started to be elucidated by gene targeting and by the identification of laminin defects in acquired or inherited human diseases. The consequent phenotypes highlight the pivotal role of laminins in determining heterogeneity in basement membrane functions. PMID:9758133

  4. Laminin 332 in junctional epidermolysis bullosa.

    PubMed

    Kiritsi, Dimitra; Has, Cristina; Bruckner-Tuderman, Leena

    2013-01-01

    Laminin 332 is an essential component of the dermal-epidermal junction, a highly specialized basement membrane zone that attaches the epidermis to the dermis and thereby provides skin integrity and resistance to external mechanical forces. Mutations in the LAMA3, LAMB3 and LAMC2 genes that encode the three constituent polypeptide chains, α3, β3 and γ2, abrogate or perturb the functions of laminin 332. The phenotypic consequences are diminished dermal-epidermal adhesion and, as clinical symptoms, skin fragility and mechanically induced blistering. The disorder is designated as junctional epidermolysis bullosa (JEB). This article delineates the signs and symptoms of the different forms of JEB, the mutational spectrum, genotype-phenotype correlations as well as perspectives for future molecular therapies. PMID:23076207

  5. Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

    SciTech Connect

    Kato, Shigemi; Ben, T.L.; De Luca, L.M. )

    1988-11-01

    The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10{sup {minus}9} and 10{sup {minus}7} M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.

  6. Olfactory Ensheathing Cells Express α7 Integrin to Mediate Their Migration on Laminin

    PubMed Central

    Ingram, Norianne T.; Khankan, Rana R.; Phelps, Patricia E.

    2016-01-01

    The unique glia located in the olfactory system, called olfactory ensheathing cells (OECs), are implicated as an attractive choice for transplantation therapy following spinal cord injury because of their pro-regenerative characteristics. Adult OECs are thought to improve functional recovery and regeneration after injury by secreting neurotrophic factors and making cell-to-cell contacts with regenerating processes, but the mechanisms are not well understood. We show first that α7 integrin, a laminin receptor, is highly expressed at the protein level by OECs throughout the olfactory system, i.e., in the olfactory mucosa, olfactory nerve, and olfactory nerve layer of the olfactory bulb. Then we asked if OECs use the α7 integrin receptor directly to promote neurite outgrowth on permissive and neutral substrates, in vitro. We co-cultured α7+/+ and α7lacZ/lacZ postnatal cerebral cortical neurons with α7+/+ or α7lacZ/lacZ OECs and found that genotype did not effect the ability of OECs to enhance neurite outgrowth by direct contact. Loss of α7 integrin did however significantly decrease the motility of adult OECs in transwell experiments. Twice as many α7+/+ OECs migrated through laminin-coated transwells compared to α7+/+ OECs on poly-L-lysine (PLL). This is in contrast to α7lacZ/lacZ OECs, which showed no migratory preference for laminin substrate over PLL. These results demonstrate that OECs express α7 integrin, and that laminin and its α7 integrin receptor contribute to adult OEC migration in vitro and perhaps also in vivo. PMID:27078717

  7. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    SciTech Connect

    Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J

    1995-04-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

  8. Laminin mediates tissue-specific gene expression in mammary epithelia

    PubMed Central

    1995-01-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta- casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain. PMID:7730398

  9. LG4-5 domains of laminin-2 binds α-dystroglycan to allow myotube attachment and prevent anoikis

    PubMed Central

    Munoz, Jesus; Zhou, YanWen; Jarrett, Harry W.

    2010-01-01

    Poly(2-hydroxyethyl methacrylate) (PolyHEMA) prevents cell attachment was used here to study anoikis, the process where cells die when unattached or attached to an inappropriate matrix, in mouse C2C12 myotubes. A method was developed to efficiently embed proteins into PolyHEMA and the effect on cultured myotubes was determined. Myotubes grown on PolyHEMA-coated plates fail to attach to the surface and remain as rounded, suspended cells, undergo dramatic increases in apoptosis and necrosis, and the number of viable cells decreases,. Incorporation of merosin (laminin-211) or the short laminin globular (LG4-5) modules of the laminin α2 chain C-terminus (called 2E3) that binds α-dystroglycan diminishes both apoptosis and necrosis and increases viability while bovine serum albumin had a much lesser effect, showing the specificity of this effect for these matrix proteins. One sarcolemma receptor for laminin-binding is α-dystroglycan. An antibody which binds α-dystroglycan but which does not block laminin-binding (VIA4) had little effect on apoptosis or viability on merosin or 2E3 embedded plates while another antibody (IIH6) which specifically blocks binding dramatically decreased viability and increased apoptosis. When merosin or 2E3 are added to culture media rather than embedded on plates these can also increase viability and decrease apoptosis even though the cells remain in suspension, though the effect is not as great as found for the embedded proteins where the cells attach. Thus, we conclude that the binding of a small LG4-5 modules of laminin-211 to α-dystroglycan is important in preventing anoikis and that attachment plus binding is necessary for maximal cell survival. PMID:19739104

  10. Agrin and Synaptic Laminin Are Required to Maintain Adult Neuromuscular Junctions

    PubMed Central

    Samuel, Melanie A.; Valdez, Gregorio; Tapia, Juan C.; Lichtman, Jeff W.; Sanes, Joshua R.

    2012-01-01

    As synapses form and mature the synaptic partners produce organizing molecules that regulate each other’s differentiation and ensure precise apposition of pre- and post-synaptic specializations. At the skeletal neuromuscular junction (NMJ), these molecules include agrin, a nerve-derived organizer of postsynaptic differentiation, and synaptic laminins, muscle-derived organizers of presynaptic differentiation. Both become concentrated in the synaptic cleft as the NMJ develops and are retained in adulthood. Here, we used mutant mice to ask whether these organizers are also required for synaptic maintenance. Deletion of agrin from a subset of adult motor neurons resulted in the loss of acetylcholine receptors and other components of the postsynaptic apparatus and synaptic cleft. Nerve terminals also atrophied and eventually withdrew from muscle fibers. On the other hand, mice lacking the presynaptic organizer laminin-α4 retained most of the synaptic cleft components but exhibited synaptic alterations reminiscent of those observed in aged animals. Although we detected no marked decrease in laminin or agrin levels at aged NMJs, we observed alterations in the distribution and organization of these synaptic cleft components suggesting that such changes could contribute to age-related synaptic disassembly. Together, these results demonstrate that pre- and post-synaptic organizers actively function to maintain the structure and function of adult NMJs. PMID:23056392

  11. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts.

    PubMed

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G

    2012-11-23

    The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

  12. The hippocampal laminin matrix is dynamic and critical for neuronal survival.

    PubMed

    Chen, Zu-Lin; Indyk, Justin A; Strickland, Sidney

    2003-07-01

    Laminins are extracellular matrix proteins that participate in neuronal development, survival, and regeneration. During excitotoxin challenge in the mouse hippocampus, neuron interaction with laminin-10 (alpha5,beta1,gamma1) protects against neuronal death. To investigate how laminin is involved in neuronal viability, we infused laminin-1 (alpha1,beta1,gamma1) into the mouse hippocampus. This infusion specifically disrupted the endogenous laminin layer. This disruption was at least partially due to the interaction of the laminin-1 gamma1 chain with endogenous laminin-10, because infusion of anti-laminin gamma1 antibody had the same effect. The disruption of the laminin layer by laminin-1 1) did not require the intact protein because infusion of plasmin-digested laminin-1 gave similar results; 2) was posttranscriptional, because there was no effect on laminin mRNA expression; and 3) occurred in both tPA(-/-) and plasminogen(-/-) mice, indicating that increased plasmin activity was not responsible. Finally, although tPA(-/-) mice are normally resistant to excitotoxin-induced neurodegeneration, disruption of the endogenous laminin layer by laminin-1 or anti-laminin gamma1 antibody renders the tPA(-/-) hippocampal neurons sensitive to kainate. These results demonstrate that neuron interactions with the deposited matrix are not necessarily recapitulated by interactions with soluble components and that the laminin matrix is a dynamic structure amenable to modification by exogenous molecules.

  13. Laminin alters Fyn regulatory mechanisms and promotes oligodendrocyte development

    PubMed Central

    Relucio, Jenne; Tzvetanova, Iva D.; Ao, Wei; Lindquist, Sabine; Colognato, Holly

    2009-01-01

    Mutations in LAMA2, the gene for the extracellular matrix protein laminin-α2, cause a severe muscular dystrophy termed MDC1A. MDC1A patients have accompanying CNS neural dysplasias and white matter abnormalities for which the underlying mechanisms remain unknown. Here we report that in laminin-deficient mice oligodendrocyte development was delayed such that oligodendrocyte progenitors accumulated inappropriately in adult brains. Conversely, laminin substrates were found to promote the transition of oligodendrocyte progenitors to newly-formed oligodendrocytes. Laminin-enhanced differentiation was Src Family Kinase –dependent and resulted in the activation of the Src Family Kinase Fyn. In laminin-deficient brains, however, increased Fyn repression was accompanied by elevated levels of the Src Family Kinase negative regulatory proteins, C-terminal Src kinase (Csk) and its transmembrane adaptor, Csk-binding protein (Cbp). These findings indicate that laminin deficiencies delay oligodendrocyte maturation by causing dysregulation of signaling pathways critical for oligodendrocyte development, and suggest that a normal role for CNS laminin is to promote the development of oligodendrocyte progenitors into myelin-forming oligodendrocytes via modulation of Fyn regulatory molecules. PMID:19776266

  14. Laminin is produced by early rat astrocytes in primary culture

    PubMed Central

    1983-01-01

    The production of laminin by early rat astrocytes in primary culture was investigated by double immunofluorescence staining for laminin and the glial fibrillary acidic protein (GFAP), a defined astrocyte marker. In early cultures (3 d in vitro; 3 DIV) cytoplasmic laminin was detected in all the GFAP-positive cells which formed the major population (80%) of the nonneuronal cells present in cultures from 20- 21-d embryonic, newborn, or 5-d-old rat brains. Monensin treatment (10 microM, 4 h) resulted in accumulation of laminin in the Golgi region, located using labeled wheat germ agglutinin. Laminin started gradually to disappear from the cells with the time in culture, was absent in star-shaped, apparently mature astrocytes, but remained as pericellular matrix deposits. The disappearance of cellular laminin was dependent on the age of the animal and the time in culture so that it started earlier in cultures from 5-d-old rat brains (5 DIV) and approximately following the in vivo age difference in cultures from newborn (12 DIV) and embryonic (14 DIV) rat brains. Our results indicate that laminin is a protein of early astrocytes and also deposited by them in primary culture, thus suggesting a role for this glycoprotein in the development of the central nervous system. PMID:6339524

  15. Laminin-Mediated Interactions in Thymocyte Migration and Development

    PubMed Central

    Savino, Wilson; Mendes-da-Cruz, Daniella Arêas; Golbert, Daiane Cristina Ferreira; Riederer, Ingo; Cotta-de-Almeida, Vinicius

    2015-01-01

    Intrathymic T-cell differentiation is a key process for the development and maintenance of cell-mediated immunity, and occurs concomitantly to highly regulated migratory events. We have proposed a multivectorial model for describing intrathymic thymocyte migration. One of the individual vectors comprises interactions mediated by laminins (LMs), a heterotrimeric protein family of the extracellular matrix. Several LMs are expressed in the thymus, being produced by microenvironmental cells, particularly thymic epithelial cells (TECs). Also, thymocytes and epithelial cells express integrin-type LM receptors. Functionally, it has been reported that the dy/dy mutant mouse (lacking the LM isoform 211) exhibits defective thymocyte differentiation. Several data show haptotactic effects of LMs upon thymocytes, as well as their adhesion on TECs; both effects being prevented by anti-LM or anti-LM receptor antibodies. Interestingly, LM synergizes with chemokines to enhance thymocyte migration, whereas classe-3 semaphorins and B ephrins, which exhibit chemorepulsive effects in the thymus, downregulate LM-mediated migratory responses of thymocytes. More recently, we showed that knocking down the ITGA6 gene (which encodes the α6 integrin chain of LM receptors) in human TECs modulates a large number of cell migration-related genes and results in changes of adhesion pattern of thymocytes onto the thymic epithelium. Overall, LM-mediated interactions can be placed at the cross-road of the multivectorial process of thymocyte migration, with a direct influence per se, as well as by modulating other molecular interactions associated with the intrathymic-trafficking events. PMID:26635793

  16. Laminin regulates PDGFRβ+ cell stemness and muscle development

    PubMed Central

    Yao, Yao; Norris, Erin H.; E. Mason, Christopher; Strickland, Sidney

    2016-01-01

    Muscle-resident PDGFRβ+ cells, which include pericytes and PW1+ interstitial cells (PICs), play a dual role in muscular dystrophy. They can either undergo myogenesis to promote muscle regeneration or differentiate into adipocytes and other cells to compromise regeneration. How the differentiation and fate determination of PDGFRβ+ cells are regulated, however, remains unclear. Here, by utilizing a conditional knockout mouse line, we report that PDGFRβ+ cell-derived laminin inhibits their proliferation and adipogenesis, but is indispensable for their myogenesis. In addition, we show that laminin alone is able to partially reverse the muscle dystrophic phenotype in these mice at the molecular, structural and functional levels. Further RNAseq analysis reveals that laminin regulates PDGFRβ+ cell differentiation/fate determination via gpihbp1. These data support a critical role of laminin in the regulation of PDGFRβ+ cell stemness, identify an innovative target for future drug development and may provide an effective treatment for muscular dystrophy. PMID:27138650

  17. Enhanced laminin adsorption on nanowires compared to flat surfaces.

    PubMed

    Hammarin, Greger; Persson, Henrik; Dabkowska, Aleksandra P; Prinz, Christelle N

    2014-10-01

    Semiconductor nanowires are widely used to interface living cells, and numerous nanowire-based devices have been developed to manipulate or sense cell behavior. We have, however, little knowledge on the nature of the cell-nanowire interface. Laminin is an extracellular matrix protein promoting cell attachment and growth. Here, we used a method based on fluorescence microscopy and measured the relative amount of laminin adsorbed on nanowires compared to flat surfaces. The amount of adsorbed laminin per surface area is up to 4 times higher on 55nm diameter gallium phosphide nanowires compared to the flat gallium phosphide surface between the nanowires. We show that this enhanced adsorption on nanowires cannot be attributed to electrostatic effects, nor to differences in surface chemistry, but possibly to pure geometrical effects, as increasing the nanowire diameter results in a decreased amount of adsorbed protein. The increased adsorption of laminin on nanowires may explain the exceptionally beneficial properties of nanowire substrates for cellular growth reported in the literature since laminin is often used as surface coating prior to cell cultures in order to promote cell growth, and also because primary cell suspensions contain endogenous laminin.

  18. The association between laminin and microglial morphology in vitro.

    PubMed

    Tam, Wing Yip; Au, Ngan Pan Bennett; Ma, Chi Him Eddie

    2016-01-01

    Microglia are immune cells in the central nervous system (CNS) that contribute to primary innate immune responses. The morphology of microglia is closely associated with their functional activities. The majority of microglial studies have focused on the ramified or amoeboid morphology; however, bipolar/rod-shaped microglia have recently received much attention. Bipolar/rod-shaped microglia form trains with end-to-end alignment in injured brains and retinae, which is proposed as an important mechanism in CNS repair. We previously established a cell culture model system to enrich bipolar/rod-shaped microglia simply by growing primary microglia on scratched poly-D-lysine (PDL)/laminin-coated surfaces. Here, we investigated the role of laminin in morphological changes of microglia. Bipolar/rod-shaped microglia trains were transiently formed on scratched surfaces without PDL/laminin coating, but the microglia alignment disappeared after 3 days in culture. Amoeboid microglia digested the surrounding laminin, and the gene and protein expression of laminin-cleaving genes Adam9 and Ctss was up-regulated. Interestingly, lipopolysaccharide (LPS)-induced transformation from bipolar/rod-shaped into amoeboid microglia increased the expression of Adam9 and Ctss, and the expression of these genes in LPS-treated amoeboid-enriched cultures remained unchanged. These results indicate a strong association between laminin and morphological transformation of microglia, shedding new light on the role of bipolar/rod-shaped microglia in CNS repair. PMID:27334934

  19. Lectin KM+-induced neutrophil haptotaxis involves binding to laminin.

    PubMed

    Ganiko, Luciane; Martins, Antônio R; Freymüller, Edna; Mortara, Renato A; Roque-Barreira, Maria-Cristina

    2005-01-18

    The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM+ with both the extracellular matrix (ECM) and neutrophils depend on the lectin ability to recognize mannose-containing glycans. Here, we report the binding of KM+ to laminin and demonstrate that this interaction potentiates the KM+-induced neutrophil migration. Labeling of lung tissue by KM+ located its ligands on the endothelial cells, in the basement membrane, in the alveolus, and in the interstitial connective tissue. Such labeling was inhibited by 400 mM D-mannose, 10 mM Manalpha1-3[Manalpha1-6]Man or 10 microM peroxidase (a glycoprotein-containing mannosyl heptasaccharide). Laminin is a tissue ligand for KM+, since both KM+ and anti-laminin antibodies not only reacted with the same high molecular mass components of a lung extract, but also determined colocalized labeling in basement membranes of the lung tissue. The relevance of the KM+-laminin interaction to the KM+ property of inducing neutrophil migration was evaluated. The inability of low concentrations of soluble KM+ to induce human neutrophil migration was reversed by coating the microchamber filter with laminin. So, the interaction of KM+ with laminin promotes the formation of a substrate-bound KM+ gradient that is able to induce neutrophil haptotaxis.

  20. Laminin degradation by plasmin regulates long-term potentiation.

    PubMed

    Nakagami, Y; Abe, K; Nishiyama, N; Matsuki, N

    2000-03-01

    Plasmin is converted from its zymogen plasminogen by tissue type or urokinase type plasminogen activator (PA) and degrades many components of the extracellular matrix (ECM). To explore the possibility that the PA-plasmin system regulates synaptic plasticity, we investigated the effect of plasmin on degradation of ECM and synaptic plasticity by using organotypic hippocampal cultures. High-frequency stimulation produced long-term potentiation (LTP) in control slices, whereas the potentiation was induced but not maintained in slices pretreated with 100 nM plasmin for 6 hr. The baseline synaptic responses were not affected by pretreatment with plasmin. The impairment of LTP maintenance was not observed in slices pretreated with 100 nM plasmin for 6 hr, washed, and then cultured for 24-48 hr in the absence of plasmin. To identify substrates of plasmin, the expression of three major components of ECM, laminin, fibronectin, and type IV collagen, was investigated by immunofluorescence imaging. The three ECM components were widely distributed in the hippocampus, and only laminin was degraded by plasmin pretreatment. The expression level of laminin returned to normal levels when the slices were cultured for 24-48 hr after washout of plasmin. Furthermore, preincubation with anti-laminin antibodies prevented both the degradation of laminin and the impairment of LTP maintenance by plasmin. These results suggest that the laminin-mediated cell-ECM interaction may be necessary for the maintenance of LTP.

  1. The association between laminin and microglial morphology in vitro

    PubMed Central

    Tam, Wing Yip; Au, Ngan Pan Bennett; Ma, Chi Him Eddie

    2016-01-01

    Microglia are immune cells in the central nervous system (CNS) that contribute to primary innate immune responses. The morphology of microglia is closely associated with their functional activities. The majority of microglial studies have focused on the ramified or amoeboid morphology; however, bipolar/rod-shaped microglia have recently received much attention. Bipolar/rod-shaped microglia form trains with end-to-end alignment in injured brains and retinae, which is proposed as an important mechanism in CNS repair. We previously established a cell culture model system to enrich bipolar/rod-shaped microglia simply by growing primary microglia on scratched poly-D-lysine (PDL)/laminin-coated surfaces. Here, we investigated the role of laminin in morphological changes of microglia. Bipolar/rod-shaped microglia trains were transiently formed on scratched surfaces without PDL/laminin coating, but the microglia alignment disappeared after 3 days in culture. Amoeboid microglia digested the surrounding laminin, and the gene and protein expression of laminin-cleaving genes Adam9 and Ctss was up-regulated. Interestingly, lipopolysaccharide (LPS)-induced transformation from bipolar/rod-shaped into amoeboid microglia increased the expression of Adam9 and Ctss, and the expression of these genes in LPS-treated amoeboid-enriched cultures remained unchanged. These results indicate a strong association between laminin and morphological transformation of microglia, shedding new light on the role of bipolar/rod-shaped microglia in CNS repair. PMID:27334934

  2. Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin

    PubMed Central

    Marshall, Jamie L.; Oh, Jennifer; Chou, Eric; Lee, Joy A.; Holmberg, Johan; Burkin, Dean J.; Crosbie-Watson, Rachelle H.

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin–glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7β1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan ‘rescue’ of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7β1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and α7β1 integrin. PMID:25504048

  3. Transient expression of laminin {alpha}1 chain in regenerating murine liver: Restricted localization of laminin chains and nidogen-1

    SciTech Connect

    Kikkawa, Yamato . E-mail: yamato@sapmed.ac.jp; Mochizuki, Yoichi; Miner, Jeffrey H.; Mitaka, Toshihiro

    2005-04-15

    Most interstitia between epithelial and endothelial cells contain basal laminae (BLs), as defined by electron microscopy. However, in liver, the sinusoidal interstitium (called space of Disse) between hepatocytes and sinusoidal endothelial cells (SECs) lacks BLs. Because laminins are major components of BLs throughout the body, whether laminins exist in sinusoids has been a controversial issue. Despite recent advances, the distribution and expression of laminin chains have not been well defined in mammalian liver. Here, using a panel of antibodies, we examined laminins in normal and regenerating mouse livers. Of {alpha} chains, {alpha}5 was widely observed in all BLs except for sinusoids, while the other {alpha} chains were variously expressed in Glisson's sheath and central veins. Laminin {gamma}1 was also distributed to all BLs except for sinusoids. Although the {beta}2 chain was observed in all BLs and sinusoids, the expression of {beta}1 chain was restricted to Glisson's sheath. Detailed analysis of regenerating liver revealed that {alpha}1 and {gamma}1 chains appeared in sinusoids and were produced by stellate cells. The staining of {alpha}1 and {gamma}1 chains reached its maximum intensity at 6 days after two-thirds partial hepatectomy (PHx). Moreover, in vitro studies showed that {alpha}1-containing laminin promoted spreading of sinusoidal endothelial cells (SECs) isolated from normal liver, but not other hepatic cells. In addition, SECs isolated from regenerating liver elongated pseudopodia on {alpha}1-containing laminin more so than did cells from normal liver. The transient expression of laminin {alpha}1 may promote formation of sinusoids after PHx.

  4. Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules

    PubMed Central

    1990-01-01

    It has been previously shown that A-chain and domain(E8)-specific antibodies to laminin that inhibit cell adhesion also interfere with the establishment of epithelial cell polarity during kidney tubule development (Klein, G., M. Langegger, R. Timpl, and P. Ekblom. 1988. Cell. 55:331-341). A monoclonal antibody specific for the integrin alpha 6 subunit, which selectively blocks cell binding to E8, was used to study the receptors involved. Immunofluorescence staining of embryonic kidneys and of organ cultures of metanephric mesenchyme demonstrated coappearance of the integrin alpha 6 subunit and the laminin A-chain in regions where nonpolarized mesenchymal cells convert into polarized epithelial cells. Both epitopes showed marked colocalization in basal areas of tubules, while an exclusive immunostaining for alpha 6 was observed in lateral and apical cell surfaces of the tubular epithelial cells. Organ culture studies demonstrated a consistent inhibition of kidney epithelium development by antibodies against the alpha 6 subunit. The data suggest that the recognition of E8 cell-binding site of laminin by a specific integrin is crucial for the formation of kidney tubule epithelium from undifferentiated mesenchymal stem cells. In some other cell types (endothelium, some ureter cells) an exclusive expression of alpha 6 with no apparent colocalization of laminin A-chain in the corresponding basement membrane was seen. Thus, in these cells, integrins possessing the alpha 6 subunit may bind to laminin isoforms that differ from those synthesized by developing tubules. PMID:2144001

  5. Quantitative analysis of laminin 5 gene expression in human keratinocytes.

    PubMed

    Akutsu, Nobuko; Amano, Satoshi; Nishiyama, Toshio

    2005-05-01

    To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms. PMID:15854126

  6. Laminin A chain: expression during Drosophila development and genomic sequence.

    PubMed Central

    Kusche-Gullberg, M; Garrison, K; MacKrell, A J; Fessler, L I; Fessler, J H

    1992-01-01

    A Drosophila laminin A chain gene was characterized as a 14 kb genomic nucleotide sequence which encodes an open reading frame of 3712 amino acids in 15 exons. Overall, this A chain is similar to its vertebrate counterparts, especially in its N- and C-terminal globular domains, but the sequence that forms the laminin A short arm is quite different and larger. Laminin messages appear in newly formed mesoderm and are later prominently expressed in hemocytes, which also synthesize basement membrane collagen IV. The composition of Drosophila basement membranes changes with development. A novel method of tandemly fused RNA probes showed that developmental increases of laminin mRNAs were primarily associated with periods of morphogenesis, and preceded those of collagen IV, a protein strongly expressed during growth. The ratio of A:B1:B2 mRNAs varied little during embryogenesis, with less mRNA for A than B chains. Staining of embryos with antibodies confirmed and extended the information provided by in situ hybridization. Homologs of the G-subdomains of this A chain, which occur in interacting regions of agrin, perlecan, laminin and sex steroid binding protein, may be involved in protein associations. Images PMID:1425586

  7. Laminin Functionalized Biomimetic Nanofibers For Nerve Tissue Engineering

    PubMed Central

    Junka, Radoslaw; Valmikinathan, Chandra M; Kalyon, Dilhan M; Yu, Xiaojun

    2013-01-01

    Large-gap peripheral nerve injuries present a significant challenge for nerve regeneration due to lack of suitable grafts, insufficient cell penetration, and repair. Biomimetic nanofibrous scaffolds, functionalized on the surface with extracellular matrix proteins, can lead to novel therapies for repair and regeneration of damaged peripheral nerves. Here, nanofibrous scaffolds electrospun from blends of poly(caprolactone) (PCL) and chitosan were fabricated. Taking advantage of the amine groups on the chitosan, the surface of the scaffolds were functionalized with laminin by carbodiimide based crosslinking. Crosslinking allowed laminin to be attached to the surfaces of the PCL-chitosan nanofibers at relatively high concentrations that were not possible using conventional adsorption methods. The nanofibrous meshes were tested for wettability, mechanical properties and cell attachment and proliferation. Blending of chitosan with PCL provided more favorable surfaces for attachment of Schwann cells due to the reduction of the contact angle in comparison to neat PCL. Proliferation rates of Schwann cells grown on PCL-chitosan scaffolds with crosslinked laminin were significantly higher than the rates for PCL-chitosan nanofibrous matrices with adsorbed laminin. PCL-chitosan scaffolds with modified surfaces via crosslinking of laminin could potentially serves as versatile substrates with excellent mechanical and surface properties for in vivo cell delivery for nerve tissue engineering applications. PMID:24083073

  8. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    SciTech Connect

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G.

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate

  9. Trichomonas vaginalis interactions with fibronectin and laminin.

    PubMed

    Crouch, M L; Alderete, J F

    1999-10-01

    The sexually transmitted protozoan Trichomonas vaginalis cytoadheres to vaginal epithelial cells and causes contact-dependent cytotoxicity which, when combined with the normal exfoliation process, leads to erosion of the epithelium, which may allow trichomonads into extracellular matrix and basement membrane sites. Therefore, the association of T. vaginalis with immobilized fibronectin (FN) and laminin (LM) on cover-slips was examined. Binding of live parasites to coated cover-slips was time- and parasite-density-dependent. Coincubation with an inhibitor of trichomonad cysteine proteinases resulted in an increased attachment of parasites to FN but had no effect on binding to LM, denoting that protease activity influenced optimal FN associations. Further, 20 h mid-exponential phase trichomonads placed in fresh culture medium for 3 h gave higher levels of binding to FN, suggesting that changes during growth in vitro to T. vaginalis organisms affect maximal levels of binding to FN. Extended incubation with substrates diminished the capacity of parasites to bind FN and LM. Treatment of live organisms with periodate reduced binding to LM but not FN, suggesting a role for carbohydrates. In addition, trypsinization of live parasites decreased numbers bound to both substrates. Placement of trypsinized parasites in medium for 2 h fully regenerated binding to FN but not LM. Incubation of trypsinized parasites with cycloheximide abrogated regeneration of attachment to FN, affirming a role for synthesized surface proteins in FN binding. Importantly, the T. vaginalis adhesin proteins that mediate cytoadherence, and iron, a factor that regulates adhesin synthesis, were not involved in FN and LM recognition. These results suggest a role for surface proteins and carbohydrates in trichomonal associations with FN and LM, respectively. PMID:10537205

  10. The complete cDNA sequence of laminin alpha 4 and its relationship to the other human laminin alpha chains.

    PubMed

    Richards, A; Al-Imara, L; Pope, F M

    1996-06-15

    We previously localised the gene (LAMA4) encoding a novel laminin alpha 4 chain to chromosome 6q21. In this study, we describe the complete coding sequence and compare the protein with the other three known human laminin alpha chains. Although closely linked to LAMA2, the LAMA4 product most closely resembles laminin alpha 3, a constituent of laminin 5. Like laminin alpha 3A, the alpha 4 chain is a truncated version of the alpha 1 and alpha 2 chains, with a much reduced short arm. While the alpha 4 molecule is most similar to alpha 3, it shares some features of the C-terminal domains G4 and G5 in common with alpha 2. Unlike the LAMA3 gene, LAMA4 appears to encode only a single transcript, as determined by 5' rapid amplification of cDNA ends. The cDNA sequence encodes 1816 amino acids, which include a 24-residue signal peptide. The gene is expressed in skin, placenta, heart, lung, skeletal muscle, and pancreas. We have also shown that the mRNA can be readily reverse transcribed and amplified from cultured dermal fibroblasts. PMID:8706685

  11. Laminin-6 assembles into multimolecular fibrillar complexes with perlecan and participates in mechanical-signal transduction via a dystroglycan-dependent, integrin-independent mechanism

    PubMed Central

    Jones, Jonathan C. R.; Lane, Kimberly; Hopkinson, Susan B.; Lecuona, Emilia; Geiger, Robert C.; Dean, David A.; Correa-Meyer, Eduardo; Gonzales, Meredith; Campbell, Kevin; Sznajder, Jacob I.; Budinger, Scott

    2010-01-01

    Summary Mechanical ventilation is a valuable treatment regimen for respiratory failure. However, mechanical ventilation (especially with high tidal volumes) is implicated in the initiation and/or exacerbation of lung injury. Hence, it is important to understand how the cells that line the inner surface of the lung [alveolar epithelial cells (AECs)] sense cyclic stretching. Here, we tested the hypothesis that matrix molecules, via their interaction with surface receptors, transduce mechanical signals in AECs. We first determined that rat AECs secrete an extracellular matrix (ECM) rich in anastamosing fibers composed of the α3 laminin subunit, complexed with β1 and γ1 laminin subunits (i.e. laminin-6), and perlecan by a combination of immunofluorescence microscopy and immunoblotting analyses. The fibrous network exhibits isotropic expansion when exposed to cyclic stretching (30 cycles per minute, 10% strain). Moreover, this same stretching regimen activates mitogen-activated-protein kinase (MAPK) in AECs. Stretch-induced MAPK activation is not inhibited in AECs treated with antagonists to α3 or β1 integrin. However, MAPK activation is significantly reduced in cells treated with function-inhibiting antibodies against the α3 laminin subunit and dystroglycan, and when dystroglycan is knocked down in AECs using short hairpin RNA. In summary, our results support a novel mechanism by which laminin-6, via interaction with dystroglycan, transduces a mechanical signal initiated by stretching that subsequently activates the MAPK pathway in rat AECs. These results are the first to indicate a function for laminin-6. They also provide novel insight into the role of the pericellular environment in dictating the response of epithelial cells to mechanical stimulation and have broad implications for the pathophysiology of lung injury. PMID:15928048

  12. Evidence That a Laminin-Like Insect Protein Mediates Early Events in the Interaction of a Phytoparasite with Its Vector's Salivary Gland

    PubMed Central

    Dias, Felipe de Almeida; dos Santos, Andre Luis Souza; Lery, Letícia Miranda Santos; Alves e Silva, Thiago Luiz; Oliveira, Mauricio Martins; Bisch, Paulo Mascarello; Saraiva, Elvira Maria; Souto-Padrón, Thaïs Cristina; Lopes, Angela Hampshire

    2012-01-01

    Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 β3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin β3 subunit. Anti-human laminin-5 β3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 β3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa. PMID:23118944

  13. The unfolding of native laminin investigated by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Nemes, Cs.; Ramsden, J. J.; Rozlosnik, N.

    2002-10-01

    Atomic force microscopy has been used to directly measure the forces required to unfold individual domains of the extracellular matrix protein laminin. The approach-retraction cycles display a characteristic saw-tooth motif. Tooth heights and separations were used to establish a statistical relation between domain unfolding force and domain extension. The extensible domains of laminin require an unfolding force intermediate between previously established values for α-helical and β-sheet domains in other proteins. The relationship between unfolding force and extension for a given domain is not smooth; discrete steps are observed, interpreted as originating from the modularity of the protein structure.

  14. Laminin-based Nanomaterials for Peripheral Nerve Tissue Engineering

    NASA Astrophysics Data System (ADS)

    Neal, Rebekah Anne

    Peripheral nerve transection occurs commonly in traumatic injury, causing motor and sensory deficits distal to the site of injury. One option for surgical repair is the nerve conduit. Conduits currently on the market are hollow tubes into which the nerve ends are sutured. Although these conduits fill the gap, they often fail due to the slow rate of regeneration over long gaps. To facilitate increased speed of regeneration and greater potential for functional recovery, the ideal conduit should provide biochemically relevant signals and physical guidance cues, thus playing an active role in peripheral nerve regeneration. In this dissertation, I fabricated laminin-1 and laminin-polycaprolactone (PCL) blend nanofibers that mimic the geometry and functionality of the peripheral nerve basement membrane. These fibers resist hydration in aqueous media and require no harsh chemical crosslinkers. Adhesion and differentiation of both neuron-like and neuroprogenitor cells is improved on laminin nanofibrous meshes over two-dimensional laminin substrates. Blend meshes with varying laminin content were characterized for composition, tensile properties, degradation rates, and bioactivity in terms of cell attachment and axonal elongation. I have established that 10% (wt) laminin content is sufficient to retain the significant neurite-promoting effects of laminin critical in peripheral nerve repair. In addition, I utilized modified collector plate design to manipulate electric field gradients during electrospinning for the fabrication of aligned nanofibers. These aligned substrates provide enhanced directional guidance cues to the regenerating axons. Finally, I replicated the clinical problem of peripheral nerve transection using a rat tibial nerve defect model for conduit implantation. When the lumens of conduits were filled with nanofiber meshes of varying laminin content and alignment, I observed significant recovery of sensory and motor function over six weeks. This recovery was

  15. Structural basis of laminin binding to the LARGE glycans on dystroglycan.

    PubMed

    Briggs, David C; Yoshida-Moriguchi, Takako; Zheng, Tianqing; Venzke, David; Anderson, Mary E; Strazzulli, Andrea; Moracci, Marco; Yu, Liping; Hohenester, Erhard; Campbell, Kevin P

    2016-10-01

    Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by α-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-β1,3-xylose-α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native α-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin-G-like (LG) domains 4 and 5 (LG4 and LG5) of laminin-α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-β1,3-xylose disaccharide repeat straddles a Ca(2+) ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca(2+)-bound water molecules. The chelating binding mode accounts for the high affinity of this protein-carbohydrate interaction. These results reveal a previously uncharacterized mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy. PMID:27526028

  16. Complex interactions between the laminin 4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo

    NASA Astrophysics Data System (ADS)

    Gonzalez, Annette M.; Gonzales, Meredith; Herron, G. Scott; Nagavarapu, Usha; Hopkinson, Susan B.; Tsuruta, Daisuke; Jones, Jonathan C. R.

    2002-12-01

    The 4 laminin subunit is a component of the basement membrane of blood vessels where it codistributes with the integrins v3, 31, and 61. An antibody against the G domain (residues 919-1207; G919-1207) of the 4 laminin subunit inhibits angiogenesis in a mouse-human chimeric model, indicating the functional importance of this domain. Additional support for the latter derives from the ability of recombinant G919-1207 to support endothelial cell adhesion. In particular, endothelial cell adhesion to G919-1207 is half-maximal at 1.4 nM, whereas residues 919-1018 and 1016-1207 of the G domain are poor cellular ligands. Function blocking antibodies against integrins v3 and 1 and a combination of antibodies against 3 and α6 integrin subunits inhibit endothelial cell attachment to G919-1207. Moreover, both αvβ3 and α3β1 integrin bind with high affinity to G919-1207. Together, our studies demonstrate that the G domain of laminin α4 chain is a specific, high affinity ligand for the αvβ3 and α3β1 integrin heterodimers and that these integrins, together with α6β1, function cooperatively to mediate endothelial cell-α4 laminin interaction and hence blood vessel development. We propose a model based on these data that reconcile apparent discrepancies in the recent literature with regard to the role of the αvβ3 integrin in angiogenesis. matrix | matrix receptor | blood vessels

  17. Fetal rat septal cells adhere to and extend processes on basement membrane, laminin, and a synthetic peptide from the laminin A chain sequence.

    PubMed

    Jucker, M; Kleinman, H K; Ingram, D K

    1991-04-01

    Responses of rat embryonic septal cells to reconstituted basement membrane, laminin, and laminin A chain-derived synthetic peptides were studied in culture. Dissociated fetal E16/17 septal cells were grown for three days on differently coated plastic substrata. Reconstituted basement membrane (Matrigel), laminin, and a 19-amino acid synthetic peptide CSRARKQAASIKVAVSADR-NH2 (PA22-2) from the laminin A chain sequence mediated cell-substratum adhesion and promoted neurite outgrowth. In contrast, cells did not attach to or form processes on uncoated plastic or on plastic substrata coated with synthetic, laminin-derived control peptides. Polyethylenimine (PEI) supported the adhesion and survival of fetal septal cells; however, when laminin was added to the medium during cell plating or 18 hr afterward, a dose-dependent increase was observed in neurite outgrowth of cells attached to this substratum. Cells grown for 6 days on PEI in the presence of laminin showed a determined increase in the number of cholinergic neurons as marked by acetylcholinesterase staining. These data suggest that the subpopulation of cholinergic septal neurons present in the septal cells studied here were also responding to laminin. The results of this in vitro study suggest potential uses for basement membrane, laminin, or synthetic peptides, such as PA22-2, in fetal septal grafts to enhance regeneration in the damaged septo-hippocampal system.

  18. Localization of a tumor cell adhesion domain of laminin by a monoclonal antibody

    SciTech Connect

    Skubitz, A.P.N.; Charonis, A.S.; Tsilibary, E.C.; Furcht, L.T. )

    1987-12-01

    Monoclonal antibodies were prepared to localize the domain(s) of laminin to which tumor cells adhere. Rat Y3-Ag 1.2.3 myeloma cells were fused with spleen cells from a rat immunized with a purified 440-kDa fragment of chymotrypsin-digested laminin. Three monoclonal antibodies (AL-1 to AL-3) that bound to intact laminin in a solid-phase radioimmunoassay were chosen for further analysis. The epitopes recognized by these antibodies were characterized by radioimmunoassays, immunoblotting, radioimmunoprecipitation, and immunoaffinity chromatography. In cell adhesion assays, monoclonal antibody AL-2 inhibited the highly metastatic melanoma cell line, K-1735-M4, to both intact laminin and the 440-kDa fragment of laminin. Electron microscopic examination of laminin-monoclonal antibody interactions showed that monoclonal antibody AL-2 reacted with the long arm of laminin directly below the cross-region. Two monoclonal antibodies that failed to inhibit tumor cell adhesion to laminin reacted with epitopes on the lateral short arms or cross-region of laminin as seen by electron microscopy. These results suggest that a new tumor cell binding domain of laminin may be located close to the cross-region on the long arm of laminin.

  19. The Laminin 511/521 Binding Site on the Lutheran Blood Group Glycoprotein is Located at theFlexible Junction of Ig Domains 2 and 3

    SciTech Connect

    Mankelow, Tosti J.; Burton, Nicholas; Stedansdottir, Fanney O.; Spring, Frances A.; Parsons, Stephen F.; Pesersen, Jan S.; Oliveira, Cristiano L.P.; Lammie, Donna; Wess, Timothy; Mohandas, Narla; Chasis, Joel A.; Brady, R. Leo; Anstee, David J.

    2007-07-01

    The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the {alpha}5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vasocclusive events that are an important cause of morbidity in sickle cell disease. Using X-ray crystallography, small angle X-ray scattering and site directed mutagenesis we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.

  20. The Laminin 511/521–binding site on the Lutheran blood group glycoprotein is located at the flexible junction of Ig domains 2 and 3

    PubMed Central

    Burton, Nicholas; Stefansdottir, Fanney O.; Spring, Frances A.; Parsons, Stephen F.; Pedersen, Jan S.; Oliveira, Cristiano L. P.; Lammie, Donna; Wess, Timothy; Mohandas, Narla; Chasis, Joel Anne; Anstee, David J.

    2007-01-01

    The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the α5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vaso-occlusive events that are an important cause of morbidity in sickle cell disease. Using x-ray crystallography, small-angle x-ray scattering, and site-directed mutagenesis, we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin-binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease. PMID:17638854

  1. Spontaneous growth of a laminin-apatite nano-composite in a metastable calcium phosphate solution.

    PubMed

    Oyane, Ayako; Uchida, Masaki; Onuma, Kazuo; Ito, Atsuo

    2006-01-01

    We have previously reported that a laminin-apatite composite layer is formed on an ethylene-vinyl alcohol copolymer (EVOH) in a laminin-containing calcium phosphate (LCP) solution. In this work, the stability of the LCP solution and growth process of the laminin-apatite composite layer have been investigated. Dynamic light scattering technique revealed that the LCP solution was stable for periods as long as 24 h; it did not induce homogeneous precipitation of laminin or calcium phosphates in the solution. Analysis of the EVOH surface and the LCP solution showed that the laminin-apatite composite layer was formed via coprecipitation of laminin and apatite on the EVOH plate, i.e., spontaneous growing of apatite and simultaneous immobilization of laminin molecules or laminin-calcium phosphate nano-complexes onto its surface. Transmission electron microscopy also revealed that the laminin molecules in the resulting composite layer were not localized or aggregated, but were dispersed on a nano-scale in the entire layer. Because of this nano-composite structure, a large number of laminin molecules were stably immobilized on the EVOH plate. This may be responsible for the excellent cell adhesion properties of this type of composite material.

  2. Differential expression of laminin isoforms and alpha 6-beta 4 integrin subunits in the developing human and mouse intestine.

    PubMed

    Simon-Assmann, P; Duclos, B; Orian-Rousseau, V; Arnold, C; Mathelin, C; Engvall, E; Kedinger, M

    1994-09-01

    The intestinal tissue is characterized by important morphogenetic movements during development as well as by a continuous dynamic crypt to villus epithelial cell migration leading to differentiation of specialized cells. In this study, we have examined the spatio-temporal distribution of laminin A and M chains as well as of alpha 6 and beta 4 integrin subunits in adult and developing human and mouse intestine by indirect immunofluorescence. Selective expression of the constituent polypeptides of laminin isoforms (A and M chains) was demonstrated. In the mature human intestine, A and M chains were found to be complementary, the M chain being restricted to the base of crypts and the A chain lining the villus basement membrane. In the developing human intestine, M chain expression was delayed as compared to that of A chain; as soon as the M chain was visualized, it exhibited the typical localization in the crypt basement membrane. A somewhat different situation was found in the adult mouse intestine, since both M and A chains were found in the crypts. During mouse intestinal development the delayed expression of the M chain as compared to that of the A chain was also obvious. The absence of M chain expression in mutant dy mouse did not impair intestinal morphogenesis nor cell differentiation. The expression of alpha 6 and beta 4 subunits was not coordinated. In both species the alpha 6 expression preceded that of beta 4. Furthermore, while beta 4 staining in adult mouse intestine was detected at the basal surface of all cells lining the crypt-villus, that of alpha 6 was mainly confined to the crypt cell compartment. An overall similarity of location between alpha 6 integrin subunit and laminin A chain at the epithelial/stromal interface was noted. These data indicate that the spatial and temporal distribution of laminin variants in the developing intestine may be characteristic for each species and that interactions of laminin variants with particular receptors may be

  3. Intermolecular forces and enthalpies in the adhesion of Streptococcus mutans and an antigen I/II-deficient mutant to laminin films.

    PubMed

    Busscher, Henk J; van de Belt-Gritter, Betsy; Dijkstra, Rene J B; Norde, Willem; Petersen, Fernanda C; Scheie, Anne A; van der Mei, Henny C

    2007-04-01

    The antigen I/II family of surface proteins is expressed by most oral streptococci, including Streptococcus mutans, and mediates specific adhesion to, among other things, salivary films and extracellular matrix proteins. In this study we showed that antigen I/II-deficient S. mutans isogenic mutant IB03987 was nearly unable to adhere to laminin films under flow conditions due to a lack of specific interactions (0.8 x 10(6) and 1.1 x 10(6) cells cm(-2) at pH 5.8 and 6.8, respectively) compared with parent strain LT11 (21.8 x 10(6) and 26.1 x 10(6) cells cm(-2)). The adhesion of both the parent and mutant strains was slightly greater at pH 6.8 than at pH 5.8. In addition, atomic force microscopy (AFM) experiments demonstrated that the parent strain experienced less repulsion when it approached a laminin film than the mutant experienced. Upon retraction, combined specific and nonspecific adhesion forces were stronger for the parent strain (up to -5.0 and -4.9 nN at pH 5.8 and 6.8, respectively) than for the mutant (up to -1.5 and -2.1 nN), which was able to interact only through nonspecific interactions. Enthalpy was released upon adsorption of laminin to the surface of the parent strain but not upon adsorption of laminin to the surface of IB03987. A comparison of the adhesion forces in AFM with the adhesion forces reported for specific ligand-receptor complexes resulted in the conclusion that the number of antigen I/II binding sites for laminin on S. mutans LT11 is on the order of 6 x 10(4) sites per organism and that the sites are probably arranged along exterior surface structures, as visualized here by immunoelectron microscopy.

  4. A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins

    PubMed Central

    1995-01-01

    To examine the function of the alpha 6 beta 4 integrin we have determined its ligand-binding ability and overexpressed two potentially dominant negative mutant beta 4 subunits, lacking either the cytoplasmic or extracellular domain, in bladder epithelial 804G cells. The results of cell adhesion and radioligand-binding assays showed that alpha 6 beta 4 is a receptor for several laminin isoforms, including laminin 1, 2, 4, and 5. Overexpression of the tail-less or head-less mutant beta 4 subunit did not suppress alpha 6 beta 4-mediated adhesion to laminins, as both types of transfectants adhered to these ligands in the presence of blocking anti-beta 1 antibodies as well as the controls. However, immunofluorescence experiments indicated that the endogenous alpha 6 beta 4 integrin and other hemidesmosomal markers were not concentrated in hemidesmosomes in cells overexpressing tail- less beta 4, while the distribution of these molecules was not altered in cells overexpressing the head-less subunit. Electron microscopic studies confirmed that cells overexpressing tail-less beta 4 had a drastically reduced number of hemidesmosomes, while cells expressing the head-less subunit had a normal number of these structures. Thus, expression of a tail-less, but not a head-less mutant beta 4 subunit leads to a dominant negative effect on hemidesmosome assembly without suppressing initial adhesion to laminins. We conclude that the alpha 6 beta 4 integrin binds to several laminins and plays an essential role in the assembly and/or stability of hemidesmosomes, that alpha 6 beta 4- mediated adhesion and hemidesmosome assembly have distinct requirements, and that it is possible to use a dominant negative approach to selectively interfere with a specific function of an integrin. PMID:7721947

  5. Short Laminin Peptide for Improved Neural Stem Cell Growth

    PubMed Central

    Li, Xiaowei; Liu, Xiaoyan; Josey, Benjamin; Chou, C. James; Tan, Yu; Zhang, Ning

    2014-01-01

    Human neural stem/progenitor cells (hNSCs) are very difficult to culture and require human or animal source extracellular matrix molecules, such as laminin or collagen type IV, to support attachment and to regulate their survival and proliferation. These extracellular matrix molecules are difficult to purify from human or animal tissues, have high batch-to-batch variability, and may cause an immune response if used in clinical applications. Although several laminin- and collagen IV-derived peptides are commercially available, they do not support long-term hNSC attachment and growth. To solve this problem, we developed a novel peptide sequence with only 12 amino acids based on the Ile-Lys-Val-Ala-Val, or IKVAV, sequence: Ac-Cys-Cys-Arg-Arg-Ile-Lys-Val-Ala-Val-Trp-Leu-Cys. This short peptide sequence, similar to tissue-derived full laminin molecules, supported hNSCs to attach and proliferate to confluence for continuous passage and subculture. This short peptide also directed hNSCs to differentiate into neurons. When conjugated to poly(ethylene glycol) hydrogels, this short peptide benefited hNSC attachment and proliferation on the surface of hydrogels and promoted cell migration inside the hydrogels with maximum enhancement at a peptide density of 10 μM. This novel short peptide shows great promise in artificial niche development for supporting hNSC culture in vitro and in vivo and for promoting hNSC transplantation in future clinical therapy. PMID:24692587

  6. Biological activities of the homologous loop regions in the laminin α chain LG modules.

    PubMed

    Katagiri, Fumihiko; Hara, Toshihiro; Yamada, Yuji; Urushibata, Shunsuke; Hozumi, Kentaro; Kikkawa, Yamato; Nomizu, Motoyoshi

    2014-06-10

    Each laminin α chain (α1-α5 chains) has chain-specific diverse biological functions. The C-terminal globular domain of the α chain consists of five laminin-like globular (LG1-5) modules and plays a critical role in biological activities. The LG modules consist of a 14-stranded β-sheet (A-N) sandwich structure. Previously, we described the chain-specific biological activities of the loop regions between the E and F strands in the LG4 modules using five homologous peptides (G4EF1-G4EF5). Here, we further analyze the biological activities of the E-F strands loop regions in the rest of LG modules. We designed 20 homologous peptides (approximately 20 amino acid length), and 17 soluble peptides were used for the cell attachment assay. Thirteen peptides promoted cell attachment activity with different cell morphologies. Cell attachment to peptides G1EF1, G1EF2, G2EF1, G3EF4, and G5EF4 was inhibited by heparin, and peptides G1EF1, G1EF2, and G2EF1 specifically bound to syndecan-overexpressing cells. Cell attachment to peptides G2EF3, G3EF1, G3EF3, G5EF1, G5EF3, and G5EF5 was inhibited EDTA. Further, cell attachment to peptides G3EF3, G5EF1, and G5EF5 was inhibited by both anti-integrin α2 and β1 antibodies, whereas cell attachment to peptide G5EF3 was inhibited by only anti-integrin β1 antibody. Cell attachment to peptides G1EF4, G3EF4, and G5EF4 was inhibited by both heparin and EDTA and was not inhibited by anti-integrin antibodies. The active peptide sequence alignments suggest that the syndecan-binding peptides contain a "basic amino acid (BAA)-Gly-BAA" motif in the middle of the molecule and that the integrin-binding peptides contain an "acidic amino acid (AAA)"-Gly-BAA motif. Core-switched peptide analyses suggested that the "BAA-Gly-BAA" motif is critical for binding to syndecans and that the "AAA-Gly-BAA" motif has potential to recognize integrins. These findings are useful for understanding chain-specific biological activities of laminins and to evaluate

  7. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells.

    PubMed

    Pook, Martin; Teino, Indrek; Kallas, Ade; Maimets, Toivo; Ingerpuu, Sulev; Jaks, Viljar

    2015-01-01

    Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain-145 kDa-accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation. PMID:26378917

  8. Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Pook, Martin; Teino, Indrek; Kallas, Ade; Maimets, Toivo; Ingerpuu, Sulev; Jaks, Viljar

    2015-01-01

    Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation. PMID:26378917

  9. Laminin in the cutaneous basement membrane as a potential target in lewisite vesication.

    PubMed

    King, J R; Peters, B P; Monteiro-Riviere, N A

    1994-05-01

    The epidermal-dermal junction has a complex molecular architecture, with numerous components playing key roles in adhesion of the epidermis to the dermis. The purpose of this study was to examine structural components of the epidermal-dermal junction as potential targets for toxicity by lewisite (dichloro(2-chlorovinyl)arsine). This was accomplished by (1) immunocytochemical mapping of laminin, type IV collagen, and bullous pemphigoid antigen (BPA) in lewisite-treated isolated perfused porcine skin flaps (IPPSF), (2) evaluation of protease activity in IPPSF blister fluid against laminin substrate from murine EHS tumor and human keratinocytes, and (3) examination of human keratinocyte laminin for direct chemical modification by lewisite. Lewisite-induced epidermal-dermal separation was localized to the lamina lucida. Localization of the separation suggested that laminin, a cysteine-rich and highly protease-sensitive adhesive glycoprotein, is a potential target for lewisite action. It was hypothesized that chemical modification of laminin directly (via chemical alkylation of laminin thiols by the arsenical) or indirectly (due to lewisite-induced cytotoxic release of proteases) could result in blister formation. Employing sensitive methodology, no evidence of proteolytic activity against EHS tumor laminin or human keratinocyte laminin was identified in the blister fluid. In addition, no evidence for direct chemical modification of laminin by lewisite was demonstrated. However, up to 36% of the thiol groups in human keratinocyte laminin immunoprecipitates was potentially available for reaction with alkylating agents. While these studies did not demonstrate a lewisite-induced chemical modification of laminin, they do not rule out the possibility that other adhesive molecules of the basement membrane are targets for lewisite action. Further evaluation of the molecular role that these binding modalities play in vesicant-induced separation may provide new insights into

  10. Responses of cultured neural retinal cells to substratum-bound laminin and other extracellular matrix molecules.

    PubMed

    Adler, R; Jerdan, J; Hewitt, A T

    1985-11-01

    The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described. Retinal cell suspensions in serum-free medium containing the "N1" supplement (J. E. Bottenstein, S. D. Skaper, S. Varon, and J. Sato, 1980, Exp. Cell Res. 125, 183-190) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested. Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin. Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN. In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN. No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells. Laminin effects on retinal neurons were clearly substratum dependent. When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development. PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule. Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development. Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina.

  11. Overexpression of laminin alpha1 chain in colonic cancer cells induces an increase in tumor growth.

    PubMed

    De Arcangelis, A; Lefebvre, O; Méchine-Neuville, A; Arnold, C; Klein, A; Rémy, L; Kedinger, M; Simon-Assmann, P

    2001-10-01

    Laminins represent a growing family of glycoproteins constituting the basement membrane. They are known to direct many biological processes. With respect to carcinogenesis, laminins play an important role in cell adhesion, mitogenesis, differentiation and even metastasis. To further study the biological significance of laminin-1 (composed of alpha1, beta1 and gamma1 chains) in intestinal cell differentiation or tumorigenesis, an alpha1-laminin expression vector was introduced into the HT29 colonic cancer cells, in which laminin alpha1 chain is not expressed. Upon transfection of the alpha1 chain, the alpha1beta1gamma1 trimer was found secreted in the media along with free alpha1 chain as assessed by immunoprecipitation. The presence of the laminin alpha1 chain did not significantly modify the levels of the other laminin chains nor the integrins expressed by the HT29 cells. In spite of similar growth properties with the control cells in vitro (plastic dish, soft agar), the laminin alpha1 transfectants showed a significantly increased tumor growth when injected in nude mice. Histologic and immunohistochemic examination of the laminin alpha1-expressing tumors points to an increased recruitment of the host stromal and vascular cells, without modification in the differentiation profile and invasion potential. In parallel, a clear accumulation of laminin-10 (alpha5beta1gamma1) at the carcinoma/stromal interface and a segregation of the integrin beta4 subunit at the basal pole of the cancer cells occurred, compared to control tumors. Overall, our observations emphasize the importance of laminin-1 as a chemoattractant of both stromal and vascular cells and in epithelial/stromal cell interactions for the organization of the basement membrane and segregation of integrins leading to an epithelial cell growth signal. Such a sequence of events is reminiscent of what occurs during development.

  12. Responses of cultured neural retinal cells to substratum-bound laminin and other extracellular matrix molecules.

    PubMed

    Adler, R; Jerdan, J; Hewitt, A T

    1985-11-01

    The responses of cultured chick embryo retinal neurons to several extracellular matrix molecules are described. Retinal cell suspensions in serum-free medium containing the "N1" supplement (J. E. Bottenstein, S. D. Skaper, S. Varon, and J. Sato, 1980, Exp. Cell Res. 125, 183-190) were seeded on tissue culture plastic surfaces pretreated with polyornithine (PORN) and with one of the factors to be tested. Substantial cell survival could be observed after 72 hr in vitro on PORN pretreated with serum or laminin, whereas most cells appeared to be degenerating on untreated PORN, PORN-fibronectin, and PORN-chondronectin. Cell attachment, although quantitatively similar for all these substrata, was temperature-dependent on serum and laminin but not on fibronectin or untreated PORN. In a short-term bioassay, neurite development was abundant on laminin, scarce on serum and fibronectin, and absent on PORN. No positive correlation between cell spreading and neurite production could be seen: cell spreading was more extensive on PORN and fibronectin than on laminin or serum, while on laminin-treated dishes, spreading was similar for neurite-bearing and non-neurite-bearing cells. Laminin effects on retinal neurons were clearly substratum dependent. When bound to tissue culture plastic, laminin showed a dose-dependent inhibitory effect on cell attachment and did not stimulate neurite development. PORN-bound laminin, on the other hand, did not affect cell attachment but caused marked stimulation of neurite development, suggesting that laminin conformation and/or the spatial distribution of active sites play an important role in the neurite-promoting function of this extracellular matrix molecule. Investigation of the embryonic retina with ELISA and immunocytochemical methods showed that laminin is present in this organ during development. Therefore, in vivo and in vitro observations are consistent with the possibility that laminin might influence neuronal development in the retina

  13. Lung-specific loss of the laminin α3 subunit confers resistance to mechanical injury.

    PubMed

    Urich, Daniela; Eisenberg, Jessica L; Hamill, Kevin J; Takawira, Desire; Chiarella, Sergio E; Soberanes, Saul; Gonzalez, Angel; Koentgen, Frank; Manghi, Tomas; Hopkinson, Susan B; Misharin, Alexander V; Perlman, Harris; Mutlu, Gokhan M; Budinger, G R Scott; Jones, Jonathan C R

    2011-09-01

    Laminins are heterotrimeric glycoproteins of the extracellular matrix that are secreted by epithelial cells and which are crucial for the normal structure and function of the basement membrane. We have generated a mouse harboring a conditional knockout of α3 laminin (Lama3(fl/fl)), one of the main laminin subunits in the lung basement membrane. At 60 days after intratracheal treatment of adult Lama3(fl/fl) mice with an adenovirus encoding Cre recombinase (Ad-Cre), the protein abundance of α3 laminin in whole lung homogenates was more than 50% lower than that in control-treated mice, suggesting a relatively long half-life for the protein in the lung. Upon exposure to an injurious ventilation strategy (tidal volume of 35 ml per kg of body weight for 2 hours), the mice with a knockdown of the α3 laminin subunit had less severe injury, as shown by lung mechanics, histology, alveolar capillary permeability and survival when compared with Ad-Null-treated mice. Knockdown of the α3 laminin subunit resulted in evidence of lung inflammation. However, this did not account for their resistance to mechanical ventilation. Rather, the loss of α3 laminin was associated with a significant increase in the collagen content of the lungs. We conclude that the loss of α3 laminin in the alveolar epithelium results in an increase in lung collagen, which confers resistance to mechanical injury. PMID:21878500

  14. Laminin: a possible role as a mediator of natural cell-mediated functions

    SciTech Connect

    Laybourn, K.A.

    1986-01-01

    Natural Cell-mediated cytotoxicity is clearly important in regulating host response during tumorgenicity. Natural killer (NK), and Natural Cytotoxic (NC) lymphocytes responsible for mediating cytolysis, are subpopulations of non-B, non-T, nonphagocytic, nonadherent lymphocytes capable of spontaneously lysing a variety of tumor targets. Evidence is presented that laminin, a high molecular weight glycoprotein, is present on the surface of murine NK cells. In addition, NK sensitive tumor targets are able to bind laminin. This suggest that laminin participates as a ligand in the binding of NK cells to their tumor targets. The saturable binding of laminin by NK and NC sensitive tumor targets was shown by laminin-induced cell-cell aggregation/adherence, and /sup 125/I-laminin binding studies. Exogenous laminin inhibited NK cytotoxicity but not CTL cytotoxicity in vitro. In comparison, NK-resistant tumor cells bound little, if any exogenous laminin. Modulating NK activity in vivo prior to challenging mice with an inoculation of a murine fibrosarcoma, showed that elimination or activation of NK activity resulted in an increase or decrease in pulmonary metastases, respectively.

  15. Upregulation of gamma-2 laminin-332 in the mouse ear vesicant wound model.

    PubMed

    Chang, Yoke-Chen; Sabourin, Carol L K; Lu, Shou-En; Sasaki, Takako; Svoboda, Kathy K H; Gordon, Marion K; Riley, David J; Casillas, Robert P; Gerecke, Donald R

    2009-01-01

    Epithelial cell migration during wound healing is regulated in part by enzymatic processing of laminin-332 (formerly LN-5), a heterodimer formed from alpha, beta, and gamma polypeptide chains. Under static conditions, laminin-332 is secreted into the extracellular matrix as a proform and has two chains processed to smaller forms, allowing it to anchor epithelial cells to the basement membrane of the dermis. During incisional wounding, laminin gamma2 chains in particular are processed to smaller sizes and function to promote epithelial sheet migration over the wound bed. The present study examines whether this same function occurs following chemical injury. The mouse ear vesicant model (MEVM) was used to follow the pathology in the ear and test whether processed laminin-332 enhances epithelial cell migration. Skin biopsies of sulfur mustard (SM) exposed ears for several time points were analyzed by histology, immunohistochemistry, real-time PCR, and Western blot analysis. SM exposure greatly increased mRNA levels for laminin-gamma2 in comparison to the other two chains. Protein production of laminin-gamma2 was upregulated, and there was an increase in the processed forms. Protein production was in excess of the amount required to form heterotrimeric laminin-332 and was associated with the migrating epithelial sheet, suggesting a potential role in wound healing for monomeric laminin-gamma2.

  16. Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

    PubMed

    Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-08-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.

  17. Purification, crystallization and preliminary crystallographic analysis of Streptococcus pyogenes laminin-binding protein Lbp

    SciTech Connect

    Linke, Christian; Caradoc-Davies, Tom T.; Proft, Thomas; Baker, Edward N.

    2008-02-01

    The S. pyogenes laminin-binding protein Lbp, which is essential for adhesion to human laminin, has been expressed, purified and crystallized. The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 Å, β = 106.27°, and diffracted to 2.5 Å resolution.

  18. Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin.

    PubMed

    Chen, Z L; Strickland, S

    1997-12-26

    Excess excitatory amino acids can provoke neuronal death in the hippocampus, and the extracellular proteases tissue plasminogen activator (tPA) and plasmin (ogen) have been implicated in this death. To investigate substrates for plasmin that might influence neuronal degeneration, extracellular matrix (ECM) protein expression was examined. Laminin is expressed in the hippocampus and disappears after excitotoxin injection. Laminin disappearance precedes neuronal death, is spatially coincident with regions that exhibit neuronal loss, and is blocked by either tPA-deficiency or infusion of a plasmin inhibitor, both of which also block neuronal degeneration. Preventing neuron-laminin interaction by infusion of anti-laminin antibodies into tPA-deficient mice restores excitotoxic sensitivity to their hippocampal neurons. These results indicate that disruption of neuron-ECM interaction via tPA/plasmin catalyzed degradation of laminin sensitizes hippocampal neurons to cell death.

  19. Co-expression of laminin β3 and γ2 chains and epigenetic inactivation of laminin α3 chain in gastric cancer.

    PubMed

    Ii, Masanori; Yamamoto, Hiroyuki; Taniguchi, Hiroaki; Adachi, Yasushi; Nakazawa, Mayumi; Ohashi, Hirokazu; Tanuma, Tokuma; Sukawa, Yasutaka; Suzuki, Hiromu; Sasaki, Shigeru; Imai, Kohzoh; Shinomura, Yasuhisa

    2011-09-01

    Laminin-332 (LM-332, formerly termed laminin-5) is a heterotrimeric glycoprotein that regulates cell adhesion and migration. Molecular alterations of LM-332 are involved in cancer progression. The aim of this study was to clarify alterations of LM-332 in gastric carcinoma. The expression of LM-332 subunits in 10 gastric carcinoma cell lines was investigated by RT-PCR, Western blotting, and immuno-cytochemical/immunofluorescent analyses. The promoter methylation status of LM-332-encoding genes (LAMA3, LAMB3 and LAMC2) was analyzed by methylation-specific PCR (MSP). The relationship between cell migration and LM-332 expression was assessed by the scratch assay. The expression of LM-332 was analyzed immunohistochemically in 90 gastric cancer tissues. Co-expression of laminin β3 and γ2 chains was often observed in gastric carcinoma cell lines at mRNA and protein levels. In contrast, there was no expression of laminin α3 at either the mRNA or protein levels. Extra-cellular secretion of laminin β3 and γ2 chains was found in 2 of the 10 cell lines. The LAMA3 gene was transcriptionally silenced by methylation of the promoter CpG islands in all of the cell lines, while the LAMB3 and LAMC2 genes were silenced in several cell lines. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), restored expression of the LM-332-encoding genes. Methylation frequency of LAMA3 was higher than those of the LAMB2 and LAMC2 genes in gastric cancer tissues. Migration distances were significantly correlated with cytoplasmic laminin γ2 chain expression. Immunohistochemistry showed frequent co-expression of laminin β3 and γ2 chains in gastric carcinoma cells, which was significantly correlated with depth of invasion and advanced tumor stage. The results suggest that the laminin β3 and γ2 chains accumulate intracellularly and play a role in gastric cancer progression, while epigenetic silencing of the laminin α3 chain may lead to inability to synthesize the basement

  20. Calcium channels link the muscle-derived synapse organizer laminin β2 to Bassoon and CAST/Erc2 to organize presynaptic active zones.

    PubMed

    Chen, Jie; Billings, Sara E; Nishimune, Hiroshi

    2011-01-12

    Synapse formation requires the organization of presynaptic active zones, the synaptic vesicle release sites, in precise apposition to postsynaptic neurotransmitter receptor clusters; however, the molecular mechanisms responsible for these processes remain unclear. Here, we show that P/Q-type and N-type voltage-dependent calcium channels (VDCCs) play essential roles as scaffolding proteins in the organization of presynaptic active zones. The neuromuscular junction of double knock-out mice for P/Q- and N-type VDCCs displayed a normal size but had significantly reduced numbers of active zones and docked vesicles and featured an attenuation of the active-zone proteins Bassoon, Piccolo, and CAST/Erc2. Consistent with this phenotype, direct interactions of the VDCC β1b or β4 subunits and the active zone-specific proteins Bassoon or CAST/Erc2 were confirmed by immunoprecipitation. A decrease in the number of active zones caused by a loss of presynaptic VDCCs resembled the pathological conditions observed in the autoimmune neuromuscular disorder Lambert-Eaton myasthenic syndrome. At the synaptic cleft of double knock-out mice, we also observed a decrease of the synaptic organizer laminin β2 protein, an extracellular ligand of P/Q- and N-type VDCCs. However, the transcription level of laminin β2 did not decrease in double knock-out mice, suggesting that the synaptic accumulation of laminin β2 protein required its interaction with presynaptic VDCCs. These results suggest that presynaptic VDCCs link the target-derived synapse organizer laminin β2 to active-zone proteins and function as scaffolding proteins to anchor active-zone proteins to the presynaptic membrane.

  1. Laminin isoforms: biological roles and effects on the intracellular distribution of nuclear proteins in intestinal epithelial cells

    SciTech Connect

    Turck, Natacha; Gross, Isabelle; Gendry, Patrick; Stutzmann, Jeanne; Freund, Jean-Noel; Kedinger, Michele; Simon-Assmann, Patricia; Launay, Jean-Francois . E-mail: Jean-Francois.Launay@inserm.u-strasbg.fr

    2005-02-15

    Laminins are structurally and functionally major components of the extracellular matrix. Four isoforms of laminins (laminin-1, -2, -5 and -10) are expressed in a specific pattern along the crypt-villus axis of the intestine. Previous works indicated that expression of these isoforms is developmentally regulated and that laminins could modulate the behaviour of intestinal cells, but the exact role of each isoform remained unclear. Here, we report the first systematic analysis of the cellular functions of the four isoforms using the human colon adenocarcinoma Caco2/TC7 cell line as a model. We compared the respective abilities of each isoform to modulate adhesion, proliferation and differentiation of intestinal epithelial cells. We found that the isoforms were functionally distinct, with laminin-10 being the most adhesive substratum, laminin-2, laminin-5 and laminin-10 enhancing cellular proliferation and at the opposite, laminin-1 stimulating intestinal cell differentiation. To begin to characterise the molecular events induced by the different isoforms, we examined by immunofluorescence the intracellular distribution of several nuclear proteins, recently highlighted by a nuclear proteomic approach. We observed clear nucleocytoplasmic redistribution of these proteins, which depended on the laminin isoform. These results provide evidence for a distinct functional role of laminins in intestinal cell functions characterised by specific localisation of nuclear proteins.

  2. The impact of laminin on 3D neurite extension in collagen gels

    NASA Astrophysics Data System (ADS)

    Swindle-Reilly, Katelyn E.; Papke, Jason B.; Kutosky, Hannah P.; Throm, Allison; Hammer, Joshua A.; Harkins, Amy B.; Kuntz Willits, Rebecca

    2012-08-01

    The primary goal of this research was to characterize the effect of laminin on three-dimensional (3D) neurite growth. Gels were formed using type I collagen at concentrations of 0.4-2.0 mg mL-1 supplemented with laminin at concentrations of 0, 1, 10, or 100 µg mL-1. When imaged with confocal microscopy, laminin was shown to follow the collagen fibers; however, the addition of laminin had minimal effect on the stiffness of the scaffolds at any concentration of collagen. Individual neurons dissociated from E9 chick dorsal root ganglia were cultured in the gels for 24 h, and neurite lengths were measured. For collagen gels without laminin, a typical bimodal response of neurite outgrowth was observed, with increased growth at lower concentrations of collagen gel. However, alteration of the chemical nature of the collagen gel by the laminin additive shifted, or completely mitigated, the bimodal neurite growth response seen in gels without laminin. Expression of integrin subunits, α1, α3, α6 and β1, were confirmed by PCR and immunolabeling in the 3D scaffolds. These results provide insight into the interplay between mechanical and chemical environment to support neurite outgrowth in 3D. Understanding the relative impact of environmental factors on 3D nerve growth may improve biomaterial design for nerve cell regeneration.

  3. A Review on the Potential Role of Basement Membrane Laminin in the Pathogenesis of Psoriasis.

    PubMed

    McFadden, J P; Kimber, I

    2016-01-01

    We have previously reviewed alterations to basement membrane laminin in psoriasis and how disruption of this layer could lead to at least some of the pathological changes observed. We here postulate that basement membrane laminin is the key antigen in driving psoriasis, inducing a T cell-mediated autoimmune response. For laminin to be considered as the key autoantigen in psoriasis, it would be reasonable to expect the following to be demonstrable: (1) that autoantigens are present in psoriatic inflammation; (2) that basement membrane laminin is perturbed in involved and uninvolved skin, and that some of the pathological changes associated with psoriasis could be predicted as a sequel to this; (3) that disruption of the basement membrane is among the earliest events in the evolution of psoriatic lesions; (4) that as streptococcal pharyngitis is the most clearly defined event to trigger or exacerbate psoriasis, then a T cell-mediated autoimmune response to laminin should be anticipated as a potential sequelae to streptococcal pharyngitis; (5) that T cells in psoriasis can be shown to react to peptides with homology to laminin; (6) that HLACw6, as the most closely related gene associated with psoriasis and which is involved in antigen expression, should be preferentially expressed within lesional psoriasis towards the basement membrane, together with other proximal associated immune activity; and (7) that there is some association between antilaminin pemphigoid, a humorally mediated autoimmune disease to skin basement membrane laminin, and psoriasis. We here review the data relevant to each of these requirements.

  4. Drosophila laminins act as key regulators of basement membrane assembly and morphogenesis

    PubMed Central

    Urbano, Jose M.; Torgler, Catherine N.; Molnar, Cristina; Tepass, Ulrich; López-Varea, Ana; Brown, Nicholas H.; de Celis, Jose F.; Martín-Bermudo, Maria D.

    2009-01-01

    Laminins are heterotrimeric molecules found in all basement membranes. In mammals, they have been involved in diverse developmental processes, from gastrulation to tissue maintenance. The Drosophila genome encodes two laminin α chains, one β and one Γ, which form two distinct laminin trimers. So far, only mutations affecting one or other trimer have been analysed. In order to study embryonic development in the complete absence of laminins, we mutated the gene encoding the sole laminin β chain in Drosophila, LanB1, so that no trimers can be made. We show that LanB1 mutant embryos develop until the end of embryogenesis. Electron microscopy analysis of mutant embryos reveals that the basement membranes are absent and the remaining extracellular material appears disorganised and diffuse. Accordingly, abnormal accumulation of major basement membrane components, such as Collagen IV and Perlecan, is observed in mutant tissues. In addition, we show that elimination of LanB1 prevents the normal morphogenesis of most organs and tissues, including the gut, trachea, muscles and nervous system. In spite of the above structural roles for laminins, our results unravel novel functions in cell adhesion, migration and rearrangement. We propose that while an early function of laminins in gastrulation is not conserved in Drosophila and mammals, their function in basement membrane assembly and organogenesis seems to be maintained throughout evolution. PMID:19906841

  5. Laminin modified infection-preventing collagen membrane containing silver sulfadiazine-hyaluronan microparticles.

    PubMed

    Lee, Jong-Eun; Park, Jong-Chul; Lee, Kwang Hoon; Oh, Sang Ho; Suh, Hwal

    2002-06-01

    The newly developed laminin modified infection-preventing collagen membrane consists of a 3 component laminate, comprising 2 outer collagen layers and a central laminin layer. The 2 outer collagen layers (dense and porous layers) were fabricated by air-drying and freeze drying, respectively, and the laminin layer was formed by a straightforward liquid coating method. In addition, hyaluronan based microparticles containing silver sulfadiazine (AgSD) were incorporated into the 2 collagen layers (AgSD content 50 microg/cm2). Laminin coated collagen surfaces did not promote fibroblast attachment but showed a retarded fibroblast proliferation rate and an increased rate of collagen synthesis versus pure collagen surfaces. In an animal study, a laminin coating on a nonmedicated collagen membrane significantly increased both wound size reduction and vessel proliferation 7 days after application versus polyurethane film. Interestingly, the laminin coated AgSD medicated collagen membrane demonstrated higher wound size reduction and vessel proliferation and lower inflammation than the polyurethane control, suggesting that the laminin AgSD medicated collagen membrane substantially improves dermal wound healing.

  6. The E8 subfragment of laminin promotes locomotion of myoblasts over extracellular matrix

    PubMed Central

    1989-01-01

    The locomotion of murine myoblasts over the extracellular matrix components laminin and fibronectin was analyzed using quantitative videomicroscopy, and the organization of the cytoskeleton was observed in parallel immunofluorescence studies. Cells plated on the laminin- nidogen complex locomoted twice as fast as on laminin alone. The main form of translocation on laminin was a jerky cycle of prolonged lamellipod extension followed by rapid (approximately 200- less than 500 microh h-1) movement of the cell body into the extended lamellipod. The locomotion-stimulating activity of laminin resides in the elastase digestion fragment E8, part of the laminin long arm, while the E1-4 fragment containing the three short arms is inactive. Myoblasts moved poorly over fibronectin irrespective of whether high, intermediate, or low coating concentrations were used (approximately 5,000- approximately 10 fmol cm-2). In contrast, the locomotory responses both to laminin and to E8 peaked sharply at coating concentrations approximately 20-50 fmol cm-2 and decreased at higher concentrations. This response corresponds to that expected for a haptotactic stimulant. When cells locomoted over a mixed substrate of laminin and fibronectin, the fibronectin effects appeared to predominate. The cytoskeleton has been implicated in many cellular motile processes. Within 6 h on fibronectin many cells expressed vinculin-containing focal contacts, elaborated stress fibers and had periodically organized alpha actinin, whereas on laminin, most cells showed diffuse vinculin and alpha actinin and a fine meshlike actin cytoskeleton. We conclude that the poor locomotion of cells over fibronectin is because of the cytoskeletal stabilization it induces. PMID:2503526

  7. Merosin and laminin in myogenesis; specific requirement for merosin in myotube stability and survival

    PubMed Central

    1996-01-01

    Laminin (laminin-1; alpha 1-beta 1-gamma 1) is known to promote myoblast proliferation, fusion, and myotube formation. Merosin (laminin- 2 and -4; alpha 2-beta 1/beta 2-gamma 1) is the predominant laminin variant in skeletal muscle basement membranes; genetic defects affecting its structure or expression are the causes of some types of congenital muscular dystrophy. However, the precise nature of the functions of merosin in muscle remain unknown. We have developed an in vitro system that exploits human RD and mouse C2C12 myoblastic cell lines and their clonal variants to study the roles of merosin and laminin in myogenesis. In the parental cells, which fuse efficiently to multinucleated myotubes, merosin expression is upregulated as a function of differentiation while laminin expression is downregulated. Cells from fusion-deficient clones do not express either protein, but laminin or merosin added to the culture medium induced their fusion. Clonal variants which fuse, but form unstable myotubes, express laminin but not merosin. Exogenous merosin converted these myotubes to a stable phenotype, while laminin had no effect. Myotube instability was corrected most efficiently by transfection of the merosin-deficient cells with the merosin alpha 2 chain cDNA. Finally, merosin appears to promote myotube stability by preventing apoptosis. Hence, these studies identify novel biological functions for merosin in myoblast fusion and muscle cell survival; furthermore, these explain some of the pathogenic events observed in congenital muscular dystrophy caused by merosin deficiency and provide in vitro models to further investigate the molecular mechanisms of this disease. PMID:8830776

  8. Stable, Covalent Attachment of Laminin to Microposts Improves the Contractility of Mouse Neonatal Cardiomyocytes

    PubMed Central

    2015-01-01

    The mechanical output of contracting cardiomyocytes, the muscle cells of the heart, relates to healthy and disease states of the heart. Culturing cardiomyocytes on arrays of elastomeric microposts can enable inexpensive and high-throughput studies of heart disease at the single-cell level. However, cardiomyocytes weakly adhere to these microposts, which limits the possibility of using biomechanical assays of single cardiomyocytes to study heart disease. We hypothesized that a stable covalent attachment of laminin to the surface of microposts improves cardiomyocyte contractility. We cultured cells on polydimethylsiloxane microposts with laminin covalently bonded with the organosilanes 3-glycidoxypropyltrimethoxysilane and 3-aminopropyltriethoxysilane with glutaraldehyde. We measured displacement of microposts induced by the contractility of mouse neonatal cardiomyocytes, which attach better than mature cardiomyocytes to substrates. We observed time-dependent changes in contractile parameters such as micropost deformation, contractility rates, contraction and relaxation speeds, and the times of contractions. These parameters were affected by the density of laminin on microposts and by the stability of laminin binding to micropost surfaces. Organosilane-mediated binding resulted in higher laminin surface density and laminin binding stability. 3-glycidoxypropyltrimethoxysilane provided the highest laminin density but did not provide stable protein binding with time. Higher surface protein binding stability and strength were observed with 3-aminopropyltriethoxysilane with glutaraldehyde. In cultured cardiomyocytes, contractility rate, contraction speeds, and contraction time increased with higher laminin stability. Given these variations in contractile function, we conclude that binding of laminin to microposts via 3-aminopropyltriethoxysilane with glutaraldehyde improves contractility observed by an increase in beating rate and contraction speed as it occurs during the

  9. Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and beta1 integrin.

    PubMed

    Gama-de-Souza, Letícia N; Cyreno-Oliveira, Elaine; Freitas, Vanessa M; Melo, Edielle S; Vilas-Boas, Vanessa F; Moriscot, Anselmo S; Jaeger, Ruy G

    2008-06-01

    We studied the induction of protease activity by the laminin alpha1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M1 cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells.

  10. Transient laminin beta 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration.

    PubMed

    Chen, Chen-Hui; Merriman, Alexander F; Savage, Jeremiah; Willer, Jason; Wahlig, Taylor; Katsanis, Nicholas; Yin, Viravuth P; Poss, Kenneth D

    2015-08-01

    The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis. PMID:26305099

  11. Transient laminin beta 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration

    PubMed Central

    Chen, Chen-Hui; Merriman, Alexander F.; Savage, Jeremiah; Willer, Jason; Wahlig, Taylor; Katsanis, Nicholas; Yin, Viravuth P.; Poss, Kenneth D.

    2015-01-01

    The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis. PMID:26305099

  12. Lens Extrusion from Laminin Alpha 1 Mutant Zebrafish

    PubMed Central

    Semina, Elena V.; Duncan, Melinda K.

    2014-01-01

    We report analysis of the ocular lens phenotype of the recessive, larval lethal zebrafish mutant, lama1a69/a69. Previous work revealed that this mutant has a shortened body axis and eye defects including a defective hyaloid vasculature, focal corneal dysplasia, and loss of the crystalline lens. While these studies highlight the importance of laminin α1 in lens development, a detailed analysis of the lens defects seen in these mutants was not reported. In the present study, we analyze the lenticular anomalies seen in the lama1a69/a69 mutants and show that the lens defects result from the anterior extrusion of lens material from the eye secondary to structural defects in the lens capsule and developing corneal epithelium associated with basement membrane loss. Our analysis provides further insights into the role of the lens capsule and corneal basement membrane in the structural integrity of the developing eye. PMID:24526906

  13. Combinatorial Fibronectin and Laminin Signaling Promote Highly Efficient Cardiac Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Sa, Silin; Wong, Lian

    2014-01-01

    Abstract Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies—primarily based on the optimization of growth factors—have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4+ and ITGB5+ cells. PMID:25126479

  14. Localisation of laminin within Plasmodium berghei oocysts and the midgut epithelial cells of Anopheles stephensi

    PubMed Central

    Nacer, Adéla; Walker, Karen; Hurd, Hilary

    2008-01-01

    Background Oocysts of the malaria parasite form and develop in close proximity to the mosquito midgut basal lamina and it has been proposed that components of this structure play a crucial role in the development and maturation of oocysts that produce infective sporozoites. It is further suggested that oocysts incorporate basal lamina proteins into their capsule and that this provides them with a means to evade recognition by the mosquito's immune system. The site of production of basal lamina proteins in insects is controversial and it is still unclear whether haemocytes or midgut epithelial cells are the main source of components of the mosquito midgut basal lamina. Of the multiple molecules that compose the basal lamina, laminin is known to interact with a number of Plasmodium proteins. In this study, the localisation of mosquito laminin within the capsule and cytoplasm of Plasmodium berghei oocysts and in the midgut epithelial cells of Anopheles stephensi was investigated. Results An ultrastructural examination of midgut sections from infected and uninfected An. stephensi was performed. Post-embedded immunogold labelling demonstrated the presence of laminin within the mosquito basal lamina. Laminin was also detected on the outer surface of the oocyst capsule, incorporated within the capsule and associated with sporozoites forming within the oocysts. Laminin was also found within cells of the midgut epithelium, providing support for the hypothesis that these cells contribute towards the formation of the midgut basal lamina. Conclusion We suggest that ookinetes may become coated in laminin as they pass through the midgut epithelium. Thereafter, laminin secreted by midgut epithelial cells and/or haemocytes, binds to the outer surface of the oocyst capsule and that some passes through and is incorporated into the developing oocysts. The localisation of laminin on sporozoites was unexpected and the importance of this observation is less clear. PMID:18808667

  15. Laminin α5 influences the architecture of the mouse small intestinal mucosa

    PubMed Central

    Mahoney, Zhen X.; Stappenbeck, Thaddeus S.; Miner, Jeffrey H.

    2008-01-01

    Summary The mammalian intestine displays two distinct patterns of mucosal organization. The small intestine contains mucosal epithelial invaginations called crypts of Lieberkühn that are continuous with evaginations into the lumen called villi. The colon also contains crypts, but its epithelial surface is lined by flat surface cuffs. The epithelial cells of both organs communicate with the underlying mesenchyme through a basement membrane that is composed of a variety of extracellular matrix proteins, including members of the laminin family. The basement membranes of the small intestine and colon contain distinct laminin subtypes; notably, the villus basement membrane is rich in laminin α5. Here we show that diminution of laminin α5 in a mouse model led to a compensatory deposition of colonic laminins that resulted in a transformation from a small intestinal to a colonic mucosal architecture. The alteration in mucosal architecture was associated with reduced levels of nuclear p27Kip1, a cell cycle regulator, and altered intestinal epithelial cell proliferation, migration, and differentiation. Our results suggest that laminin α5 plays a crucial role in establishing and maintaining the specific mucosal pattern of the mouse small intestine. PMID:18628307

  16. Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces

    PubMed Central

    Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

    2016-01-01

    The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces. PMID:26996815

  17. Borrelia burgdorferi BmpA Is a Laminin-Binding Protein▿

    PubMed Central

    Verma, Ashutosh; Brissette, Catherine A.; Bowman, Amy; Stevenson, Brian

    2009-01-01

    The Borrelia burgdorferi BmpA outer surface protein plays a significant role in mammalian infection by the Lyme disease spirochete and is an important antigen for the serodiagnosis of human infection. B. burgdorferi adheres to host extracellular matrix components, including laminin. The results of our studies indicate that BmpA and its three paralogous proteins, BmpB, BmpC, and BmpD, all bind to mammalian laminin. BmpA did not bind mammalian type I or type IV collagens or fibronectin. BmpA-directed antibodies significantly inhibited the adherence of live B. burgdorferi to laminin. The laminin-binding domain of BmpA was mapped to the carboxy-terminal 80 amino acids. Solubilized collagen inhibited BmpA-laminin binding, suggesting interactions through the collagen-binding domains of laminin. These results, together with previous data, indicate that BmpA and its paralogs are targets for the development of preventative and curative therapies for Lyme disease. PMID:19703983

  18. Tendon chitosan tubes covalently coupled with synthesized laminin peptides facilitate nerve regeneration in vivo.

    PubMed

    Suzuki, Masumi; Itoh, Soichiro; Yamaguchi, Isamu; Takakuda, Kazuo; Kobayashi, Hisatoshi; Shinomiya, Kenichi; Tanaka, Junzo

    2003-06-01

    We have developed tendon chitosan tubes having the ability to bind peptides covalently, and the effectiveness of laminin peptides coupled to these tubular wall on nerve regeneration was examined in vivo. Bridge graft implantation (15 mm) into the sciatic nerve of SD rats was carried out using chitosan tubes having a triangular cross section containing either covalently bound intact laminin or the laminin peptides CDPGYIGSR or CSRARKQAASIKVAVSAD or being nontreated (N = 20 in each group). As a control, isografting (N = 5) was carried out. Three rats in each experimental group were sacrificed for histology observations after 1, 2, 4, 6, and 8 weeks. The total area of regenerating tissue in the tube and the length of the area where regenerating tissue attached to the inner surface of the tube were measured. In five rats from each experimental and control group, the latency quotient between the implanted and the nontreated site was determined 12 weeks after implantation. Furthermore, the percentage of myelinated axon area was measured at a 10-mm distance from the distal anastomosed site. Histological findings suggest that the immobilized laminin, confirmed by immunostaining as long as 12 weeks postoperatively, as well as laminin oligopeptides may effectively assist nerve tissue extension. According to statistical analysis of the percentage neural tissue found in relation to evoked action potentials, the sequential treatments with YIGSR first followed by IKVAV matched the effectiveness of intact laminin in enhancing nerve regeneration. However, when compared with that after isografting, the enhancement of regenerated axon growth was less sufficient.

  19. Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces

    NASA Astrophysics Data System (ADS)

    Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

    2016-03-01

    The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces.

  20. Effects of penicillin and erythromycin on adherence of invasive and noninvasive isolates of Streptococcus pyogenes to laminin.

    PubMed

    Šmitran, Aleksandra; Vuković, Dragana; Gajić, Ina; Marinković, Jelena; Ranin, Lazar

    2015-08-01

    This study investigated the possible relationship between the invasiveness of group A Streptococcus (GAS) strains and their abilities to adhere to laminin and assessed the effects of subinhibitory concentrations of penicillin and erythromycin on the ability of GAS to adhere to laminin. The adherence of noninvasive and highly invasive isolates of GAS to laminin was significantly higher than the adherence displayed by isolates of low invasiveness. Antibiotic treatment caused significant reductions in adherence to laminin in all three groups of strains. Penicillin was more successful in reducing the adherence abilities of the tested GAS strains than erythromycin.

  1. Analysis of adhesive binding forces between laminin-1 and C2C12 muscle cell membranes measured via high resolution force spectroscopy

    NASA Astrophysics Data System (ADS)

    Gluck, George; Gilbert, Richard; Ortiz, Christine

    2002-03-01

    Laminins are a family of glycoproteins that regulate cell differentiation, shape, and motility through interactions with various cell surface receptors. Here, we have directly measured the biomolecular adhesive binding forces between a cantilever / probe tip that was covalently attached with laminin-1 and membrane receptors on C2C12 muscle cells using the technique of high-resolution force spectroscopy (HRFS). On retraction of the probe tip away from the membrane surface, discrete, long-range adhesive unbinding events were always observed. Statistical analysis of the data revealed an initial broad distribution of heterogeneous unbinding events (occurring at separation distances, D=0-2µm from the point of maximum compression) of magnitude 92.23±37.87pN followed by a narrow distribution of homogeneous unbinding events (occurring at D > 2µm) of magnitude 38.16±9.10pN, which is suggestive of an individual biomolecular adhesive interaction. On-going studies include loading rate dependence and effect of dystroglycan mutation.

  2. Novel receptors for bacterial protein toxins.

    PubMed

    Schmidt, Gudula; Papatheodorou, Panagiotis; Aktories, Klaus

    2015-02-01

    While bacterial effectors are often directly introduced into eukaryotic target cells by various types of injection machines, toxins enter the cytosol of host cells from endosomal compartments or after retrograde transport via Golgi from the ER. A first crucial step of toxin-host interaction is receptor binding. Using optimized protocols and new methods novel toxin receptors have been identified, including metalloprotease ADAM 10 for Staphylococcus aureus α-toxin, laminin receptor Lu/BCAM for Escherichia coli cytotoxic necrotizing factor CNF1, lipolysis stimulated lipoprotein receptor (LSR) for Clostridium difficile transferase CDT and low-density lipoprotein receptor-related protein (LRP) 1 for Clostridium perfringens TpeL toxin.

  3. Functional regeneration of the transected recurrent laryngeal nerve using a collagen scaffold loaded with laminin and laminin-binding BDNF and GDNF

    PubMed Central

    Wang, Baoxin; Yuan, Junjie; Chen, Xinwei; Xu, Jiafeng; Li, Yu; Dong, Pin

    2016-01-01

    Recurrent laryngeal nerve (RLN) injury remains a challenge due to the lack of effective treatments. In this study, we established a new drug delivery system consisting of a tube of Heal-All Oral Cavity Repair Membrane loaded with laminin and neurotrophic factors and tested its ability to promote functional recovery following RLN injury. We created recombinant fusion proteins consisting of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) fused to laminin-binding domains (LBDs) in order to prevent neurotrophin diffusion. LBD-BDNF, LBD-GDNF, and laminin were injected into a collagen tube that was fitted to the ends of the transected RLN in rats. Functional recovery was assessed 4, 8, and 12 weeks after injury. Although vocal fold movement was not restored until 12 weeks after injury, animals treated with the collagen tube loaded with laminin, LBD-BDNF and LBD-GDNF showed improved recovery in vocalisation, arytenoid cartilage angles, compound muscle action potentials and regenerated fibre area compared to animals treated by autologous nerve grafting (p < 0.05). These results demonstrate the drug delivery system induced nerve regeneration following RLN transection that was superior to that induced by autologus nerve grafting. It may have potential applications in nerve regeneration of RLN transection injury. PMID:27558932

  4. Functional regeneration of the transected recurrent laryngeal nerve using a collagen scaffold loaded with laminin and laminin-binding BDNF and GDNF.

    PubMed

    Wang, Baoxin; Yuan, Junjie; Chen, Xinwei; Xu, Jiafeng; Li, Yu; Dong, Pin

    2016-01-01

    Recurrent laryngeal nerve (RLN) injury remains a challenge due to the lack of effective treatments. In this study, we established a new drug delivery system consisting of a tube of Heal-All Oral Cavity Repair Membrane loaded with laminin and neurotrophic factors and tested its ability to promote functional recovery following RLN injury. We created recombinant fusion proteins consisting of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) fused to laminin-binding domains (LBDs) in order to prevent neurotrophin diffusion. LBD-BDNF, LBD-GDNF, and laminin were injected into a collagen tube that was fitted to the ends of the transected RLN in rats. Functional recovery was assessed 4, 8, and 12 weeks after injury. Although vocal fold movement was not restored until 12 weeks after injury, animals treated with the collagen tube loaded with laminin, LBD-BDNF and LBD-GDNF showed improved recovery in vocalisation, arytenoid cartilage angles, compound muscle action potentials and regenerated fibre area compared to animals treated by autologous nerve grafting (p < 0.05). These results demonstrate the drug delivery system induced nerve regeneration following RLN transection that was superior to that induced by autologus nerve grafting. It may have potential applications in nerve regeneration of RLN transection injury. PMID:27558932

  5. Lung-specific loss of α3 laminin worsens bleomycin-induced pulmonary fibrosis.

    PubMed

    Morales-Nebreda, Luisa I; Rogel, Micah R; Eisenberg, Jessica L; Hamill, Kevin J; Soberanes, Saul; Nigdelioglu, Recep; Chi, Monica; Cho, Takugo; Radigan, Kathryn A; Ridge, Karen M; Misharin, Alexander V; Woychek, Alex; Hopkinson, Susan; Perlman, Harris; Mutlu, Gokhan M; Pardo, Annie; Selman, Moises; Jones, Jonathan C R; Budinger, G R Scott

    2015-04-01

    Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3(fl/fl)). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-β was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-β. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression.

  6. Lung-specific loss of α3 laminin worsens bleomycin-induced pulmonary fibrosis.

    PubMed

    Morales-Nebreda, Luisa I; Rogel, Micah R; Eisenberg, Jessica L; Hamill, Kevin J; Soberanes, Saul; Nigdelioglu, Recep; Chi, Monica; Cho, Takugo; Radigan, Kathryn A; Ridge, Karen M; Misharin, Alexander V; Woychek, Alex; Hopkinson, Susan; Perlman, Harris; Mutlu, Gokhan M; Pardo, Annie; Selman, Moises; Jones, Jonathan C R; Budinger, G R Scott

    2015-04-01

    Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3(fl/fl)). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-β was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-β. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression. PMID:25188360

  7. Lung-Specific Loss of α3 Laminin Worsens Bleomycin-Induced Pulmonary Fibrosis

    PubMed Central

    Morales-Nebreda, Luisa I.; Rogel, Micah R.; Eisenberg, Jessica L.; Hamill, Kevin J.; Soberanes, Saul; Nigdelioglu, Recep; Chi, Monica; Cho, Takugo; Radigan, Kathryn A.; Ridge, Karen M.; Misharin, Alexander V.; Woychek, Alex; Hopkinson, Susan; Perlman, Harris; Mutlu, Gokhan M.; Pardo, Annie; Selman, Moises; Jones, Jonathan C. R.

    2015-01-01

    Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3fl/fl). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-β was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-β. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression. PMID:25188360

  8. Targeted inactivation of murine laminin gamma2-chain gene recapitulates human junctional epidermolysis bullosa.

    PubMed

    Meng, Xianmin; Klement, John F; Leperi, Dominic A; Birk, David E; Sasaki, Takako; Timpl, Rupert; Uitto, Jouni; Pulkkinen, Leena

    2003-10-01

    Junctional forms of epidermolysis bullosa (JEB) are associated with mutations in six distinct genes expressed in the cutaneous basement membrane zone; these include LAMA3, LAMB3, and LAMC2, which encode laminin 5 subunit polypeptides, the alpha3-, beta3-, and gamma2-chains, respectively. Here we generated a mouse model for JEB by inactivating the laminin gamma2-chain gene by targeted frameshift deletion of exon 8 in Lamc2. Heterozygous mice were phenotypically normal, whereas the majority of Lamc2-/- mice showed blistering phenotype on days 1 to 2 and died within 5 days of birth. The Lamc2-/- mice demonstrated absent expression of laminin gamma2-chain on the basement membrane zone as well as attenuated expression of alpha3- and beta3-chains of laminin. Transmission electron microscopy revealed rudimentary, poorly developed hemidesmosomes. The epidermis of the Lamc2-/- mice revealed induced apoptosis in the basal cells of the blistered skin, suggesting that cell-matrix adhesion provided by laminin 5 plays a role in cell survival in vivo. Cultured Lamc2-/- keratinocytes demonstrated slightly positive staining with gamma2-chain-specific antibodies, which could be explained by the presence of a transcript with partial restoration of the reading frame owing to alternative splicing in vitro. These cells proliferated in different matrices and attached to type IV collagen and Matrigel as efficiently as the wild-type keratinocytes, whereas their attachment on plastic and laminin was significantly weaker. In summary, Lamc2-/- mouse recapitulates human JEB and provides novel insight into the role of laminin 5 in keratinocyte biology. PMID:14632187

  9. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin

    PubMed Central

    1993-01-01

    The dystrophin-glycoprotein complex was tested for interaction with several components of the extracellular matrix as well as actin. The 156-kD dystrophin-associated glycoprotein (156-kD dystroglycan) specifically bound laminin in a calcium-dependent manner and was inhibited by NaCl (IC50 = 250 mM) but was not affected by 1,000-fold (wt/wt) excesses of lactose, IKVAV, or YIGSR peptides. Laminin binding was inhibited by heparin (IC50 = 100 micrograms/ml), suggesting that one of the heparin-binding domains of laminin is involved in binding dystroglycan while negatively charged oligosaccharide moieties on dystroglycan were found to be necessary for its laminin-binding activity. No interaction between any component of the dystrophin- glycoprotein complex and fibronectin, collagen I, collagen IV, entactin, or heparan sulfate proteoglycan was detected by 125I-protein overlay and/or extracellular matrix protein-Sepharose precipitation. In addition, laminin-Sepharose quantitatively precipitated purified dystrophin-glycoprotein complex, demonstrating that the laminin-binding site is accessible when dystroglycan is associated with the complex. Dystroglycan of nonmuscle tissues also bound laminin. However, the other proteins of the striated muscle dystrophin-glycoprotein complex appear to be absent, antigenically dissimilar or less tightly associated with dystroglycan in nonmuscle tissues. Finally, we show that the dystrophin-glycoprotein complex cosediments with F-actin but does not bind calcium or calmodulin. Our results support a role for the striated muscle dystrophin-glycoprotein complex in linking the actin- based cytoskeleton with the extracellular matrix. Furthermore, our results suggest that dystrophin and dystroglycan may play substantially different functional roles in nonmuscle tissues. PMID:8349731

  10. Laminin chain expression suggests that laminin-10 is a major isoform in the mouse hippocampus and is degraded by the tissue plasminogen activator/plasmin protease cascade during excitotoxic injury.

    PubMed

    Indyk, J A; Chen, Z L; Tsirka, S E; Strickland, S

    2003-01-01

    Laminins are important components of the extracellular matrix, and participate in neuronal development, survival and regeneration. The tissue plasminogen activator/plasmin extracellular protease cascade and downstream laminin degradation are implicated in excitotoxin-induced neuronal degeneration. To determine which specific laminin chains are involved, we investigated the expression of laminins in the hippocampus, and the cell types expressing them. Reverse transcription-PCR demonstrated that the messenger RNAs for all laminin chains could be detected in the hippocampus. To determine the localization of laminin chain expression, immunostaining was used. This method showed that alpha5, beta1 and gamma1 are most highly expressed in the neuronal cell layers. Immunoblotting confirmed the hippocampal expression of the chains alpha5, beta1 and gamma1, and RNA in situ hybridization showed a neuronal expression pattern of alpha5, beta1 and gamma1. At early time points following intrahippocampal injection of kainate, alpha5, beta1 and gamma1 chain immunoreactivities were lost. In addition, tissue plasminogen activator-deficient mice, which are resistant to kainate-induced neuronal death, show no significant change in laminins alpha5, beta1 and gamma1 after intrahippocampal kainate injection. Taken together, these results suggest that laminin-10 (alpha5-beta1-gamma1) comprises a major neuronal laminin in the mouse hippocampus, and is degraded before neuronal death during excitotoxic injury by the tissue plasminogen activator/plasmin protease cascade. By identifying a neuronal laminin (laminin-10) that participates in neuronal degeneration after excitotoxic injury, this study clarifies the molecular definition of the extracellular matrix in the hippocampus and further defines a pathway for mechanisms of neuronal death.

  11. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

    PubMed

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  12. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins

    PubMed Central

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  13. Laminin-5 mutational analysis in an Italian cohort of patients with junctional epidermolysis bullosa.

    PubMed

    Posteraro, Patrizia; De Luca, Naomi; Meneguzzi, Guerrino; El Hachem, May; Angelo, Corrado; Gobello, Tommaso; Tadini, Gianluca; Zambruno, Giovanna; Castiglia, Daniele

    2004-10-01

    Junctional epidermolysis bullosa (JEB) is a rare genodermatosis characterized by dermal-epidermal separation that is caused by mutations in the genes encoding hemidesmosomal components and laminin-5, the major epithelial adhesion ligand. Here, we report on the mutational analysis of LAMA3, LAMB3, and LAMC2 genes encoding laminin-5 chains in 19 Italian patients, 11 affected with the severe Herlitz (H JEB) and eight with the mild non-Herlitz variant of JEB (non-H JEB). Eighteen mutations, seven of which were novel, were identified and their consequences analyzed at the mRNA and protein level. Premature termination codon mutations in both alleles of LAMB3 or LAMC2 genes were found in nine of the 11 H JEB patients, with a prevalence of mutations in LAMC2. In one case, a homozygous frameshift mutation in LAMB3 was associated to illegitimate splicing leading to non-H JEB. One H JEB patient showed a large intragenic duplication within LAMC2, a genetic defect so far uncovered in laminin-5 genes. Splicing or missense mutations, were prevalent in non-H JEB patients. Collectively, five mutations appeared to be frequent in laminin-5 JEB patients: R635X, 29insC, E210K, W143X in LAMB3 and R95X in LAMC2. These recurrent mutations account for approximately 44% of laminin-5 JEB alleles in Italian patients. PMID:15373767

  14. Immobilization of Cell-Adhesive Laminin Peptides in Degradable PEGDA Hydrogels Influences Endothelial Cell Tubulogenesis

    PubMed Central

    Ali, Saniya; Saik, Jennifer E.; Gould, Dan J.; Dickinson, Mary E.

    2013-01-01

    Abstract Attachment, spreading, and organization of endothelial cells into tubule networks are mediated by interactions between cells in the extracellular microenvironment. Laminins are key extracellular matrix components and regulators of cell adhesion, migration, and proliferation. In this study, laminin-derived peptides were conjugated to poly(ethylene glycol) (PEG) monoacrylate and covalently incorporated into degradable PEG diacrylate (PEGDA) hydrogels to investigate the influence of these peptides on endothelial cellular adhesion and function in organizing into tubule networks. Degradable PEGDA hydrogels were synthesized by incorporating a matrix metalloproteinase (MMP)–sensitive peptide, GGGPQGIWGQGK (abbreviated PQ), into the polymer backbone. The secretion of MMP-2 and MMP-9 by endothelial cells promotes polymer degradation and consequently cell migration. We demonstrate the formation of extensive networks of tubule-like structures by encapsulated human umbilical vein endothelial cells in hydrogels with immobilized synthetic peptides. The resulting structures were stabilized by pericyte precursor cells (10T1/2s) in vitro. During tubule formation and stabilization, extracellular matrix proteins such as collagen IV and laminin were deposited. Tubules formed in the matrix of metalloproteinase sensitive hydrogels were visualized from 7 days to 4 weeks in response to different combination of peptides. Moreover, hydrogels functionalized with laminin peptides and transplanted in a mouse cornea supported the ingrowth and attachment of endothelial cells to the hydrogel during angiogenesis. Results of this study illustrate the use of laminin-derived peptides as potential candidates for modification of biomaterials to support angiogenesis. PMID:23914330

  15. Role of laminin in maintenance of type II pneumocyte morphology and function

    SciTech Connect

    Rannels, S.R.; Yarnell, J.A.; Fisher, C.S.; Fabisiak, J.P.; Rannels, D.E. )

    1987-12-01

    Loss of differentiated function by type II pneumocytes plated on plastic surfaces was demonstrated by decreased lamellar body content, increased cellular protein, and rapid cellular flattening, changes that were retarded modestly by plating cells on laminin-coated surfaces. Laminin surfaces also inhibited ({sup 3}H)thymidine (THM) incorporation into cellular DNA by 40% compared with plastic at 40 h, but did not alter an additional mitogenic effect of rat serum over fetal calf serum. In contrast, cells plated on the laminin-rich basement membrane-like gel formed from an extract of EHS mouse sarcoma, matrix gel (MG), maintained a high content of intracellular lipids in lamellar inclusions and retained a rounded morphology for at least 3 days. MG markedly inhibited THM incorporation and morphological changes when cells were cultured on this surface of when MG was formed over cells initially plated on plastic for various intervals. The importance of the laminin component of MG was demonstrated when these surfaces were pretreated with a highly specific antilaminin serum. Type II cells commenced flattening on the treated MG surface, and THM incorporation increased with the same time course as did control cells on plastic. The data suggest that short-term culture and study of differentiated type II pneumocytes may require a laminin-rich substratum. THM incorporation into type II cell DNA provides an important early and sensitive index of cell-basement membrane interaction and subsequent maintenance of function.

  16. Construction of a fucoidan/laminin functional multilayer to direction vascular cell fate and promotion hemocompatibility.

    PubMed

    Ye, Changrong; Wang, Yan; Su, Hong; Yang, Ping; Huang, Nan; F Maitz, Manfred; Zhao, Anshan

    2016-07-01

    Surface biofunctional modification of cardiovascular stents is a versatile approach to reduce the adverse effects after implantation. In this work, a novel multifunctional coating was fabricated by coimmobilization of the sulfated polysaccharide of brown algae fucoidan and laminin to biomimic the vascular intimal conditions in order to support rapid endothelialization, prevent restenosis and improve hemocompatibility. The surface properties of the coating such as hydrophilicity, bonding density of biomolecules and stability were evaluated and optimized. According to the biocompatibility tests, the fucoidan/laminin multilayer coated surface displayed less platelet adhesion with favorable anticoagulant property. In addition, the fucoidan/laminin complex showed function to selectively regulate vascular cells growth behavior. The proliferation of endothelial cells (ECs) on the fucoidan/laminin biofunctional coating was significantly promoted. For the smooth muscle cells (SMCs), inhibitory effects on cell adhesion and proliferation were observed. In conclusion, the fucoidan/laminin biofunctional coating was successfully fabricated with desirable anticoagulant and endothelialization properties which show a promising application in the vascular devices such as vascular stents or grafts surface modification. PMID:27127049

  17. Construction of a fucoidan/laminin functional multilayer to direction vascular cell fate and promotion hemocompatibility.

    PubMed

    Ye, Changrong; Wang, Yan; Su, Hong; Yang, Ping; Huang, Nan; F Maitz, Manfred; Zhao, Anshan

    2016-07-01

    Surface biofunctional modification of cardiovascular stents is a versatile approach to reduce the adverse effects after implantation. In this work, a novel multifunctional coating was fabricated by coimmobilization of the sulfated polysaccharide of brown algae fucoidan and laminin to biomimic the vascular intimal conditions in order to support rapid endothelialization, prevent restenosis and improve hemocompatibility. The surface properties of the coating such as hydrophilicity, bonding density of biomolecules and stability were evaluated and optimized. According to the biocompatibility tests, the fucoidan/laminin multilayer coated surface displayed less platelet adhesion with favorable anticoagulant property. In addition, the fucoidan/laminin complex showed function to selectively regulate vascular cells growth behavior. The proliferation of endothelial cells (ECs) on the fucoidan/laminin biofunctional coating was significantly promoted. For the smooth muscle cells (SMCs), inhibitory effects on cell adhesion and proliferation were observed. In conclusion, the fucoidan/laminin biofunctional coating was successfully fabricated with desirable anticoagulant and endothelialization properties which show a promising application in the vascular devices such as vascular stents or grafts surface modification.

  18. The dermal-epidermal junction of human skin contains a novel laminin variant

    PubMed Central

    1992-01-01

    We report the identification of a novel laminin variant that appears to be unique to a subset of epithelial basement membranes. The variant contains two chains electrophoretically and immunologically identical to the B1 and B2 chains. Epitopes contained in the laminin A chain are absent from the molecule, and a 190-kD chain substitutes for the A chain. V8 protease analysis and Western blotting studies indicate that the variant 190-kD chain shows structural and immunological similarity to the 200-kD chain of kalinin. Rotary shadowing analysis indicates that the 190-kD chain contributes a large globular structure to the variant long arm, but lacks the short arm contributed to laminin by the A chain. The variant is produced by cultured skin explants, human keratinocytes and a squamous cell carcinoma line, and is present in human amniotic fluid. Polyclonal antibodies raised to kalinin, a recently characterized novel component of anchoring filaments, and mAb BM165 which recognizes a subunit of kalinin (Rousselle et al., 1991) cross react with the variant under nonreducing conditions. Immunohistological surveys of human tissues using the crossreacting antikalinin antiserum indicate that the distribution of this laminin variant is at least restricted to anchoring filament containing basement membranes. We propose the name K-laminin for this variant. PMID:1383241

  19. Laminin deficits induce alterations in the development of dopaminergic neurons in the mouse retina

    PubMed Central

    Dénes, Viktória; Witkovsky, Paul; Koch, Manuel; Hunter, Dale D.; Pinzón-Duarte, Germán; Brunken, William J.

    2010-01-01

    Genetically modified mice lacking the β2 laminin chain (β2null), the γ3 laminin chain (γ3 null), or both β2/γ3 chains (compound null) were produced. The development of tyrosine hydroxylase (TH) immunoreactive neurons in these mouse lines was studied between birth and postnatal day (P) 20. Compared to wild type mice, no alterations were seen in γ3 null mice. In β2 null mice, however, the large, type I TH neurons appeared later in development, were at a lower density and had reduced TH immunoreactivity, although TH process number and size were not altered. In the compound null mouse, the same changes were observed together with reduced TH process outgrowth. Surprisingly, in the smaller, type II TH neurons, TH immunoreactivity was increased in laminin-deficient compared to wild type mice. Other retinal defects we observed were a patchy disruption of the inner limiting retinal basement membrane and a disoriented growth of Müller glial cells. Starburst and AII type amacrine cells were not apparently altered in laminin-deficient relative to wild type mice. We postulate that laminin-dependent developmental signals are conveyed to TH amacrine neurons through intermediate cell types, perhaps the Müller glial cell and/or the retinal ganglion cell. PMID:17711601

  20. [Effect of laminin on structural karyotype variability of kangaroo rat kidney cell lines].

    PubMed

    Polianskaia, G G; Goriachaia, T S; Pinaev, G P

    2003-01-01

    The structural karyotypic variability has been investigated in the "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, and in cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, there is a significant increase in the frequency of chromosomal aberrations, both chromosomal breaks and dicentrics (telomeric associations). Different sensitivity of individual chromosomes to inducing chromosomal breaks was observed in addition to a preferential involvement of some chromosomes in dicentric formation. Structural instability of chromosomes at cultivation on laminin demonstrates nonspecific reaction of the "markerless" cell lines to unfavourable factors of the environment. We discuss possible reasons of differences in the character of karyotypic variability between a cell line of the Indian muntjac skin fibroblasts and epithelial-like Rat kangaroo kidney cell lines cultivated on laminin.

  1. TOPICAL APPLICATION OF LAMININ-322 TO DIABETIC MOUSE WOUNDS

    PubMed Central

    Sullivan, Stephen R.; Underwood, Robert A.; Sigle, Randall O.; Fukano, Yuko; Muffley, Lara A.; Usui, Marcia L.; Gibran, Nicole S.; Antezana, Marcos A.; Carter, William G.; Olerud, John E.

    2007-01-01

    Background Keratinocyte migration is essential for wound healing and diabetic wound keratinocytes migrate poorly. Keratinocyte migration and anchorage appears to be mediated by laminin-332 (LM-332). Impaired diabetic wound healing may be due to defective LM-332 mediated keratinocyte migration. Objective To evaluate LM-332 expression in diabetic (db/db) and control (db/-) mice and to test LM-332 wound healing effects when applied to mouse wounds. Methods LM-332 expression in mouse wounds was evaluated using immunohistochemistry. LM-332 wound healing effects were evaluated by directly applying soluble LM-332, a LM-332 biomaterial, or a control to mouse wounds. Percent wound closure and histology score, based on healing extent, were measured. Results Precursor LM-332 expression was markedly reduced in db/db when compared to db/- mice. In vitro, soluble LM-332 and LM-332 biomaterial demonstrated significant keratinocyte adhesion. In vivo, soluble LM-332 treated wounds had the highest histology score, but significant differences were not found between wound treatments (p>0.05). No differences in percentage wound closure between treatment and control wounds were found (p>0.05). Conclusion The db/db wounds express less precursor LM-332 when compared to db/-. However, LM-332 application did not improve db/db wound healing. LM-332 purified from keratinocytes was primarily physiologically cleaved LM-332 and may not regulate keratinocyte migration. Application of precursor LM-332 rather than cleaved LM-332 may be necessary to improve wound healing, but this isoform is not currently available in quantities sufficient for testing. PMID:17719208

  2. The Laminin Response in Inflammatory Bowel Disease: Protection or Malignancy?

    PubMed Central

    Spenlé, Caroline; Lefebvre, Olivier; Lacroute, Joël; Méchine-Neuville, Agnès; Barreau, Frédérick; Blottière, Hervé M.; Duclos, Bernard; Arnold, Christiane; Hussenet, Thomas; Hemmerlé, Joseph; Gullberg, Donald; Kedinger, Michèle; Sorokin, Lydia; Orend, Gertraud; Simon-Assmann, Patricia

    2014-01-01

    Laminins (LM), basement membrane molecules and mediators of epithelial-stromal communication, are crucial in tissue homeostasis. Inflammatory Bowel Diseases (IBD) are multifactorial pathologies where the microenvironment and in particular LM play an important yet poorly understood role in tissue maintenance, and in cancer progression which represents an inherent risk of IBD. Here we showed first that in human IBD colonic samples and in murine colitis the LMα1 and LMα5 chains are specifically and ectopically overexpressed with a concomitant nuclear p53 accumulation. Linked to this observation, we provided a mechanism showing that p53 induces LMα1 expression at the promoter level by ChIP analysis and this was confirmed by knockdown in cell transfection experiments. To mimic the human disease, we induced colitis and colitis-associated cancer by chemical treatment (DSS) combined or not with a carcinogen (AOM) in transgenic mice overexpressing LMα1 or LMα5 specifically in the intestine. We demonstrated that high LMα1 or LMα5 expression decreased susceptibility towards experimentally DSS-induced colon inflammation as assessed by histological scoring and decrease of pro-inflammatory cytokines. Yet in a pro-oncogenic context, we showed that LM would favor tumorigenesis as revealed by enhanced tumor lesion formation in both LM transgenic mice. Altogether, our results showed that nuclear p53 and associated overexpression of LMα1 and LMα5 protect tissue from inflammation. But in a mutation setting, the same LM molecules favor progression of IBD into colitis-associated cancer. Our transgenic mice represent attractive new models to acquire knowledge about the paradoxical effect of LM that mediate either tissue reparation or cancer according to the microenvironment. In the early phases of IBD, reinforcing basement membrane stability/organization could be a promising therapeutic approach. PMID:25347196

  3. Convergence of bone morphogenetic protein and laminin-1 signaling pathways promotes proliferation and colony formation by fetal mouse pancreatic cells

    SciTech Connect

    Jiang Fangxu . E-mail: jiang@wehi.edu.au; Harrison, Leonard C.

    2005-08-01

    We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its {alpha}{sub 6} integrin and {alpha}-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells.

  4. Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits.

    PubMed Central

    Engvall, E; Earwicker, D; Haaparanta, T; Ruoslahti, E; Sanes, J R

    1990-01-01

    The distribution of subunits of the basement membrane proteins laminin and merosin in human and rabbit tissue was studied by immunofluorescence using monoclonal antibodies. The laminin A chain is present in epithelial, endothelial, and smooth muscle basement membranes. Merosin, as defined by its heavy chain M, is present in striated muscle and peripheral nerve. The A subunit colocalizes with at least two B subunits: B2 plus either B1 or the recently discovered B1 homologue S. The M subunit most often colocalizes with B1 and B2. Exceptions include the myotendinous junction, where M colocalizes with S, and the trophoblast basement membrane, where the M subunit colocalizes with S as well as B1. The presence of all five known subunits of the laminin family in placenta allowed isolation of their parent molecules in native form by the use of monoclonal antibodies in affinity chromatography. Four different heterotrimeric proteins could be identified: B1 chain-containing laminin (A-B1-B2), S chain-containing laminin (A-S-B2), B1-containing merosin (M-B1-B2), and S-containing merosin (M-S-B2). The data show that the proteins in the laminin family are heterotrimers composed of one heavy and two light chains; that most basement membranes contain predominantly one protein of the laminin family; and that laminin, as defined by the A subunit, has a much more restricted distribution than previously thought. Images PMID:2099832

  5. Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse.

    PubMed Central

    Xu, H; Christmas, P; Wu, X R; Wewer, U M; Engvall, E

    1994-01-01

    M-laminin is a major member of the laminin family of basement membrane proteins. It is prominently expressed in striated muscle and peripheral nerve. M-laminin is deficient in patients with the autosomal recessive Fukuyama congenital muscular dystrophy but is normal in patients with the sex-linked Duchenne and Becker muscular dystrophies. We have examined M-laminin expression in mice with autosomal recessive muscular dystrophy caused by the mutation dy. The heavy chain of M-laminin was undetectable in skeletal muscle, heart muscle, and peripheral nerve by immunofluorescence and immunoblotting in homozygous dystrophic dy/dy mice but was normal in heterozygous and wild-type nondystrophic mice. Immunofluorescence confirmed the presence of other major basement membrane proteins in the dystrophic mice. Very low levels of M-laminin heavy chain mRNA were detected by Northern blotting of muscle and heart tissue from dy/dy mice, suggesting that M-laminin heavy-chain mRNA may be produced at very low levels or is unstable. Information about the chromosomal localization of the M heavy-chain in human and mouse suggests that a mutation in the M-chain gene causes the muscular dystrophy in dy/dy mice. The dy mouse may provide a model for autosomal muscular dystrophies in humans and facilitate studies of functions of M-laminin. Images PMID:8202529

  6. Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse.

    PubMed

    Xu, H; Christmas, P; Wu, X R; Wewer, U M; Engvall, E

    1994-06-01

    M-laminin is a major member of the laminin family of basement membrane proteins. It is prominently expressed in striated muscle and peripheral nerve. M-laminin is deficient in patients with the autosomal recessive Fukuyama congenital muscular dystrophy but is normal in patients with the sex-linked Duchenne and Becker muscular dystrophies. We have examined M-laminin expression in mice with autosomal recessive muscular dystrophy caused by the mutation dy. The heavy chain of M-laminin was undetectable in skeletal muscle, heart muscle, and peripheral nerve by immunofluorescence and immunoblotting in homozygous dystrophic dy/dy mice but was normal in heterozygous and wild-type nondystrophic mice. Immunofluorescence confirmed the presence of other major basement membrane proteins in the dystrophic mice. Very low levels of M-laminin heavy chain mRNA were detected by Northern blotting of muscle and heart tissue from dy/dy mice, suggesting that M-laminin heavy-chain mRNA may be produced at very low levels or is unstable. Information about the chromosomal localization of the M heavy-chain in human and mouse suggests that a mutation in the M-chain gene causes the muscular dystrophy in dy/dy mice. The dy mouse may provide a model for autosomal muscular dystrophies in humans and facilitate studies of functions of M-laminin.

  7. Abnormalities of dystrophin, the sarcoglycans, and laminin alpha2 in the muscular dystrophies.

    PubMed Central

    Jones, K J; Kim, S S; North, K N

    1998-01-01

    Abnormalities of dystrophin, the sarcoglycans, and laminin alpha2 are responsible for a subset of the muscular dystrophies. In this study we aim to characterise the nature and frequency of abnormalities of these proteins in an Australian population and to formulate an investigative algorithm to aid in approaching the diagnosis of the muscular dystrophies. To reduce ascertainment bias, biopsies with dystrophic (n=131) and non-dystrophic myopathic (n=71) changes were studied with antibodies to dystrophin, alpha, beta, and gamma sarcoglycan, beta dystroglycan, and laminin alpha2, and results were correlated with clinical phenotype. Abnormalities of dystrophin, the sarcoglycans, or laminin alpha2 were present in 61/131 (47%) dystrophic biopsies and in 0/71 myopathic biopsies, suggesting that immunocytochemical study of dystrophin, the sarcoglycans, and laminin alpha2 may, in general, be restricted to patients with dystrophic biopsies. Two patients with mutations identified in gamma sarcoglycan had abnormal dystrophin (by immunocytochemistry and immunoblot), showing that abnormalities of dystrophin may be a secondary phenomenon. Therefore, biopsies should not be excluded from sarcoglycan analysis on the basis of abnormal dystrophin alone. The diagnostic yield was highest in those with severe, rapidly progressive limb-girdle weakness (92%). Laminin alpha2 deficiency was identified in 5/131 (4%) patients; 215 patients presented after infancy, indicating that abnormalities of laminin alpha2 are not limited to the congenital muscular dystrophy phenotype. Overall patterns of immunocytochemistry and immunoblotting provided a guide to mutation analysis and, on the basis of this study, we have formulated a diagnostic algorithm to guide the investigation of patients with muscular dystrophy. Images PMID:9610800

  8. Terminal short arm domains of basement membrane laminin are critical for its self-assembly

    PubMed Central

    1990-01-01

    Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity. PMID:2307709

  9. Nidogen-1 regulates laminin-1-dependent mammary-specific gene expression

    SciTech Connect

    Pujuguet, Philippe; Simian, Marina; Liaw, Jane; Timpl, Rupert; Werb, Zena; Bissell, Mina J..

    2000-02-01

    Nidogen-1 (entactin) acts as a bridge between the extracellular matrix molecules laminin-1 and type IV collagen, and thus participates in the assembly of basement membranes. To investigate the role of nidogen-1 in regulating cell-type-specific gene expression in mammary epithelium, we designed a culture microecosystem in which each component, including epithelial cells, mesenchymal cells, lactogenic hormones and extracellular matrix, could be controlled. We found that primary and established mesenchymal and myoepithelial cells synthesized and secreted nidogen-1, whereas expression was absent in primary and established epithelial cells. In an epithelial cell line containing mesenchymal cells, nidogen-1 was produced by the mesenchymal cells but deposited between the epithelial cells. In this mixed culture, mammary epithelial cells express b-casein in the presence of lactogenic hormones. Addition of either laminin-1 plus nidogen-1, or laminin-1 alone to mammary epithelial cells induced b- casein production. We asked whether recombinant nidogen-1 alone could signal directly for b-casein. Nidogen-1 did not induce b-casein synthesis in epithelial cells, but it augmented the inductive capacity of laminin-1. These data suggest that nidogen-1 can cooperate with laminin-1 to regulate b-casein expression. Addition of full length nidogen-1 to the mixed cultures had no effect on b-casein gene expression; however, a nidogen-1 fragment containing the laminin-1 binding domain, but lacking the type IV collagen-binding domain, had a dominant negative effect on b-casein expression. These data point to a physiological role for nidogen-1 in the basement membrane-induced gene expression by epithelial cells.

  10. Peroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin.

    PubMed

    Degendorfer, Georg; Chuang, Christine Y; Hammer, Astrid; Malle, Ernst; Davies, Michael J

    2015-12-01

    Basement membranes (BM) are specialized extracellular matrices underlying endothelial cells in the artery wall. Laminin, the most abundant BM glycoprotein, is a structural and biologically active component. Peroxynitrous acid (ONOOH), a potent oxidizing and nitrating agent, is formed in vivo at sites of inflammation from superoxide and nitric oxide radicals. Considerable data supports ONOOH formation in human atherosclerotic lesions, and an involvement of this oxidant in atherosclerosis development and lesion rupture. These effects may be mediated, at least in part, via extracellular matrix damage. In this study we demonstrate co-localization of 3-nitrotyrosine (a product of tyrosine damage by ONOOH) and laminin in human atherosclerotic lesions. ONOOH-induced damage to BM was characterized for isolated murine BM, and purified murine laminin-111. Exposure of laminin-111 to ONOOH resulted in dose-dependent loss of protein tyrosine and tryptophan residues, and formation of 3-nitrotyrosine, 6-nitrotryptophan and the cross-linked material di-tyrosine, as detected by amino acid analysis and Western blotting. These changes were accompanied by protein aggregation and fragmentation as detected by SDS-PAGE. Endothelial cell adhesion to isolated laminin-111 exposed to 10 μM or higher levels of ONOOH was significantly decreased (~25%) compared to untreated controls. These data indicate that laminin is oxidized by equimolar or greater concentrations of ONOOH, with this resulting in structural and functional changes. These modifications, and resulting compromised cell-matrix interactions, may contribute to endothelial cell dysfunction, a weakening of the structure of atherosclerotic lesions, and an increased propensity to rupture.

  11. Hydroxyapatite-coated tendon chitosan tubes with adsorbed laminin peptides facilitate nerve regeneration in vivo.

    PubMed

    Itoh, Soichiro; Yamaguchi, Isamu; Suzuki, Masumi; Ichinose, Shizuko; Takakuda, Kazuo; Kobayashi, Hisatoshi; Shinomiya, Kenichi; Tanaka, Junzo

    2003-12-12

    On the inner surface of tendon chitosan tubes having a triangular shape and a hydroxyapatite coating (t-chitosan/HAp tube), laminin-1 and laminin peptides (YIGSR, IKVAV) have been adsorbed in order to develop nerve growth conduits. The mechanical property, biocompatibility and efficacy of these tubes for nerve regeneration were examined. Step-1: bridge grafting (15 mm) into the sciatic nerve of Sprague-Dawley (SD) rats was carried out using either t-chitosan or t-chitosan/HAp tubes having either a circular or triangular cross section (N=12 in each group). Specimens were taken after 2-, 4-, 6- and 8-week post-implantation (N=3 in each group) for histology determinations. Step-2: t-chitosan/HAp tubes having a triangular cross section with adsorbed laminin-1, CDPGYIGSR or CSRARKQAASIKVAVSAD, as well as control tubes without pre-adsorption were used for implantation (N=18 in each group). Isografting was also carried out (N=6). Histological evaluation was carried out similarly as in Step-1. Furthermore, evoked muscle and sensory nerve action potentials were recorded, and the percentage of myelinated axon area measured at 10 mm distance of the distal anastomosed site in the experimental, control and isograft groups after 12 weeks (N=6 in each group). The results of histological findings, as well as mechanical properties, suggest that a triangular tube shape with a HAp coating benefits nerve regeneration. The effect of laminin peptides (YIGSR, followed by IKVAV) to enhance the growth of regenerating axons has been found comparable with intact laminin-1. Although histological regeneration in both the YIGSR- and laminin-1-treated t-chitosan/HAp tubes matches the isografts, the functional recovery is however delayed.

  12. Conformation Analysis of Peptides Derived from Laminin Alpha 1-2 Chain Using Molecular Dynamics Simulation

    NASA Astrophysics Data System (ADS)

    Yamada, Hironao; Fukuda, Masaki; Miyakawa, Takeshi; Morikawa, Ryota; Takasu, Masako

    Laminin is one of the components of the basement membrane and has diverse biological activities. Several functional peptides (EF1-EF5) are identified from LG4 modules of laminin alpha 1-5 chains. Thus, we perform conformation analysis of EF1 and EF2 using molecular dynamics simulations. In this study, we perform structure sampling with NPT ensemble (300 K, 1 bar). Our results show that EF1 peptide has β-sheet structure in water, and EF2 peptide does not have. Likewise, the EF2 peptide has unstable structure compared with the EF1 peptide in water.

  13. Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

    PubMed

    Zaitsev, Boris N; Benedetti, Fabrizio; Mikhaylov, Andrey G; Korneev, Denis V; Sekatskii, Sergey K; Karakouz, Tanya; Belavin, Pavel A; Netesova, Nina A; Protopopova, Elena V; Konovalova, Svetlana N; Dietler, Giovanni; Loktev, Valery B

    2014-12-01

    The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. PMID:25319621

  14. Laminin 332 Deposition is Diminished in Irradiated Skin in an Animal Model of Combined Radiation and Wound Skin Injury

    PubMed Central

    Jourdan, M. M.; Lopez, A.; Olasz, E. B.; Duncan, N. E.; Demara, M.; Kittipongdaja, W.; Fish, B. L.; Mäder, M.; Schock, A.; Morrow, N. V.; Semenenko, V. A.; Baker, J. E.; Moulder, J. E.; Lazarova, Z.

    2011-01-01

    Skin exposure to ionizing radiation affects the normal wound healing process and greatly impacts the prognosis of affected individuals. We investigated the effect of ionizing radiation on wound healing in a rat model of combined radiation and wound skin injury. Using a soft X-ray beam, a single dose of ionizing radiation (10–40 Gy) was delivered to the skin without significant exposure to internal organs. At 1 h postirradiation, two skin wounds were made on the back of each rat. Control and experimental animals were euthanized at 3, 7, 14, 21 and 30 days postirradiation. The wound areas were measured, and tissue samples were evaluated for laminin 332 and matrix metalloproteinase (MMP) 2 expression. Our results clearly demonstrate that radiation exposure significantly delayed wound healing in a dose-related manner. Evaluation of irradiated and wounded skin showed decreased deposition of laminin 332 protein in the epidermal basement membrane together with an elevated expression of all three laminin 332 genes within 3 days postirradiation. The elevated laminin 332 gene expression was paralleled by an elevated gene and protein expression of MMP2, suggesting that the reduced amount of laminin 332 in irradiated skin is due to an imbalance between laminin 332 secretion and its accelerated processing by elevated tissue metalloproteinases. Western blot analysis of cultured rat keratinocytes showed decreased laminin 332 deposition by irradiated cells, and incubation of irradiated keratinocytes with MMP inhibitor significantly increased the amount of deposited laminin 332. Furthermore, irradiated keratinocytes exhibited a longer time to close an artificial wound, and this delay was partially corrected by seeding keratinocytes on laminin 332-coated plates. These data strongly suggest that laminin 332 deposition is inhibited by ionizing radiation and, in combination with slower keratinocyte migration, can contribute to the delayed wound healing of irradiated skin. PMID

  15. Increased mRNA expression of a laminin-binding protein in human colon carcinoma: Complete sequence of a full-length cDNA encoding the protein

    SciTech Connect

    Yow, Hsiukang; Wong, Jau Min; Chen, Hai Shiene; Lee, C.; Steele, G.D. Jr.; Chen, Lanbo

    1988-09-01

    Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers the authors constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here they report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is /approx/9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant and highly negatively charged.

  16. Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa.

    PubMed

    Dellambra, E; Vailly, J; Pellegrini, G; Bondanza, S; Golisano, O; Macchia, C; Zambruno, G; Meneguzzi, G; De Luca, M

    1998-06-10

    Laminin-5 is composed of three distinct polypeptides, alpha3, beta3, and gamma2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta3 polypeptide. In vitro, beta3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta3 cDNA was used to transduce primary beta3-null keratinocytes. Clonogenic beta3-null keratinocytes were transduced with an efficiency of 100%. Beta3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses. PMID:9650620

  17. Proteolytic disruption of laminin-integrin complexes on muscle cells during synapse formation.

    PubMed Central

    Anderson, M J; Shi, Z Q; Zackson, S L

    1996-01-01

    To explore whether a neural modulation of muscle integrins' extracellular ligand interactions contributes to synapse induction, we compared the distributions of beta1-integrins and basal lamina proteins on Xenopus myotomal myocytes developing in culture. beta1-Integrins formed numerous organized aggregates scattered over the entire muscle surface, with particularly dense accumulations at specialized sites resembling myotendinous and neuromuscular junctions. Integrin aggregates on muscle cells differed from those on surrounding fibroblasts and epithelial cells, both in their lack of response to cross-linking by multivalent ligands and in their consistent association with the cells' own extracellular matrices. Muscle integrin clusters were usually associated with congruent basal lamina accumulations containing laminin and a heparan sulfate proteoglycan (HSPG), sometimes including fibronectin and vitronectin acquired from the surrounding medium. Immediately prior to synaptic differentiation, any existing laminin and HSPG accumulations along the path of cell contact were eliminated, disrupting otherwise stable laminin-integrin complexes. This apparently proteolytic modulation of integrins' extracellular ligand interactions was soon followed by the accumulation of new congruent accumulations of laminin and HSPG in the developing synaptic basal lamina. Combining these results with earlier findings, we consider the possibility that postsynaptic differentiation is induced, at least in part, by the proteolytic disruption of integrin-ligand complexes at sites of nerve-muscle contact. PMID:8756656

  18. Regulation of laminin and entactin mRNA levels by retinoic acid and dibutyryl cyclic AMP

    SciTech Connect

    Durkin, M.E.; Phillips, S.L.; Carlin, B.E.; Merlie, J.P.; Chung, A.E.

    1986-05-01

    Retinoic acid and dibutyryl cAMP induced F9 embryonal carcinoma cells to differentiate to parietal endoderm; the morphological changes were accompanied by the increased synthesis of the basement membrane glycoproteins laminin and entactin. cDNA clones have been isolated for the A (400 kD), B1 (220 kD), and B2 (205 kD) chains of laminin. Northern blot analysis indicated that the A, B1, and B2 chains were encoded by RNA species of 9.8, 6.0, and 8.0 kb, respectively. The kinetics of induction of the laminin mRNAs were studied by dot-blotting dilutions of RNA extracted from F9 cells cultured in retinoic acid and dibutyryl cAMP for increasing amounts of time and hybridizing to /sup 32/P-labeled recombinant plasmids. Very low levels of the A and B chain RNAs were found in uninduced cells, and a large increase occurred between 48 and 72 hr of growth in retinoic acid and dibutyryl cAMP. A cDNA clone was also obtained for entactin, a 150 kD glycoprotein that forms a complex with laminin. Retinoic acid and dibutyryl cAMP treatment also increased the amount of entactin RNA in F9 cells. These results suggested that a common mechanism may exist for the coordinate regulation of the 4 basement membrane protein genes during differentiation.

  19. Laminin α4 deficient mice exhibit decreased capacity for adipose tissue expansion and weight gain.

    PubMed

    Vaicik, Marcella K; Thyboll Kortesmaa, Jill; Movérare-Skrtic, Sofia; Kortesmaa, Jarkko; Soininen, Raija; Bergström, Göran; Ohlsson, Claes; Chong, Li Yen; Rozell, Björn; Emont, Margo; Cohen, Ronald N; Brey, Eric M; Tryggvason, Karl

    2014-01-01

    Obesity is a global epidemic that contributes to the increasing medical burdens related to type 2 diabetes, cardiovascular disease and cancer. A better understanding of the mechanisms regulating adipose tissue expansion could lead to therapeutics that eliminate or reduce obesity-associated morbidity and mortality. The extracellular matrix (ECM) has been shown to regulate the development and function of numerous tissues and organs. However, there is little understanding of its function in adipose tissue. In this manuscript we describe the role of laminin α4, a specialized ECM protein surrounding adipocytes, on weight gain and adipose tissue function. Adipose tissue accumulation, lipogenesis, and structure were examined in mice with a null mutation of the laminin α4 gene (Lama4-/-) and compared to wild-type (Lama4+/+) control animals. Lama4-/- mice exhibited reduced weight gain in response to both age and high fat diet. Interestingly, the mice had decreased adipose tissue mass and altered lipogenesis in a depot-specific manner. In particular, epididymal adipose tissue mass was specifically decreased in knock-out mice, and there was also a defect in lipogenesis in this depot as well. In contrast, no such differences were observed in subcutaneous adipose tissue at 14 weeks. The results suggest that laminin α4 influences adipose tissue structure and function in a depot-specific manner. Alterations in laminin composition offers insight into the roll the ECM potentially plays in modulating cellular behavior in adipose tissue expansion.

  20. Bortezomib partially improves laminin α2 chain-deficient muscular dystrophy.

    PubMed

    Körner, Zandra; Fontes-Oliveira, Cibely C; Holmberg, Johan; Carmignac, Virginie; Durbeej, Madeleine

    2014-05-01

    Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. We have recently demonstrated that proteasome activity is increased in laminin α2 chain-deficient muscle and that treatment with the nonpharmaceutical proteasome inhibitor MG-132 reduces muscle pathology in laminin α2 chain-deficient dy(3K)/dy(3K) mice. Here, we explore the use of the selective and therapeutic proteasome inhibitor bortezomib (currently used for treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3K)/dy(3K) mice and in congenital muscular dystrophy type 1A muscle cells. Outcome measures included quantitative muscle morphology, gene and miRNA expression analyses, proteasome activity, motor activity, and survival. Bortezomib improved several histological hallmarks of disease, partially normalized miRNA expression (miR-1 and miR-133a), and enhanced body weight, locomotion, and survival of dy(3K)/dy(3K) mice. In addition, bortezomib reduced proteasome activity in congenital muscular dystrophy type 1A myoblasts and myotubes. These findings provide evidence that the proteasome inhibitor bortezomib partially reduces laminin α2 chain-deficient muscular dystrophy. Investigation of the clinical efficacy of bortezomib administration in congenital muscular dystrophy type 1A clinical trials may be warranted.

  1. Sulfhydryl-dependent attachment of Treponema denticola to laminin and other proteins.

    PubMed Central

    Haapasalo, M; Singh, U; McBride, B C; Uitto, V J

    1991-01-01

    Attachment of Treponema denticola ATCC 35405 to laminin, a major basement membrane protein, and to other proteins was studied. Microdilution plates were coated with the proteins, and the attachment of T. denticola was measured by the enzyme-linked immunosorbent assay technique. Compared with bovine serum albumin (BSA), T. denticola had a high affinity to laminin, fibronectin, fibrinogen, and gelatin, as well as to type I and type IV collagens. Attachment to RGD peptide (Gly-Arg-Gly-Asp-Ser, the integrin recognition sequence) was only about 30% of that to laminin and was comparable to attachment to BSA. Tests with laminin fragments obtained through elastase digestion showed that the spirochetes attached well to an A-chain 140-kDa fragment involved in eukaryote cell attachment but did not attach to a 50-kDa fragment that includes the heparin binding site. Pretreatment of T. denticola with soluble laminin, fibronectin, gelatin, BSA, or fibrinogen had no effect on the attachment of the bacteria to laminin or fibronectin. A wide variety of compounds were tested for their possible inhibitory actions on the attachment. While most treatments of T. denticola ATCC 35405 had little or no effect on the attachment to proteins, sulfhydryl reagents p-chloromercuribenzoic acid (pCMBA) and oxidized glutathione inhibited the attachment by 70 to 99%, depending on the protein. When T. denticola was first allowed to attach to proteins, addition of pCMBA or oxidized glutathione could no longer reverse the attachment. Heat treatment of the spirochetes also markedly reduced the attachment to laminin, gelatin, and fibrinogen but not to BSA. Mixed glycosidase treatment of the spirochetes inhibited the attachment by 20 to 80%. None of the above treatments of the substrate proteins had any marked effect on the spirochete attachment. The results indicate that T. denticola has the capacity to bind to many different kinds of proteins by utilizing specific attachment mechanisms. The binding

  2. Selective overexpression of gamma1 laminin in astrocytes in amyotrophic lateral sclerosis indicates an involvement in ALS pathology.

    PubMed

    Wiksten, Markus; Väänänen, Antti; Liesi, Päivi

    2007-07-01

    Our earlier studies indicate that the KDI tripeptide of gamma1 laminin reverts paralysis and protects adult rat CNS from excitotoxicity of glutamate and from oxidative stress. Here we show that gamma1 laminin is selectively overexpressed in reactive astrocytes of the amyotrophic lateral sclerosis (ALS) spinal cord, with both gray and white matter astrocytes overexpressing gamma1 laminin. Intensely gamma1 laminin-positive, aggressive-looking reactive astrocytes of the lateral columns of both cervical and thoracic spinal cord surround the lateral ventral horns and roots and extend into the area of the lateral corticospinal tract. In the cervical ALS spinal cord, large numbers of strongly gamma1 laminin-immunoreactive astrocytes are also present in the dorsal columns of the ascending sensory pathways. No other laminin or any other ALS-associated protein localizes in this manner. This unique distribution of gamma1 laminin-immunoreactive astrocytes in the ALS white matter together with our recent results on the efficacy of the KDI domain as a neuronal protector strongly suggest that gamma1 laminin may be expressed by astrocytes of the ALS spinal cord as a protective measure intended to aid neuronal survival. Further comparative studies on ALS spinal cord tissues and those of the animal models of ALS are needed to clarify the specific role of gamma1 laminin and its KDI domain in ALS and its putative interactions with the additional ALS-associated factors, such as excitotoxicity, oxidative stress, and neurofilament accumulation. Most importantly, further studies are urgently needed to test the potential of the KDI tripeptide as a therapeutic treatment for ALS.

  3. The extracellular matrix proteins laminin and fibronectin contain binding domains for human plasminogen and tissue plasminogen activator.

    PubMed

    Moser, T L; Enghild, J J; Pizzo, S V; Stack, M S

    1993-09-01

    This study describes the binding of plasminogen and tissue-type plasminogen activator (t-PA) to the extracellular matrix proteins fibronectin and laminin. Plasminogen bound specifically and saturably to both fibronectin and laminin immobilized on microtiter wells, with Kd(app) values of 115 and 18 nM, respectively. Limited proteolysis by endoproteinase V8 coupled with ligand blotting analysis showed that both plasminogen and t-PA preferentially bind to a 55-kDa fibronectin fragment and a 38-kDa laminin fragment. Amino acid sequence analysis demonstrated that the 5-kDa fragment originates with the fibronectin amino terminus whereas the laminin fragment was derived from the carboxyl-terminal globular domain of the laminin A chain. Ligand blotting experiments using isolated plasminogen domains were also used to identify distinct regions of the plasminogen molecule involved in fibronectin and laminin binding. Solution phase fibronectin binding to immobilized plasminogen was mediated primarily via lysine binding site-dependent interactions with plasminogen kringles 1-4. Lysine binding site-dependent binding of soluble laminin to immobilized plasminogen kringles 1-5 as well as an additional lysine binding site-independent interaction between mini-plasminogen and the 38-kDa laminin A chain fragment were also observed. These studies demonstrate binding of plasminogen and tissue-type plasminogen activator to specific regions of the extracellular matrix glycoproteins laminin and fibronectin and provide further insight into the mechanism of regulation of plasminogen activation by components of the extracellular matrix. PMID:8360181

  4. Kruppel-like factor 4 regulates laminin alpha 3A expression in mammary epithelial cells.

    PubMed

    Miller, K A; Eklund, E A; Peddinghaus, M L; Cao, Z; Fernandes, N; Turk, P W; Thimmapaya, B; Weitzman, S A

    2001-11-16

    Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated alpha3A, beta3, and gamma2), each encoded by a separate gene. Laminin-5 is markedly down-regulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin alpha3A chain (LAMA3A) and investigated its regulation in functionally normal MCF10A breast epithelial cells and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A cells, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZF) are required for expression driven by the LAMA3A promoter. Electrophoretic mobility shift assays reveal absence of KLF4 binding activity in extracts from T47D, MDA-MB 231, ZR75-1, MDA-MB 436, and MCF7 breast cancer cells. Transient transfection of a plasmid expressing KLF4 activates transcription from the LAMA3A promoter in breast cancer cells. A reporter vector containing duplicate KLF4-binding sites in its promoter is expressed at high levels in MCF10A cells but at negligible levels in breast cancer cells. Thus, KLF4 is required for LAMA3A expression and absence of laminin alpha3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.

  5. Computational Modeling of Laminin N-Terminal Domains Using Sparse Distance Constraints from Disulfide Bonds and Chemical Cross-Linking

    PubMed Central

    Kalkhof, Stefan; Haehn, Sebastian; Paulsson, Mats; Smyth, Neil; Meiler, Jens; Sinz, Andrea

    2016-01-01

    Basement membranes are thin extracellular protein layers, which separate endothelial and epithelial cells from the underlying connecting tissue. The main non-collagenous components of basement membranes are laminins, trimeric glycoproteins, which form polymeric networks by interactions of their N-terminal (LN) domains; however, no high-resolution structure of laminin LN domains exists so far. To construct models for laminin β1 and γ1 LN domains 14 potentially suited template structures were determined using fold recognition methods. For each target/template-combination comparative models were created with Rosetta. Final models were selected based on their agreement with experimentally obtained distance constraints from natural cross-links, i.e., disulfide bonds as well as chemical cross-links obtained from reactions with two amine-reactive cross-linkers. We predict that laminin β1 and γ1 LN domains share the galactose-binding domain-like fold. PMID:20939100

  6. Regeneration of Aplysia Bag Cell Neurons is Synergistically Enhanced by Substrate-Bound Hemolymph Proteins and Laminin

    NASA Astrophysics Data System (ADS)

    Hyland, Callen; Dufrense, Eric R.; Forscher, Paul

    2014-04-01

    We have investigated Aplysia hemolymph as a source of endogenous factors to promote regeneration of bag cell neurons. We describe a novel synergistic effect between substrate-bound hemolymph proteins and laminin. This combination increased outgrowth and branching relative to either laminin or hemolymph alone. Notably, the addition of hemolymph to laminin substrates accelerated growth cone migration rate over ten-fold. Our results indicate that the active factor is either a high molecular weight protein or protein complex and is not the respiratory protein hemocyanin. Substrate-bound factor(s) from central nervous system-conditioned media also had a synergistic effect with laminin, suggesting a possible cooperation between humoral proteins and nervous system extracellular matrix. Further molecular characterization of active factors and their cellular targets is warranted on account of the magnitude of the effects reported here and their potential relevance for nervous system repair.

  7. The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype

    PubMed Central

    Santos-Valle, Patricia; Guijarro-Muñoz, Irene; Cuesta, Ángel M.; Alonso-Camino, Vanesa; Villate, Maider; Álvarez-Cienfuegos, Ana; Blanco, Francisco J.; Sanz, Laura; Álvarez-Vallina, Luis

    2012-01-01

    Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling. PMID:22723936

  8. Abortion in mice induced by intravenous injections of antibodies to type IV collagen or laminin.

    PubMed

    Foidart, J M; Yaar, M; Figueroa, A; Wilk, A; Brown, K S; Liotta, L A

    1983-03-01

    Purified antibodies to laminin or Type IV collagen administered intravenously to pregnant mice were found to localize in the basement membranes of all maternal tissues as well as the parietal and visceral yolk sacs and trophoblast basement membranes but not in embryonic tissues. Antibodies to Type IV collagen induced a higher incidence of abortions, retroplacental hematomas and fetal deaths. When administered intraamniotically, both antiserums were embryotoxic. The functional consequences of the attachment of antibodies to these specific basement membrane antigens appear to be hemorrhage within the parietal and visceral yolk sacs and separation of fetal from maternal tissues. Complement activation appears to play an important role in the interruptions of pregnancy, because this was not observed in strains of mice lacking C5, the fifth component of complement, in mice depleted of C3 by administration of cobra venom factor, or in mice injected with the F(ab) fragments of antibody to Type IV collagen or laminin.

  9. Basement membrane proteins in salivary gland tumours. Distribution of type IV collagen and laminin.

    PubMed

    Skalova, A; Leivo, I

    1992-01-01

    Immunohistochemical localization of type IV collagen and laminin in normal salivary glands and in salivary gland tumours of various types was studied using rabbit antisera. In normal salivary glands, type IV collagen and laminin were co-localized in basement membranes surrounding acini, ducts, fat cells and peripheral nerves. In salivary gland tumours, three main patterns of co-expression of these basement membrane proteins were distinguished. Linear basement membrane-like staining was detected in duct-cell-derived benign salivary gland tumours and in acinic cell carcinomas. In invasive lesions, however, these basement membrane proteins were distributed in an irregular, interrupted manner, and in many cases they were completely absent. Both benign and malignant salivary gland tumours which have a prominent myoepithelial cell component display a particular deposition of basement membrane molecules adjacent to the modified myoepithelial cells, and at the margins of extracellular matrix deposits within these tumours.

  10. The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

    PubMed

    deHart, Gregory W; Healy, Kevin E; Jones, Jonathan C R

    2003-02-01

    Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the

  11. Laminin gene LAMB4 is somatically mutated and expressionally altered in gastric and colorectal cancers.

    PubMed

    Choi, Mi Ryoung; An, Chang Hyeok; Yoo, Nam Jin; Lee, Sug Hyung

    2015-01-01

    Laminins are important in tumor invasion and metastasis as well as in maintenance of normal epithelial cell structures. However, mutation status of laminin chain-encoding genes remains unknown in cancers. Aim of this study was to explore whether laminin chain genes are mutated and expressionally altered in gastric (GC) and colorectal cancers (CRC). In a public database, we found that laminin chain genes LAMA1, LAMA3, LAMB1 and LAMB4 had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with microsatellite instability (MSI). We analyzed the genes in 88 GC and 139 CRC [high MSI (MSI-H) or stable MSI/low MSI (MSS/MSI-L)] by single strand conformation polymorphism analysis and DNA sequencing. In the present study, we found LAMB4 (11.8% of GC and 7.6% of CRC with MSI-H), LAMA3 (2.9% of GC and 2.5 of CRC with MSI-H), LAMA1 (5.9% of GC with MSI-H) and LAMB1 frameshift mutations (1.3% of CRC with MSI-H). These mutations were not found in MSS/MSI-L (0/114). We also analyzed LAMB4 expression in GC and CRC by immunohistochemistry. Loss of LAMB4 expression was identified in 17-32% of the GC and CRC. Of note, the loss expression was more common in the cancers with LAMB4 mutation or those with MSI-H. Our data show that frameshift mutations of LAMA1, LAMA3, LAMB1 and LAMB4, and loss of LAMB4 may be features of GC and CRC with MSI-H. PMID:25257191

  12. A Laminin G-EGF-Laminin G module in Neurexin IV is essential for the apico-lateral localization of Contactin and organization of septate junctions.

    PubMed

    Banerjee, Swati; Paik, Raehum; Mino, Rosa E; Blauth, Kevin; Fisher, Elizabeth S; Madden, Victoria J; Fanning, Alan S; Bhat, Manzoor A

    2011-01-01

    Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons.

  13. Immobilized laminin concentration gradients on electrospun fiber scaffolds for controlled neurite outgrowth.

    PubMed

    Zander, Nicole E; Beebe, Thomas P

    2014-03-01

    Neuronal process growth is guided by extrinsic environmental cues such as extracellular matrix (ECM) proteins. Recent reports have described that the growth cone extension is superior across gradients of the ECM protein laminin compared to growth across uniformly distributed laminin. In this work, the authors have prepared gradients of laminin on aligned electrospun nanofibers for use as substrates for neuronal growth. The substrates therefore presented both topographical and chemical guidance cues. Step gradients were prepared by the controlled robotic immersion of plasma-treated polycaprolactone fibers reacted with N-hydroxysuccinimide into the protein solution. The gradients were analyzed using x-ray photoelectron spectroscopy and confocal laser scanning microscopy. Gradients with a dynamic range of protein concentrations were successfully generated and neurite outgrowth was evaluated using neuronlike pheochromocytoma cell line 12 (PC12) cells. After 10 days of culture, PC12 neurite lengths varied from 32.7 ± 14.2 μm to 76.3 ± 9.1 μm across the protein concentration gradient. Neurite lengths at the highest concentration end of the gradient were significantly longer than neurite lengths observed for cells cultured on samples with uniform protein coverage. Gradients were prepared both in the fiber direction and transverse to the fiber direction. Neurites preferentially aligned with the fiber direction in both cases indicating that fiber alignment has a more dominant role in controlling neurite orientation, compared to the chemical gradient. PMID:24739010

  14. Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

    SciTech Connect

    Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi; Nelson, Celeste M.; Mroue, Rana; Spencer, Virginia A.; Brownfield, Doug; Radisky, Derek C.; Bustamante, Carlos; Bissell, Mina J.

    2008-10-20

    In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for {beta}-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of {beta}-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of {beta}-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.

  15. Cutaneous myxidiosis in European eel, Anguilla anguilla (Linnaeus, 1758): histopathology, histochemistry and laminin immunohistochemistry.

    PubMed

    Manera, M; Borreca, C; Dezfuli, B S

    2016-07-01

    Histopathology, histochemistry and immunohistochemistry of the integument of European eel, Anguilla anguilla (Linnaeus, 1758), infected by Myxidium sp. are reported. Skin samples from affected and unaffected eels were dissected, formalin fixed, paraffin embedded, sectioned and stained with H&E, Periodic acid-Schiff's staining method, Alcian Blue 8 GX pH 2.5/Periodic acid-Schiff's and McCallum-Goodpasture's Gram stain. Moreover, immunohistochemistry was performed using a primary polyclonal laminin antibody. Histologically, cysts (diameter 2-3 mm) were observed mainly under the scale pockets, encircled by a thin collagen layer, lined by elongated, flattened fibroblasts and containing bipolar, PAS- and Gram-positive spores with opposite polar capsules. The epidermis stretched by the underlying cyst appeared dysplastic, thinned with a significant reduction in mucous cells number. Only inconsistent and aspecific inflammatory reaction was noted around the cysts at the dermis/epidermis interface. Intense laminin-like protein immunolabel was documented in the plasmodial ectoplasm and related to host anergia. This was the first report of laminin immunolabel in a member of the Myxozoa. Epidermal dysplasia represents likely an aspecific response against the underlying tensile force exerted by the developing parasite cyst, while fibroblast and collagen encapsulation denote a parasite-driven host response protecting, rather than harming, the encircled parasite.

  16. Reproduction and sera embryotoxicity after immunization of monkeys with the laminin peptides YIGSR, RGD, and IKVAV.

    PubMed Central

    Chambers, B J; Klein, N W; Conrad, S H; Ruppenthal, G C; Sackett, G P; Weeks, B S; Kleinman, H K

    1995-01-01

    Monkeys with excellent reproductive histories were immunized with the laminin peptides YIGSR, RGD, IKVAV, and YD, a control sequence with no known biological function. Sera from the YIGSR-immunized monkey became toxic, causing neural tube defects in whole rat embryo cultures, and this monkey experienced fetal loss after immunization. Sera from the RGD-immunized monkey also became embryotoxic in culture after immunization, but this monkey appeared to become infertile as she failed to initiate a pregnancy for at least 2 years after immunization. In contrast, embryos cultured on sera from the IKVAV- or YD-immunized monkeys were predominantly normal and both monkeys completed successful pregnancies. Antibody levels to the respective peptides or to laminin were not predictive of embryotoxicity, but antibody binding to homogenized yolk sacs as well as to yolk sacs of cultured embryos was associated with sera embryotoxicity and reproductive outcomes in vivo. These observations suggested that the laminin sequences YIGSR and RGD may play a role in immune-mediated reproductive failure by reacting directly with embryonic tissue and could provide a basis for identifying individuals at risk for both spontaneous abortion and infertility. Images Fig. 3 Fig. 4 PMID:7624326

  17. Induction of a putative laminin-binding protein of Streptococcus gordonii in human infective endocarditis.

    PubMed Central

    Sommer, P; Gleyzal, C; Guerret, S; Etienne, J; Grimaud, J A

    1992-01-01

    There is evidence to suggest that the virulence of Streptococcus strains in infective endocarditis might be due to the expression of binding sites for the extracellular matrix proteins of damaged valves. In this communication, we draw attention to one laminin-binding protein from a strain of Streptococcus gordonii isolated from a patient with human endocarditis. This 145-kDa protein was found on the cell wall of the bacterium. The level of expression of this binding protein might be regulated by the presence of extracellular matrix proteins: the protein was lacking after in vitro selection of laminin, collagen I, and fibronectin nonbinding variants, and it was recovered after growth of the variants when laminin or collagen I was added to the growth medium. It was also missing after 10 subcultures in minimal medium, indicating some positive control. Furthermore, the 145-kDa protein was recognized as a major antigen by sera from patients treated for streptococcal infective endocarditis, while sera from patients with valvulopathies gave only slight recognition, suggesting an increase of the expression of this protein during infective endocarditis. It was also shown that the 145-kDa protein carried a collagen I-like determinant detected with anti-human collagen I antibodies. Images PMID:1530927

  18. Immobilization of laminin peptide in molecularly aligned chitosan by covalent bonding.

    PubMed

    Matsuda, Atsushi; Kobayashi, Hisatoshi; Itoh, Soichiro; Kataoka, Kazunori; Tanaka, Junzo

    2005-05-01

    We developed a new biomaterial effective for nerve regeneration consisting of molecularly aligned chitosan with laminin peptides bonded covalently. Molecularly aligned chitosan was prepared from crab (Macrocheira kaempferi) tendons by ethanol treatment and 4 wt%-NaOH aqueous solutions to remove proteins and calcium phosphate, followed by deacetyl treatment using a 50 wt%-NaOH aqueous solution at 100 degrees C. Molecularly aligned tendon chitosan was chemically thiolated by reacting 4-thiobutyrolactone with the chitosan amino group. The introduction of thiol groups and their distribution to tendon chitosan and chitosan cast film were confirmed using ATR FT-IR, (1)H-NMR, and EDS. The 1.24 micromol/g of thiol groups introduced on the surface of tendon chitosan and the chitosan cast film was confirmed using ultraviolet (UV) spectra. Thiol groups of cysteine located at the end of synthetic laminin peptides were then reacted chemically with thiolated chitosan to form chitosan-S-S-laminin peptide. YIGSR estimated at 0.92 micromol/g and IKVAV estimated at 0.28 micromol/g on thiolated tendon chitosan were confirmed using UV spectra. YIGSR was estimated at 0.85 micromol/g and IKVAV was estimated at 0.34 micromol/g on the thiolated chitosan cast film.

  19. Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia

    PubMed Central

    Alcaraz, Jordi; Xu, Ren; Mori, Hidetoshi; Nelson, Celeste M; Mroue, Rana; Spencer, Virginia A; Brownfield, Doug; Radisky, Derek C; Bustamante, Carlos; Bissell, Mina J

    2008-01-01

    In the mammary gland, epithelial cells are embedded in a ‘soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for β-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of β-casein expression required both laminin signalling and a ‘soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of β-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues' unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis. PMID:18843297

  20. Developmental characterization of the gene for laminin alpha-chain in sea urchin embryos.

    PubMed

    Benson, S; Page, L; Ingersoll, E; Rosenthal, E; Dungca, K; Signor, D

    1999-03-01

    We describe the isolation and characterization of a cDNA clone encoding a region of the carboxy terminal globular domain (G domain) of the alpha-1 chain of laminin from the sea urchin, Strongylocentrotus purpuratus. Sequence analysis indicates that the 1.3 kb cDNA (spLAM-alpha) encodes the complete G2 and G3 subdomains of sea urchin a-laminin. The 11 kb spLAM-alpha mRNA is present in the egg and declines slightly in abundance during development to the pluteus larva. The spLAM-alpha gene is also expressed in a variety of adult tissues. Whole mount in situ hybridization of gastrula stage embryos indicates that ectodermal and endodermal epithelia and mesenchyme cells contain the spLAM-alpha mRNA. Immunoprecipitation experiments using an antibody made to a recombinant fusion protein indicates spLAM-alpha protein is synthesized continuously from fertilization as a 420 kDa protein which accumulates from low levels in the egg to elevated levels in the pluteus larva. Light and electron microscopy identify spLAM-alpha as a component of the basal lamina. Blastocoelic microinjection of an antibody to recombinant spLAM-alpha perturbs gastrulation and skeleton formation by primary mesenchyme cells suggesting an important role for laminin in endodermal and mesodermal morphogenesis. PMID:10330483

  1. Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells

    PubMed Central

    2014-01-01

    Background Diabetes mellitus (DM) is an incurable metabolic disease constituting a major threat to human health. Insulin-producing cells (IPCs) differentiated from mesenchymal stem cells (MSCs) hold great promise in the treatment of DM. The development of an efficient IPC induction system is a crucial step for the clinical application of IPCs for DM. Laminin 411 is a key component of the basement membrane and is involved in the regulation of cell differentiation; however, little is known about a role of laminin 411 in the regulation of IPC differentiation from human MSCs. Methods MSCs were isolated from human umbilical cord (UC-MSCs) and expanded in an in vitro culture system. UC-MSCs were then cultured in the IPC induction and differentiation medium in the presence of laminin 411. Flow cytometry, Quantitative realtime PCR, immunofluorescence staining, ELISA, Western blotting and other techniques were applied to determine IPC generation, insulin expression and related mechanisms. To evaluate potential therapeutic efficacy of IPCs induced from UC-MSCs, a type-1 diabetes (T1DM) rat model was generated using streptozotocin. Blood glucose, insulin levels, and survival of rats were monitored periodically following intravenous injection of the tested cells. Results Laminin 411 markedly induced the expression of the genes Foxa2 and Sox17, markers for pancreatic precursor cells, efficiently induced IPC differentiation from MSCs, and up-regulated insulin expression at both mRNA and protein levels. Furthermore, the expression of the genes known to govern insulin expression including Pdx1 and Ngn3 was markedly induced by laminin 411, which suggests that Pdx1 and Ngn3 signaling pathways are involved in laminin 411 induced-insulin expression machinery. More importantly, administration of laminin 411-induced IPCs rapidly and significantly down-regulated fasting blood glucose levels, significantly reduced the HbA1c concentration and markedly improved the symptoms and survival of

  2. Localization of the gene (LAMA4) to chromosome 6q21 and isolation of a partial cDNA encoding a variant laminin A chain

    SciTech Connect

    Richards, A.J.; Al-Imara, L.; Carter, N.P.

    1994-07-01

    Laminin is a basement membrane glycoprotein composed of three nonidentical chains, A, B1, and B2. Variant chains such as merosin and S-laminin have been found in different tissues. The authors have isolated a cDNA encoding a novel laminin A variant that hybridizes to a 6.45-kb mRNA. Using amplification of genomic DNA and flow-sorted chromosomes they have assigned the gene (LAMA4) for this new laminin A variant to chromosome 6. Fluorescence in situ hybridization of a YAC clone further localized the gene to 6q21. 19 refs., 2 figs.

  3. In vitro Evaluation of Calcium Phosphate Precipitation on Possibly Bioactive Titanium Surfaces in the Presence of Laminin

    PubMed Central

    Stenport, Victoria Franke; Currie, Fredrik; Wennerberg, Ann

    2011-01-01

    ABSTRACT Objectives The aim of the present study was to evaluate calcium phosphate precipitation and the amount of precipitated protein on three potentially bioactive surfaces when adding laminin in simulated body fluid. Material and Methods Blasted titanium discs were prepared by three different techniques claimed to provide bioactivity: alkali and heat treatment (AH), anodic oxidation (AO) or hydroxyapatite coating (HA). A blasted surface incubated in laminin-containing simulated body fuid served as a positive control (B) while a blasted surface incubated in non laminin-containing simulated body fuid served as a negative control (B-). The immersion time was 1 hour, 24 hours, 72 hours and 1 week. Surface topography was investigated by interferometry and morphology by Scanning Electron Microscopy (SEM). Analysis of the precipitated calcium and phosphorous was performed by Energy Dispersive X-ray Spectroscopy (EDX) and the adsorbed laminin was quantified by iodine (125I) labeling. Results SEM demonstrated that all specimens except for the negative control were totally covered with calcium phosphate (CaP) after 1 week. EDX revealed that B- demonstrated lower sum of Ca and P levels compared to the other groups after 1 week. Iodine labeling demonstrated that laminin precipitated in a similar manner on the possibly bioactive surfaces as on the positive control surface. Conclusions Our results indicate that laminin precipitates equally on all tested titanium surfaces and may function as a nucleation center thus locally elevating the calcium concentration. Nevertheless further studies are required to clarify the role of laminin in the interaction of biomaterials with the host bone tissue. PMID:24421995

  4. Immunohistochemical localization of basement membrane type VII collagen and laminin in neoplasms of the head and neck.

    PubMed

    Wetzels, R H; van der Velden, L A; Schaafsma, H E; Manni, J J; Leigh, I M; Vooijs, G P; Ramaekers, F C

    1992-11-01

    The distribution pattern of the basement membrane components type VII collagen and laminin, was studied immunohistochemically in normal human head and neck tissues and in a series of benign and malignant tumours from the same site. Using monoclonal antibodies, a basement membrane containing type VII collagen and laminin could be demonstrated beneath the epithelial cell layer in 16 normal head and neck tissues from different localizations. Unlike type VII collagen, laminin was also abundantly present around blood vessels and muscle fibres. With respect to 42 squamous cell carcinomas studied, type VII collagen and laminin were present in basement membranes surrounding small and large tumour fields, independent of the tumour grade. Type VII collagen was demonstrated in the cytoplasm of tumour cells in 36% of the cases, while the antibody to laminin displayed a basement membrane staining pattern mainly. Both antibodies showed a staining gradient in more than half of the cases, with strong staining in the centre of the tumour and weakening of the staining towards the tumour periphery. In a series of 22 salivary gland tumours consisting of 19 pleomorphic adenomas and three adenoid cystic carcinomas, the distribution pattern of type VII collagen and laminin was very heterogeneous. Laminin was present in 17 and type VII collagen in 10 of 19 cases of pleomorphic adenoma, mostly scattered throughout the tumour fields. In the tumours positive for type VII collagen areas with little or no positivity were also found. A correlation between type VII collagen positivity and the presence of basal cell keratin 14 positivity was noticed in the majority of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Characterization of glomerular epithelial cell matrix receptors.

    PubMed Central

    Adler, S.

    1992-01-01

    Integrin matrix receptors on glomerular epithelial cells (GEC) may play an important role in adhesion of GEC to the glomerular basement membrane (GBM) and in the maintenance of normal glomerular permeability. Therefore, the author determined the types of matrix receptors present on cultured rat GEC and examined their interactions with several components of the extracellular matrix. Beta 1 integrin matrix receptors were detected on all three glomerular cell types in rat kidney in vivo and at areas of cell-cell contact on cultured GEC. Glomerular epithelial cell adhesion to types I and IV collagen was slightly greater than to laminin and fibronectin. Adhesion to fibronectin was significantly inhibited by a synthetic peptide containing the RGD adhesion sequence. Immunoprecipitation of lysates of surface-iodinated GEC showed the presence of alpha 3 beta 1 integrin. Chromatography of lysates on immobilized collagen showed alpha 3 beta 1 integrin and a 70- to 75-kd protein band as the collagen receptors on GEC. Chromatography on the 120-kd cell-binding fragment of fibronectin disclosed only alpha 3 beta 1 as a specific fibronectin receptor. Antibody to the beta 1 integrin chain inhibited adhesion to laminin and collagen. These studies demonstrate that in vitro, as in vivo, GEC appear to express only alpha 3 beta 1 integrin. Furthermore, this matrix receptor is capable of mediating GEC adhesion to collagen, fibronectin, and laminin, components of the GBM, and presumably plays a similar role in promoting GEC adhesion to GBM in vivo. Images Figure 1 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1325740

  6. The streptococcal hemoprotein receptor: a moonlighting protein or a virulence factor?

    PubMed

    Eichenbaum, Zehava

    2012-11-15

    The β-hemolytic group A streptococcus (GAS) is a major pathogen that readily uses hemoglobin to satisfy its requirements for iron. The streptococcal hemoprotein receptor in GAS plays a central role in heme utilization and binds fibronectin and laminin in vitro. Shr inactivation attenuates the virulent M1T1 GAS strain in two murine infection models and reduces bacterial growth in blood and binding to laminin. Shr impact on the globally disseminated M1T1 strain underscores the importance of heme uptake in GAS pathogenesis and raises the possibility of targeting heme-uptake proteins in the development of new methods to combat GAS infections.

  7. Substitution of a conserved cysteine-996 in a cysteine-rich motif of the laminin {alpha}2-chain in congenital muscular dystrophy with partial deficiency of the protein

    SciTech Connect

    Nissinen, M.; Xu Zhang; Tryggvason, K.

    1996-06-01

    Congenital muscular dystrophies (CMDs) are autosomal recessive muscle disorders of early onset. Approximately half of CMD patients present laminin {alpha}2-chain (merosin) deficiency in muscle biopsies, and the disease locus has been mapped to the region of the LAMA2 gene (6q22-23) in several families. Recently, two nonsense mutations in the laminin {alpha}2-chain gene were identified in CMD patients exhibiting complete deficiency of the laminin {alpha}2-chain in muscle biopsies. However, a subset of CMD patients with linkage to LAMA2 show only partial absence of the laminin {alpha}2-chain around muscle fibers, by immunocytochemical analysis. In the present study we have identified a homozygous missense mutation in the {alpha}2-chain gene of a consanguineous Turkish family with partial laminin {alpha}2-chain deficiency. The T{r_arrow}C transition at position 3035 in the cDNA sequence results in a Cys996{r_arrow}Arg substitution. The mutation that affects one of the conserved cysteine-rich repeats in the short arm of the laminin {alpha}2-chain should result in normal synthesis of the chain and in formation and secretion of a heterotrimeric laminin molecule. Muscular dysfunction is possibly caused either by abnormal disulfide cross-links and folding of the laminin repeat, leading to the disturbance of an as yet unknown binding function of the laminin {alpha}2-chain and to shorter half-life of the muscle-specific laminin-2 and laminin-4 isoforms, or by increased proteolytic sensitivity, leading to truncation of the short arm. 42 refs., 7 figs.

  8. Identification of a novel family of laminin N-terminal alternate splice isoforms: structural and functional characterization.

    PubMed

    Hamill, Kevin J; Langbein, Lutz; Jones, Jonathan C R; McLean, W H Irwin

    2009-12-18

    The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5'-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) alpha3 and LaNt alpha5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt alpha3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt alpha3 and LaNt alpha3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt alpha3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt alpha3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro. PMID:19773554

  9. A missense mutation (G1506E) in the adhesion G domain of laminin-5 causes mild junctional epidermolysis bullosa.

    PubMed

    Scaturro, Maria; Posteraro, Patrizia; Mastrogiacomo, Alessandro; Zaccaria, Maria Letizia; De Luca, Naomi; Mazzanti, Cinzia; Zambruno, Giovanna; Castiglia, Daniele

    2003-09-12

    Laminin-5 is the major adhesion ligand for epithelial cells. Mutations in the genes encoding laminin-5 cause junctional epidermolysis bullosa (JEB), a recessive inherited disease characterized by extensive epithelial-mesenchymal disadhesion. We describe a JEB patient compound heterozygote for two novel mutations in the gene (LAMA3) encoding the laminin alpha3 chain. The maternal mutation (1644delG) generates mRNA transcripts that undergo nonsense-mediated decay. The paternal mutation results in the Gly1506-->Glu substitution (G1506E) within the C-terminal globular region of the alpha3 chain (G domain). Mutation G1506E affects the proper folding of the fourth module of the G domain and results in the retention of most of the mutated polypeptide within the endoplasmic reticulum (ER). However, scant amounts of the mutated laminin-5 are secreted, undergo physiologic extracellular maturation, and correctly localize within the cutaneous basement membrane zone in patient's skin. Our findings represent the first demonstration of an ER-retained mutant laminin-5 leading to a mild JEB phenotype. PMID:12943669

  10. Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation

    PubMed Central

    1996-01-01

    The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation. PMID:8609173

  11. Modulation of neuritogenesis by astrocyte muscarinic receptors.

    PubMed

    Guizzetti, Marina; Moore, Nadia H; Giordano, Gennaro; Costa, Lucio G

    2008-11-14

    Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.

  12. A novel laminin β gene BmLanB1-w regulates wing-specific cell adhesion in silkworm, Bombyx mori.

    PubMed

    Tong, Xiaoling; He, Songzhen; Chen, Jun; Hu, Hai; Xiang, Zhonghuai; Lu, Cheng; Dai, Fangyin

    2015-01-01

    Laminins are important basement membrane (BM) components with crucial roles in development. The numbers of laminin isoforms in various organisms are determined by the composition of the different α, β, and γ chains, and their coding genes, which are variable across spieces. In insects, only two α, one β, and one γ chains have been identified thus far. Here, we isolated a novel laminin β gene, BmLanB1-w, by positional cloning of the mutant (crayfish, cf) with blistered wings in silkworm. Gene structure analysis showed that a 2 bp deletion of the BmLanB1-w gene in the cf mutant caused a frame-shift in the open reading frame (ORF) and generated a premature stop codon. Knockdown of the BmLanB1-w gene produced individuals exhibiting blistered wings, indicating that this laminin gene was required for cell adhesion during wing development. We also identified laminin homologs in different species and showed that two copies of β laminin likely originated in Lepidoptera during evolution. Furthermore, phylogenetic and gene expression analyses of silkworm laminin genes revealed that the BmLanB1-w gene is newly evolved, and is required for wing-specific cell adhesion. This is the first report showing the tissue specific distribution and functional differentiation of β laminin in insects.

  13. Cloning of the laminin alpha 3 chain gene (LAMA3) and identification of a homozygous deletion in a patient with Herlitz junctional epidermolysis bullosa.

    PubMed

    Vidal, F; Baudoin, C; Miquel, C; Galliano, M F; Christiano, A M; Uitto, J; Ortonne, J P; Meneguzzi, G

    1995-11-20

    Laminin 5 and laminin 6 are basement membrane proteins synthesized by the basal cells of stratifying squamous epithelia. Altered expression of laminin 5 has been associated with Herlitz junctional epidermolysis bullosa (H-JEB), a severe epidermal blistering disorder inherited as an autosomal recessive disease. We have isolated cDNA clones encoding the alpha 3 chain of laminin 5 and searched for mutations in the LAMA3 gene in H-JEB patients. In one H-JEB family, an affected individual exhibited drastically reduced immunoreactivity to antibodies directed against the alpha 3 chain of laminin 5 and an impaired expression of the corresponding mRNA transcripts. RT-PCR analysis of mRNA extracted from the proband's keratinocytes identified a homozygous single basepair deletion in the transcripts encoding the laminin alpha 3A and alpha 3B isoforms. The mutation causes a frameshift and premature termination codon in both alleles of the LAMA3 gene. Inheritance of the clinical H-JEB phenotype was consistent with the segregation of the mutated allele in the family. We also report the identity of the alpha chains of laminin 5 and epiligrin and provide evidence that LAMA3 transcripts are distinct from the laminin 6 alpha chain mRNA. PMID:8586427

  14. Cloning of the laminin {alpha}3 chain gene (LAMA3) and identification of a homozygous deletion in a patient with Herlitz junctional epidermolysis bullosa

    SciTech Connect

    Vidal, F.; Ortonne, J.P. |; Galliano, M.F.

    1995-11-20

    Laminin 5 and laminin 6 are basement membrane proteins synthesized by the basal cells of stratifying squamous epithelia. Altered expression of laminin 5 has been associated with Herlitz junctional epidermolysis bullosa (H-JEB), a severe epidermal blistering disorder inherited as an autosomal recessive disease. We have isolated cDNA clones encoding the {alpha}3 chain of laminin 5 and searched for mutations in the LAMA3 gene in H-JEB patients. In one H-JEB family, an affected individual exhibited drastically reduced immunoreactivity to antibodies directed against the {alpha}3 chain of laminin 5 and an impaired expression of the corresponding mRNA transcripts. RT-PCR analysis of mRNA extracted from the proband`s keratinocytes identified a homozygous single basepair deletion in the transcripts encoding the laminin {alpha}3A and {alpha}3B isoforms. The mutation causes a frameshift and premature termination codon in both alleles of the LAMA3 gene. Inheritance of the clinical H-JEB phenotype was consistent with the segregation of the mutated allele in the family. We also report the identity of the {alpha} chains of laminin 5 and epiligrin and provide evidence that LAMA3 transcripts are distinct from the laminin 6 {alpha} chain mRNA. 35 refs., 5 figs., 1 tab.

  15. A homozygous nonsense mutation in the {beta}3 chain gene of laminin 5 (LAMB3) in herlitz junctional epidermolysis bullosa

    SciTech Connect

    Pulkkinen, L.; Christiano, A.M.; Uitto, J.

    1994-11-15

    Herlitz junctional epidermolysis bullosa (H-JEB) is a severe autosomal recessive disorder characterized by blister formation within the dermal-epidermal basement membrane. Based on immunofluorescence analysis recognizing laminin 5 epitopes (previously known as nicein/kalinin), the genes for this lamina lucida protein have been proposed as candidate genes in H-JEB. Amplification of mRNA by RT-PCR, followed by direct nucleotide sequencing, revealed a homozygous C-to T transition resulting in a premature termination codon (CGA{r_arrow}TGA) on both alleles. This mutation was verified at the genomic DNA level, and both parents were shown to be heterozygous carriers of the same mutation. This is the first description of a mutation in the laminin {beta}3 chain gene (LAMB3) of laminin 5 in an H-JEB patient. 15 refs., 2 figs.

  16. Autoantibodies to the laminin P1 fragment in HgCl2-induced membranous glomerulopathy.

    PubMed Central

    Aten, J.; Veninga, A.; Coers, W.; Sonnenberg, A.; Timpl, R.; Claessen, N.; van Eendenburg, J. D.; de Heer, E.; Weening, J. J.

    1995-01-01

    Exposure to mercuric chloride induces the development of a membranous glomerulopathy with high proteinuria in DZB rats, in which immunoglobulin (Ig)G1 and IgG2a bound in the glomeruli were previously found to react with laminin of the EHS tumor and several unidentified glomerular basement membrane components. Monoclonal antibodies were prepared by fusing cervical and mandibular lymph node cells from a HgCl2-treated DZB rat with a nonsecreting mouse myeloma. Monoclonal antibodies were screened for reactivity with collagenase-digested glomerular basement membrane and kidney sections; upon subcloning, eight stable hybridomas were obtained, named MEC1 to MEC8. MEC2 (IgG1, kappa), MEC3 (IgM, kappa), and MEC5 (IgG1, kappa), as well as the polyclonal glomerular eluate, reacted preferentially with the P1 fragment of the laminin-1 (alpha 1 beta 1 gamma 1) isoform. MEC8 (IgM, kappa) reacted with the P1 and the E4 fragment of laminin. Both MEC6 (IgM, kappa) and MEC8 bound to actin and to various other, unidentified cellular antigens, indicating that MEC6 and MEC8 are polyreactive antibodies. MEC7 (IgM, kappa) bound to a cytoskeleton-linked cell membrane antigen, present on various epithelial cells and between heart muscle fibers and associated with small peripheral, intramuscular nerves. Several of the MEC monoclonal antibodies bound in vivo along the glomerular capillary wall. Although discrete electron-dense subepithelial immune aggregates were not detected and proteinuria was not induced, MEC3 localization changed from a continuous pattern into a fine granular pattern along the glomerular basement membrane, and focally along the TBM, upon passive transfer into naive DZB rats. These findings suggest a pathogenetic role for the P1 fragment of laminin either in the induction phase of HgCl2-induced membranous glomerulopathy as an immunogen or in the effector phase as a target antigen. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 PMID:7778685

  17. Polarity determination in breast tissue: Desmosomal adhesion, myoepit helial cells, and laminin 1

    SciTech Connect

    Bissell, Mina J.; Bilder, David

    2003-06-05

    In all epithelial organs, apicobasal polarity determines functional integrity and contributes to the maintenance of tissue and organ specificity. In the breast, the functional unit is a polar double-layered tube consisting of luminal epithelial cells surrounded by myoepithelial cells and a basement membrane. It is far from clear how this double-layered structure is established and how polarity is maintained. Two recent papers have shed some light onto this intriguing problem in mammary gland biology. The results point to desmosomes and laminin 1 as having crucial roles. However, some questions remain.

  18. [Effect of laminin on numerical karyotype variability of kangaroo rat kidney cell lines].

    PubMed

    Polianskaia, G G; Goriachaia, T S; Mikhaĭlova, N A; Pinaev, G P

    2003-01-01

    The numerical karyotypic variability has been investigated in "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-11 and NBL-3-17 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, the character of numerical karyotypic variability has changed. In 2 days the general character of cell distribution for the chromosome number did not change, but the frequency of cells with modal number of chromosomes decreases significantly, while that of cells with lower chromosome number show a tendency to increase. At a prolongation of cultivation time to 4 and 12 days, the numerical karyotypic heterogeneity in cell population increases due to a significant change in the general character of cell distribution for the chromosome number, which is caused by a significant decrease in the frequency of cells with the modal number of chromosomes, and by an increase in the frequency of cells with lower chromosome number. The analysis of distribution of individual chromosomes showed that the number of types of additional structural variants of the karyotype (SVK) increases significantly on cultivation on laminin for 2-12 days. In cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, the character of numerical karyotypic variability did not change compared to control variants. Possible reasons of the observed changes of numerical karyotypic variability in cell line NBL-3-17 is discussed. The reason of differences in the character of numerical karyotypic variability between cell lines NBL-3-11 and NBL-3-17 possibly consists in the change of gene expression, namely in a dose of certain functioning genes. The polymerase chain reaction with arbitrary primers revealed no differences between DNA patterns of cell lines NBL-3-17 and NBL-3-11. This can reflect a similarity in the primary DNA structure of both cell lines. Hence, these lines differ only in the number of homologous

  19. Extracellular collagenous spherules in salivary gland tumors. Immunohistochemical analysis of laminin and various types of collagen.

    PubMed

    Skalova, A; Leivo, I

    1992-06-01

    Collagenous spherulosis is a benign breast lesion involving lobular acini and ductules and containing eosinophilic spherules measuring up to 100 microns in diameter. We present an immunohistochemical analysis of similar collagen-rich spherules that are also found in salivary gland tumors. These collagenous spherules contain varying amounts of acidic mucins, elastin, basement membrane proteins including type IV collagen and laminin, and considerable amounts of interstitial collagen types I and III. Types II and VI collagen were not detected in collagenous spherules of salivary gland tumors. The cells surrounding these collagenous spherules expressed muscle actin, S100 protein, vimentin, and cytokeratins 8, 18, and 19, indicating that these cells have myoepithelial characteristics.

  20. Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle.

    PubMed

    Cação-Benedini, L O; Ribeiro, P G; Prado, C M; Chesca, D L; Mattiello-Sverzut, A C

    2014-06-01

    Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres. PMID:24820070

  1. Domain-specific antibodies against the B2 chain of laminin inhibit neuronal migration in the neonatal rat cerebellum.

    PubMed

    Liesi, P; Hager, G; Dodt, H U; Seppälä, I; Zieglgänsberger, W

    1995-02-01

    Although the spatial and temporal patterns of neuronal migration have been analyzed in great detail, little direct evidence is available as to what extracellular matrix molecules are involved. Because there is indirect evidence implicating the extracellular matrix protein laminin in neuronal migration, we investigated the effects of antibodies against a synthetic peptide derived from a neurite outgrowth domain of the B2 chain of laminin on neuronal migration in living cerebellar slices. We show by using infrared video microscopy that divalent Fab2 fragments of these antibodies inhibit granule neuronal movement in living slices of (P8) rat cerebellum. This inhibition of neuronal movement manifests itself by cessation of both radial and horizontal translocations of nuclei inside the granule neuronal processes. Fab2 fragments of antibodies against the intact (native) laminin molecule or Fab2 fragments from the preimmune serum do not affect nuclear translocation. Immunocytochemistry shows binding of the divalent Fab2 fragments of the B2 chain-specific antibodies to the Purkinje and Bergmann glial cell areas, and as punctate deposits in between the cells of the external granule cell layer. Native laminin antibodies bind to the basement membranes, and binding of the Fab2 fragments from the preimmune sera cannot be demonstrated. These results indicate that neuronal migration in the postnatal rat cerebellum in vivo involves nuclear translocation that can be inhibited by antibodies against a neurite outgrowth domain of the B2 chain of laminin. Thus, migration of cerebellar granule neurons may depend on the interaction between a neurite outgrowth domain of the B2 chain of laminin and neuronal cytoskeleton involved in nuclear movement. PMID:7745613

  2. Human {beta}2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

    SciTech Connect

    Wewer, U.M.; Durkin, M.E.; Albrechtsen, R.

    1994-11-15

    Overlapping cDNA clones that encode the full-length human laminin {beta}2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature {beta}2 chain polypeptide of 1765 amino acids with a calculated molecular mass of 192,389 Da. The human {beta}2 chain is predicted to have all of the seven structural domains typical of the {beta} chains of laminin, including the short cysteine-rich {alpha} region. The amino acid sequence of human {beta}2 chain showed 86.1% sequence identity to the rat {beta}2 chain, 50.0% to human {beta}1 chain, and 36.3% to the human {beta}3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the {beta}2 chain of laminin purified from human amniotic basement membrane matched the sequence predicted from the cDNA, confirming that the cDNA encodes human {beta}2 laminin. The cDNA was used to assign the gene (LAMB2) to human chromosome 3p21 by in situ hybridization. It is not linked to genes for human laminin chains {alpha}1, {beta}1, and {gamma}1 or other known laminin genes. Immunostaining showed that the {beta}2 chain is localized to the smooth muscle basement membranes of the arteries, while the homologous {beta}1 chain is confined to the subendothelial basement membranes. The {beta}2 chain was found in the basement membranes of ovarian carcinomas but not colon carcinomas. These results indicate that the expression of the {beta}2 chain gene is tightly regulated in normal human tissues and in disease. 43 refs., 6 figs., 1 tab.

  3. Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles

    NASA Technical Reports Server (NTRS)

    Costes, S.; Streuli, C. H.; Barcellos-Hoff, M. H.

    2000-01-01

    We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions.

  4. Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle

    PubMed Central

    Cação-Benedini, L.O.; Ribeiro, P.G.; Prado, C.M.; Chesca, D.L.; Mattiello-Sverzut, A.C.

    2014-01-01

    Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres. PMID:24820070

  5. Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle.

    PubMed

    Cação-Benedini, L O; Ribeiro, P G; Prado, C M; Chesca, D L; Mattiello-Sverzut, A C

    2014-06-01

    Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres.

  6. [Congenital muscular dystrophy with laminin-a2 deficiency in early infancy: diagnosis and long-term follow-up].

    PubMed

    Panteliadis, C; Karatza, E; Xinias, I; Flaris, N; Tzitiridou, M; Ramantani, G

    2005-01-01

    Congenital muscular dystrophy (CMD) is a heterogeneous group of neuromuscular disorders characterized by muscle weakness and hypotonia at birth or within the first few months of life. It is inherited in an autosomal recessive pattern. About half of the patients have a deficiency of the alpha-2-chain of laminin (merosin). We describe a case of congenital muscular dystrophy in an infant with laminin-a2-chain deficiency, which appeared hypotonia in early infancy. Diagnosis was made by clinical features and the histological and immunohistochemical studies on muscle biopsy.

  7. Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells

    SciTech Connect

    Miyazaki, Takamichi; Futaki, Sugiko; Hasegawa, Kouichi; Kawasaki, Miwa; Sanzen, Noriko; Hayashi, Maria; Kawase, Eihachiro; Sekiguchi, Kiyotoshi Nakatsuji, Norio; Suemori, Hirofumi

    2008-10-10

    Human embryonic stem cells (hESCs) are thought to be a promising cell source for cell transplantation therapy. For such a clinical application, the hESCs should be manipulated using appropriate and qualified materials. In this study, we examined the efficacy of recombinant human laminin (rhLM) isoforms on the undifferentiated growth of hESCs. We first determined the major integrins expressed on the hESCs to reveal the preference of the hESCs for rhLMs, and found that the hESCs mainly expressed integrin {alpha}6{beta}1, which binds predominantly to laminin-111, -332 and -511/-521. When the hESCs were seeded onto rhLMs, the cells indeed adhered markedly to rhLM-332, and to rhLM-511 and rhLM-111 to a lesser extent. The hESCs proliferated on these three rhLMs for several passages while preserving their pluripotency. These results show that rhLM-111, -332, and -511 are good substrates to expand undifferentiated hESCs due to their high affinity to integrin {alpha}6{beta}1 expressed on hESCs.

  8. Histochemical Analysis of Laminin α Chains in Diethylstilbestrol-Induced Prolactinoma in Rats.

    PubMed

    Ramadhani, Dini; Tofrizal, Alimuddin; Tsukada, Takehiro; Yashiro, Takashi

    2015-04-28

    Laminin, a major basement membrane component, is important in structural support and cell proliferation and differentiation. Its 19 isoforms are assemblies of α, β, and γ chains, and the α chains (α1-5) determine the isoform characteristics. Although our previous studies showed alterations in α chain expressions during anterior pituitary development, their expressions in pituitary tumors yet to be determined. The present study used a rat model of diethylstilbestrol (DES)-induced prolactinoma to examine α chain expressions during prolactinoma tumorigenesis (0-12 weeks of DES treatment) by in situ hybridization and immunohistochemistry. mRNA of α1, α3, and α4 chains was detected in control and after 4 weeks of DES treatment. These expressions were undetectable after 8 weeks of DES treatment and in prolactinoma (12 weeks of DES treatment). Immunohistochemistry showed that the α1 chain was localized in some anterior pituitary cells in control and after 4 weeks of treatment and in endothelial cells after 8 weeks of treatment. The α3 and α4 chains were expressed in endothelial cells, and immunoreactivity and the number of immunopositive cells decreased during DES treatment. These findings suggest that alteration of laminin α chains is related to abnormal cell proliferation and neovascularization during development of DES-induced prolactinoma.

  9. Proteasome inhibition improves the muscle of laminin α2 chain-deficient mice.

    PubMed

    Carmignac, Virginie; Quéré, Ronan; Durbeej, Madeleine

    2011-02-01

    Muscle atrophy, a significant characteristic of congenital muscular dystrophy with laminin α2 chain deficiency (also known as MDC1A), occurs by a change in the normal balance between protein synthesis and protein degradation. The ubiquitin-proteasome system (UPS) plays a key role in protein degradation in skeletal muscle cells. In order to identify new targets for drug therapy against MDC1A, we have investigated whether increased proteasomal degradation is a feature of MDC1A. Using the generated dy(3K)/dy(3K) mutant mouse model of MDC1A, we studied the expression of members of the ubiquitin-proteasome pathway in laminin α2 chain-deficient muscle, and we treated dy(3K)/dy(3K) mice with the proteasome inhibitor MG-132. We show that members of the UPS are upregulated and that the global ubiquitination of proteins is raised in dystrophic limb muscles. Also, phosphorylation of Akt is diminished in diseased muscles. Importantly, proteasome inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. Specifically, treatment with MG-132 increases lifespan, enhances locomotive activity, enlarges muscle fiber diameter, reduces fibrosis, restores Akt phosphorylation and decreases apoptosis. These studies promote better understanding of the disease process in mice and could lead to a drug therapy for MDC1A patients.

  10. Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: implications for regulation of BMP signalling

    PubMed Central

    Lopez-Escobar, Beatriz; de Felipe, Beatriz; Sanchez-Alcazar, Jose Antonio; Sasaki, Takako; Copp, Andrew J.; Ybot-Gonzalez, Patricia

    2013-01-01

    Background The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. Results We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Conclusions Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm. PMID:22911573

  11. [The effect of immobilized laminin on titanium-oxide films to the behavior of human umbilical vein endothelial cells].

    PubMed

    Ge, Shengnan; Chen, Junying; Huang, Nan

    2009-02-01

    In this study, Ti-O films were synthesized using magnetron sputtering, and were pretreated using NaOH solution for improving surface activity from hydroxyl. The laminin(LN) biomacromolecule was further immobilized to the surface through an anminosilane linker. The surface characteristics of these samples were analyzed by Fourier Transform Infrared Spectroscopy, Scanning Electron Microscopy, Atomic Force Microscopy and the contact angle method. Finally, human umbilical vein endothelial cells (HUVEC) were in vitro seeded to the modified and unmodified Ti-O films surface for evaluating the cell compatibility. Survey results suggested that the functional group of hydroxyl was presented onto Ti-O film surface after being pretreated, and laminin could be covalently immobilized to Ti-O film surface by anminosilane linker. The in vitro cell culture results reveal that the biological behaviors of ECs on biochemical modified Ti-O film surface are excellent. The adherence, growth and proliferation of ECs on laminin-immobilized surface were obviously improved when compared to control one. It implies that the laminin immobilizing is helpful to increasing the endothelialization of Ti-O films. PMID:19334564

  12. Laminin Interactions with Head and Neck Cancer Cells under Low Fluid Shear Conditions Lead to Integrin Activation and Binding*

    PubMed Central

    Fennewald, Susan M.; Kantara, Carla; Sastry, Sarita K.; Resto, Vicente A.

    2012-01-01

    Lymphatic metastasis of cancer cells involves movement from the primary tumor site to the lymph node, where the cells must be able to productively lodge and grow. It is there that tumor cells encounter cellular and non-cellular constituent elements that make up the lymph node parenchyma. Our work shows that head and neck squamous cell carcinoma (HNSCC) cell lines are able to bind to laminin, fibronectin, vitronectin, and hyaluronic acid, which are extracellular matrix elements within the lymph node parenchyma. HNSCC cell lines bound to laminin under lymphodynamic low shear stress (0.07 dynes/cm2), consistent with lymph flow via β1 integrins, including α2β1, α3β1, and α6β1. Binding occurred in the presence of shear stress and not in the absence of flow. Additionally, tumor cell binding to laminin under flow did result in calcium signaling. Our data indicate a novel role for β1 integrin-mediated binding of HNSCC cells to laminin under conditions of lymphodynamic flow that results in intracellular calcium signaling within the cancer cell. PMID:22547070

  13. Immunolocalization of intermediate filaments and laminin in the oviduct of the immature and mature Japanese quail (Coturnix coturnix japonica).

    PubMed

    Madekurozwa, M-C

    2014-06-01

    This study describes the distribution of vimentin, desmin, smooth muscle actin (SMA) and laminin in the oviduct of the immature and mature Japanese quail. The cytoskeletal proteins vimentin, desmin and SMA have been shown to be involved in cellular support, differentiation, migration and contractility. Laminin is a major component of basement membranes. Luminal epithelia in the infundibular and magnal regions of immature and mature birds exhibited strong vimentin immunoreactivity. Luminal epithelial cells exhibiting strong vimentin immunoreactivity were present in the isthmus and shell gland regions of only mature quails. Infundibular glandular grooves displayed strong vimentin immunostaining. In contrast, the glandular epithelia of the magnum, isthmus and shell gland were vimentin immunonegative. Fibroblasts and vascular endothelial cells in the lamina propria of the oviductal regions studied exhibited strong vimentin immunostaining. Smooth muscle cells forming the tunica muscularis and vascular tunica media displayed strong desmin and SMA immunostaining. Strong laminin immunostaining was demonstrated in the basement membranes associated with smooth muscle cells, as well as in the basement membranes underlying the luminal and glandular epithelia. In conclusion, this study has shown that the immunolocalization of desmin, SMA and laminin in the oviduct of the Japanese quail is similar to that in the domestic fowl. However, differences in the immunoexpression of vimentin in the LE of the two avian species were shown to exist. In addition, the study has shown that the immunolocalization of vimentin in the Japanese quail varies depending on the oviductal region, as well as the developmental stage of the oviduct.

  14. Cultured human melanoma cells biosynthesize a novel form of laminin that lacks the M/sub r/ = 400 kDa subunit

    SciTech Connect

    Peters, B.P.; Kroll, T.G.; Ruddon, R.W.

    1987-05-01

    A human melanoma cell line (A375) was found to produce an unusual type of laminin composed exclusively of M/sub r/ = 200 kDa B subunits and lacking the M/sub r/ = 400 kDa A subunit. Detergent lysates of A375 cells pulsed with (/sup 35/S)methionine contained an immunoreactive form of laminin that migrated on nonreduced SDS-PAGE with an apparent M/sub r/ = 850 kDa. The 850 kDa laminin was clearly resolved from the typical M/sub r/ = 950 kDa laminin composed of A and B subunit types that is produced by a variety of cultured cells such as human choriocarcinoma (JAR) cells. Upon reduction of the intersubunit disulfide bonds of the A375 laminin, a pair of polypeptides co-migrated on SDS-PAGE with the M/sub r/ = 200 kDa B subunit doublet of JAR laminin. The A-deficient A375 laminin does not seem to be a result of the proteolysis of A subunit in cell lysates. No degradation of (/sup 35/S)methionine-labeled JAR laminin was observed upon incubation with nonradioactive A375 cell lysate supplemented in the usual manner with ten protease inhibitors. Furthermore, A subunit did not appear transiently in A375 cells at any chase time (0-4 hr) following a 10-min biosynthetic pulse with (/sup 35/S)methionine. The authors observations suggest that A375 cells biosynthesize principally the B subunits of laminin and assemble them to form a disulfide-linked macromolecule, possibly a B/sub 4/ tetramer.

  15. Loss of β2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction

    PubMed Central

    Chand, Kirat K; Lee, Kah Meng; Schenning, Mitja P; Lavidis, Nickolas A; Noakes, Peter G

    2015-01-01

    β2-laminin is a key mediator in the differentiation and formation of the skeletal neuromuscular junction. Loss of β2-laminin results in significant structural and functional aberrations such as decreased number of active zones and reduced spontaneous release of transmitter. In vitro β2-laminin has been shown to bind directly to the pore forming subunit of P/Q-type voltage-gated calcium channels (VGCCs). Neurotransmission is initially mediated by N-type VGCCs, but by postnatal day 18 switches to P/Q-type VGCC dominance. The present study investigated the changes in neurotransmission during the switch from N- to P/Q-type VGCC-mediated transmitter release at β2-laminin-deficient junctions. Analysis of the relationship between quantal content and extracellular calcium concentrations demonstrated a decrease in the calcium sensitivity, but no change in calcium dependence at β2-laminin-deficient junctions. Electrophysiological studies on VGCC sub-types involved in transmitter release indicate N-type VGCCs remain the primary mediator of transmitter release at matured β2-laminin-deficient junctions. Immunohistochemical analyses displayed irregularly shaped and immature β2-laminin-deficient neuromuscular junctions when compared to matured wild-type junctions. β2-laminin-deficient junctions also maintained the presence of N-type VGCC clustering within the presynaptic membrane, which supported the functional findings of the present study. We conclude that β2-laminin is a key regulator in development of the NMJ, with its loss resulting in reduced transmitter release due to decreased calcium sensitivity stemming from a failure to switch from N- to P/Q-type VGCC-mediated synaptic transmission. PMID:25556799

  16. Interactive properties of human glioblastoma cells with brain neurons in culture and neuronal modulation of glial laminin organization.

    PubMed

    Faria, Jane; Romão, Luciana; Martins, Sheila; Alves, Tércia; Mendes, Fabio A; de Faria, Giselle Pinto; Hollanda, Rosenilde; Takiya, Christina; Chimelli, Leila; Morandi, Veronica; de Souza, Jorge Marcondes; Abreu, Jose Garcia; Moura Neto, Vivaldo

    2006-12-01

    The harmonious development of the central nervous system depends on the interactions of the neuronal and glial cells. Extracellular matrix elements play important roles in these interactions, especially laminin produced by astrocytes, which has been shown to be a good substrate for neuron growth and axonal guidance. Glioblastomas are the most common subtypes of primary brain tumors and may be astrocytes in origin. As normal laminin-producing glial cells are the preferential substrate for neurons, and glial tumors have been shown to produce laminin, we questioned whether glioblastoma retained the same normal glial-neuron interactive properties with respect to neuronal growth and differentiation. Then, rat neurons were co-cultured onto rat normal astrocytes or onto three human glioblastoma cell lines obtained from neurosurgery. The co-culture confirmed that human glioblastoma cells as well as astrocytes maintained the ability to support neuritogenesis, but non-neural normal or tumoral cells failed to do so. However, glioblastoma cells did not distinguish embryonic from post-natal neurons in relation to neurite pattern in the co-cultures, as normal astrocytes did. Further, the laminin organization on both normal and tumoral glial cells was altered from a filamentous arrangement to a mixed punctuate/filamentous pattern when in co-culture with neurons. Together, these results suggest that glioblastoma cells could identify neuronal cells as partners, to support their growth and induce complex neurites, but they lost the normal glia property to distinguish neuronal age. In addition, our results show for the first time that neurons modulate the organization of astrocytes and glioblastoma laminin on the extracellular matrix.

  17. Magnetic Resonance Imaging Is Sensitive to Pathological Amelioration in a Model for Laminin-Deficient Congenital Muscular Dystrophy (MDC1A)

    PubMed Central

    Vohra, Ravneet; Accorsi, Anthony; Kumar, Ajay; Walter, Glenn; Girgenrath, Mahasweta

    2015-01-01

    Purpose To elucidate the reliability of MRI as a non-invasive tool for assessing in vivo muscle health and pathological amelioration in response to Losartan (Angiotensin II Type 1 receptor blocker) in DyW mice (mouse model for Laminin-deficient Congenital Muscular Dystrophy Type 1A). Methods Multiparametric MR quantifications along with histological/biochemical analyses were utilized to measure muscle volume and composition in untreated and Losartan-treated 7-week old DyW mice. Results MRI shows that DyW mice have significantly less hind limb muscle volume and areas of hyperintensity that are absent in WT muscle. DyW mice also have significantly elevated muscle levels (suggestive of inflammation and edema). Muscle T2 returned to WT levels in response to Losartan treatment. When considering only muscle pixels without T2 elevation, DyW T2 levels are significantly lower than WT (suggestive of fibrosis) whereas Losartan-treated animals do not demonstrate this decrease in muscle T2. MRI measurements suggestive of elevated inflammation and fibrosis corroborate with increased Mac-1 positive cells as well as increased Picrosirius red staining/COL1a gene expression that is returned to WT levels in response to Losartan. Conclusions MRI is sensitive to and tightly corresponds with pathological changes in DyW mice and thus is a viable and effective non-invasive tool for assessing pathological changes. PMID:26379183

  18. Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes.

    PubMed

    Cameron, Kate; Tan, Rosanne; Schmidt-Heck, Wolfgang; Campos, Gisela; Lyall, Marcus J; Wang, Yu; Lucendo-Villarin, Baltasar; Szkolnicka, Dagmara; Bates, Nicola; Kimber, Susan J; Hengstler, Jan G; Godoy, Patricio; Forbes, Stuart J; Hay, David C

    2015-12-01

    Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.

  19. Interactions of laminin peptides with phospholipids in Langmuir films and vesicles

    NASA Astrophysics Data System (ADS)

    Dhathathreyan, Aruna; Steinem, Claudia

    2008-10-01

    Peptide YIGSR from the β1 chain of laminin promotes cell adhesion, migration, inhibits angiogenesis and tumor metastasis. Properties of YIGSR and a mutated sequence LIGSR in Langmuir films interacting with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and in vesicles are presented in this work. The mixed lipid-peptide systems have been characterized using circular dichroic spectroscopy, quartz crystal microbalance and Brewster angle microscopic studies. LIGSR peptide seems to adhere strongly to POPC resulting in better organized lipid-peptide layer compared with the native YIGSR. The results suggest that even single amino acid mutations in such short sequences may be used to fine tune properties of the peptide.

  20. Propolis modulates vitronectin, laminin, and heparan sulfate/heparin expression during experimental burn healing*

    PubMed Central

    Olczyk, Paweł; Komosińska-Vassev, Katarzyna; Winsz-Szczotka, Katarzyna; Koźma, Ewa M.; Wisowski, Grzegorz; Stojko, Jerzy; Klimek, Katarzyna; Olczyk, Krystyna

    2012-01-01

    Objective: This study was aimed at assessing the dynamics of vitronectin (VN), laminin (LN), and heparan sulfate/heparin (HS/HP) content changes during experimental burn healing. Methods: VN, LN, and HS/HP were isolated and purified from normal and injured skin of domestic pigs, on the 3rd, 5th, 10th, 15th, and 21st days following thermal damage. The wounds were treated with apitherapeutic agent (propolis), silver sulfadiazine (SSD), physiological salt solution, and propolis vehicle. VN and LN were quantified using an immunoenzymatic assay and HS/HP was estimated by densitometric analysis. Results: Propolis treatment stimulated significant increases in VN, LN, and HS/HP contents during the initial phase of study, followed by a reduction in the estimated extracellular matrix molecules. Similar patterns, although less extreme, were observed after treatment with SSD. Conclusions: The beneficial effects of propolis on experimental wounds make it a potential apitherapeutic agent in topical burn management. PMID:23125086

  1. Canonical Wnt signalling regulates epithelial patterning by modulating levels of laminins in zebrafish appendages

    PubMed Central

    Nagendran, Monica; Arora, Prateek; Gori, Payal; Mulay, Aditya; Ray, Shinjini; Jacob, Tressa; Sonawane, Mahendra

    2015-01-01

    The patterning and morphogenesis of body appendages – such as limbs and fins – is orchestrated by the activities of several developmental pathways. Wnt signalling is essential for the induction of limbs. However, it is unclear whether a canonical Wnt signalling gradient exists and regulates the patterning of epithelium in vertebrate appendages. Using an evolutionarily old appendage – the median fin in zebrafish – as a model, we show that the fin epithelium exhibits graded changes in cellular morphology along the proximo-distal axis. This epithelial pattern is strictly correlated with the gradient of canonical Wnt signalling activity. By combining genetic analyses with cellular imaging, we show that canonical Wnt signalling regulates epithelial cell morphology by modulating the levels of laminins, which are extracellular matrix components. We have unravelled a hitherto unknown mechanism involved in epithelial patterning, which is also conserved in the pectoral fins – evolutionarily recent appendages that are homologous to tetrapod limbs. PMID:25519245

  2. Laminin Production and Basement Membrane Deposition by Mesenchymal Stem Cells upon Adipogenic Differentiation

    PubMed Central

    Sillat, Tarvo; Virtanen, Ismo; Ingerpuu, Sulev; Bäck, Nils; Konttinen, Yrjö T.; Korhonen, Matti

    2013-01-01

    The aim was to study laminin (LM) synthesis, integration, and deposition into the basement membrane (BM) during adipogenesis. Human bone marrow-derived mesenchymal stromal cells (MSCs) were induced along the adipogenic lineage. LM chain mRNA and protein levels were followed using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining, transmission electron microscopy (TEM), and immunoprecipitation. MSCs produced low levels of LM mRNAs but were not surrounded by BM in IF and TEM imaging. LM-α4, LM-β1, and LM-γ1 mRNAs increased during adipogenesis 3.9-, 5.8-, and 2.8-fold by day 28. LM-411 was immunoprecipitated from the ECM of the differentiated cells. Immunostaining suggested deposition of LM-411 and some LM-421. BM build-up was probably organized in part by integrin (Int) α6β1. At day 28, TEM images revealed BM-like structures around fat droplet-containing cells. The first signs of BM formation and Int α6β1 were seen using IF imaging at day 14. Laminin-411 and Int α6β1 were expressed in vivo in mature human subcutaneous fat tissue. Undifferentiated human MSCs did not organize LM subunits into BM, whereas LM-411 and some LM-421 are precipitated in the BM around adipocytes. This is the first demonstration of LM-411 precipitation during hMSC adipogenesis around adipocytes as a structural scaffold and Int-regulated signaling element. PMID:23900596

  3. Laminin 5 mutations in junctional epidermolysis bullosa: molecular basis of Herlitz vs. non-Herlitz phenotypes.

    PubMed

    Nakano, Aoi; Chao, Sheau-Chiou; Pulkkinen, Leena; Murrell, Dedee; Bruckner-Tuderman, Leena; Pfendner, Ellen; Uitto, Jouni

    2002-01-01

    Junctional epidermolysis bullosa (JEB) is a group of heritable blistering diseases in which tissue separation occurs within the lamina lucida of the cutaneous basement membrane zone. Clinically, two broad subcategories have been recognized: The Herlitz variant (H-JEB; OMIM 226700) is characterized by early demise of the affected individuals, usually within the first year of life, while non-Herlitz (nH-JEB; OMIM 226650) patients show a milder phenotype with life-long blistering, yet with normal lifespan. In this study, we have examined a cohort of 27 families, 15 with Herlitz and 12 with non-Herlitz JEB, for mutations in the candidate genes, LAMA3, LAMB3, and LAMC2, encoding the subunit polypeptides of laminin 5. The mutation detection strategy consisted of PCR amplification of all exons in these genes, followed by heteroduplex scanning and nucleotide sequencing. We were able to identify pathogenic mutations in both alleles of each proband, the majority of the mutations being in the LAMB3 gene. Examination of the mutation database revealed that most cases with Herlitz JEB harbored premature termination codon (PTC) mutations in both alleles. In non-Herlitz cases, the PTC mutation was frequently associated with a missense mutation or a putative splicing mutation in trans. In three cases with putative splicing mutations, RT-PCR analysis revealed a repertoire of splice variants in-frame, predicting the synthesis of either shortened or lengthened, yet partly functional, polypeptides. These observations would explain the relatively mild phenotype in cases with splicing mutations. Collectively, these findings, together with the global laminin 5 mutation database, contribute to our understanding of the genotype/phenotype correlations explaining the Herlitz vs non-Herlitz phenotypes. PMID:11810295

  4. Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide.

    PubMed

    Conway, Rebecca E; Rojas, Camilo; Alt, Jesse; Nováková, Zora; Richardson, Spencer M; Rodrick, Tori C; Fuentes, Julio L; Richardson, Noah H; Attalla, Jonathan; Stewart, Samantha; Fahmy, Beshoy; Barinka, Cyril; Ghosh, Mallika; Shapiro, Linda H; Slusher, Barbara S

    2016-10-01

    Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies. PMID:27387982

  5. Prostate-specific membrane antigen (PSMA)-mediated laminin proteolysis generates a pro-angiogenic peptide.

    PubMed

    Conway, Rebecca E; Rojas, Camilo; Alt, Jesse; Nováková, Zora; Richardson, Spencer M; Rodrick, Tori C; Fuentes, Julio L; Richardson, Noah H; Attalla, Jonathan; Stewart, Samantha; Fahmy, Beshoy; Barinka, Cyril; Ghosh, Mallika; Shapiro, Linda H; Slusher, Barbara S

    2016-10-01

    Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase expressed in a number of tissues. PSMA participates in various biological functions depending on the substrate available in the particular tissue; in the brain, PSMA cleaves the abundant neuropeptide N-acetyl-aspartyl-glutamate to regulate release of key neurotransmitters, while intestinal PSMA cleaves polyglutamated peptides to supply dietary folate. PSMA expression is also progressively upregulated in prostate cancer where it correlates with tumor progression as well as in tumor vasculature, where it regulates angiogenesis. The previous research determined that PSMA cleavage of small peptides generated via matrix metalloprotease-mediated proteolysis of the extracellular matrix protein laminin potently activated endothelial cells, integrin signaling and angiogenesis, although the specific peptide substrates were not identified. Herein, using enzymatic analyses and LC/MS, we unequivocally demonstrate that several laminin-derived peptides containing carboxy-terminal glutamate moieties (LQE, IEE, LNE) are bona fide substrates for PSMA. Subsequently, the peptide products were tested for their effects on angiogenesis in various models. We report that LQ, the dipeptide product of PSMA cleavage of LQE, efficiently activates endothelial cells in vitro and enhances angiogenesis in vivo. Importantly, LQE is not cleaved by an inactive PSMA enzyme containing an active site mutation (E424S). Endothelial cell activation by LQ was dependent on integrin beta-1-induced activation of focal adhesion kinase. These results characterize a novel PSMA substrate, provide a functional rationale for the upregulation of PSMA in cancer cells and tumor vasculature and suggest that inhibition of PSMA could lead to the development of new angiogenic therapies.

  6. Identification of the Integrin VLA-2 as a Receptor for Echovirus 1

    NASA Astrophysics Data System (ADS)

    Bergelson, Jeffrey M.; Shepley, Michael P.; Chan, Bosco M. C.; Hemler, Martin E.; Finberg, Robert W.

    1992-03-01

    Cell surface receptors for echovirus, a common human pathogen, were identified with monoclonal antibodies that protected susceptible cells from infection with echovirus 1. These monoclonal antibodies, which prevented virus attachment to specific receptor sites, recognized the α and β subunits of the integrin VLA-2 (α_2β_1), a receptor for collagen and laminin. RD rhabdomyosarcoma cells expressed little VLA-2, did not bind to 35S-labeled virus, and resisted infection until transfected with complementary DNA encoding the α_2 subunit of VLA-2. Thus, integrins, adhesion receptors important in interactions between cells and with the extracellular matrix, can mediate virus attachment and infection.

  7. Immunohistochemical analysis of the skin in junctional epidermolysis bullosa using laminin 5 chain specific antibodies is of limited value in predicting the underlying gene mutation.

    PubMed

    McMillan, J R; McGrath, J A; Pulkkinen, L; Kon, A; Burgeson, R E; Ortonne, J P; Meneguzzi, G; Uitto, J; Eady, R A

    1997-06-01

    The anchoring filament protein laminin 5 is composed of three polypeptide chains (alpha 3, beta 3 and gamma 2) each encoded by separate genes (LAMA3, LAMB3 and LAMC2, respectively). Mutations in any of these three genes may give rise to the autosomal recessive blistering skin disease, junctional epidermolysis bullosa. At present, there is no easy way of predicting which of these three genes might harbour the pathogenetic laminin 5 mutations in a case of junctional epidermolysis bullosa. In this study, we assessed whether immunohistochemistry might be helpful in this regard. We performed immunohistochemical labelling of the dermal-epidermal junction using alpha 3, beta 3 and gamma 2 chain-specific antibodies in 11 patients with junctional epidermolysis bullosa, in whom the laminin 5 mutations had been previously delineated. Although, labelling for the laminin 5 chain bearing the mutations was attenuated or undetectable in all cases, a complete absence of labelling or a reduction in the staining intensity for the other two chains was also seen in all cases. The results showed that immunohistochemical labelling of the dermal-epidermal junction using alpha 3, beta 3 and gamma 2 chain-specific antibodies is not a specific indicator for which of the laminin 5 chain genes contains the pathogenetic mutations, and is therefore unreliable in screening for individual laminin 5 gene mutations in cases of junctional epidermolysis bullosa. PMID:9217810

  8. Tumorigenicity and adenovirus-transformed cells: Collagen interaction and cell surface laminin are controlled by the serotype origin of the E1A and E1B genes

    SciTech Connect

    Bober, F.J.; Birk, D.E.; Raska, K. Jr. ); Shenk, T. )

    1988-02-01

    A library of cells transformed with recombinant adenoviruses was used to study tumorigenicity and interaction with extracellular matrix. Cells expressing the complete E1 region of highly oncogenic adenovirus type 12 (Ad12) are tumorigenic, adhere preferentially to type IV collagen, and express cell surface laminin. Weakly tumorigenic cells, which express the E1A oncogene of Ad12 and the E1B genes of Ad5, also attach preferentially to type IV collagen but do not contain laminin on their surface. Cells which express the E1A oncogene of Ad5 and the E1B genes of Ad12 are nontumorigenic and do not preferentially attach to type IV versus type I collagen but have laminin on their surface. There is no significant difference in the amounts of laminin secreted into the culture medium among cells expressing the E1B genes of Ad5 or Ad12. In vitro assays show that cells which express the E1B genes of Ad12, irrespective of the origin of the E1A genes, can bind three times more exogenously added {sup 125}I-laminin than cells expressing the E1B genes of nononcogenic Ad5. The interaction of adenovirus-transformed cells with collagen is controlled by the serotype origin of the E1A oncogene, whereas cell surface laminin is controlled by the serotype origin of the E1B genes.

  9. CD90-positive cells, an additional cell population, produce laminin {alpha}2 upon transplantation to dy{sup 3k}/dy{sup 3k} mice

    SciTech Connect

    Fukada, So-ichiro Yamamoto, Yukiko; Segawa, Masashi; Sakamoto, Kenta; Nakajima, Mari; Sato, Masaki; Morikawa, Daisuke; Uezumi, Akiyoshi; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi; Tsujikawa, Kazutake; Yamamoto, Hiroshi

    2008-01-01

    Laminin {alpha}2 is a component of skeletal and cardiac muscle basal lamina. A defect of the laminin {alpha}2 chain leads to severe congenital muscular dystrophy (MDC1A) in humans and dy/dy mice. Myogenic cells including myoblasts, myotubes, and myofibers in skeletal muscle are a possible source of the laminin {alpha}2 chain, and myogenic cells are thus proposed as a cell source for congenital muscular dystrophy therapy. However, we observed production of laminin {alpha}2 in non-myogenic cells of normal mice, and we could enrich these laminin {alpha}2-producing cells in CD90{sup +} cell fractions. Intriguingly, the number of CD90{sup +} cells increased dramatically during skeletal muscle regeneration in mice. This fraction did not include myogenic cells but exhibited a fibroblast-like phenotype. Moreover, these cells were resident in skeletal muscle, not derived from bone marrow. Finally, the production of laminin {alpha}2 in CD90{sup +} cells was not dependent on fusion with myogenic cells. Thus, CD90{sup +} cells are a newly identified additional cell fraction that increased during skeletal muscle regeneration in vivo and could be another cell source for therapy for lama2-deficient muscular dystrophy.

  10. The oriented emergence of axons from retinal ganglion cells is directed by laminin contact in vivo.

    PubMed

    Randlett, Owen; Poggi, Lucia; Zolessi, Flavio R; Harris, William A

    2011-04-28

    How the site of axon emergence is specified during neural development is not understood. Previous studies disagree on the relative importance of intrinsic and extrinsic mechanisms. The axons of retinal ganglion cells (RGCs) emerge basally in vivo, yet because RGCs develop from polarized neuroepithelial cells within a polarized environment, disentangling intrinsic and extrinsic influences is a challenge. We use time-lapse imaging to demonstrate that Laminin acting directly on RGCs is necessary and sufficient to orient axon emergence in vivo. Laminin contact with the basal processes of newborn RGCs prevents the cells from entering a stochastic Stage 2 phase, directs the rapid accumulation of the early axonal marker Kif5c560-YFP, and leads to the formation of axonal growth cones. These results suggest that contact-mediated cues may be critical for the site of axon emergence and account for the differences in cellular behavior observed in vitro and in vivo.

  11. Efficient and scalable culture of single dissociated human pluripotent stem cells using recombinant E8 fragments of human laminin isoforms.

    PubMed

    Miyazaki, Takamichi; Kawase, Eihachiro

    2015-01-01

    This unit describes a protocol for efficient expansion of human pluripotent stem cells (hPSCs). A key feature of this method is subculture of hPSCs by single-cell dissociation passaging on substrates coated with recombinant E8 fragments of human laminin isoforms (LM-E8s). LM-E8s, provide superior adhesion over intact laminin isoforms and Matrigel. Single hPSCs seeded on LM-E8s show accelerated migration and rapid reconstruction of clusters, resulting in robust survival and proliferation. This protocol yields 200-fold more hPSCs than conventional subculture methods in 1 month of culture. Furthermore, this protocol can be easily adapted to most hPSC lines in combination with the use of various xeno-free, defined culture media, and large-scale expansion of hPSCs is easily achievable to facilitate the practical applications of hPSCs.

  12. Structure of the human laminin {gamma}2 chain gene (LAMC2): Alternative splicing with different tissue distribution of two transcripts

    SciTech Connect

    Airenne, T.; Haakana, H.; Kallunki, T.

    1996-02-15

    This article discusses the exon-intron structure and tissue distribution of the laminin {gamma}2 chain (LAMC2) gene, which is mutated in some cases of junctional epidermolysis bullosa. The article also discusses the transcription and splicing of this gene, which result in alternative uses of the last two exons of the gene. The different tissue distributions of the transcripts indicate different functions for the gene in vivo. 36 refs., 8 figs., 3 tabs.

  13. Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains.

    PubMed

    Giovani, Priscila A; Salmon, Cristiane R; Martins, Luciane; Paes Leme, Adriana F; Rebouças, Pedro; Puppin Rontani, Regina M; Mofatto, Luciana S; Sallum, Enilson A; Nociti, Francisco H; Kantovitz, Kamila R

    2016-01-01

    It has been suggested that there are histological and functional distinctions between the periodontal ligament (PDL) of deciduous (DecPDL) and permanent (PermPDL) teeth. Thus, we hypothesized that DecPDL and PermPDL display differences in the constitutive expression of genes/proteins involved with PDL homeostasis. Primary PDL cell cultures were obtained for DecPDL (n = 3) and PermPDL (n = 3) to allow us to perform label-free quantitative secretome analysis. Although a highly similar profile was found between DecPDL and PermPDL cells, comparative secretome analysis evidenced that one of the most stickling differences involved cell adhesion molecules, including laminin subunit gamma 1 (LAMC1) and beta 2 (LAMB2). Next, total RNA and protein extracts were obtained from fresh PDL tissues of deciduous (n = 6) and permanent (n = 6) teeth, and Western blotting and qPCR analysis were used to validate our in vitro findings. Western blot analysis confirmed that LAMC1 was increased in DecPDL fresh tissues (p<0.05). Furthermore, qPCR data analysis revealed that mRNA levels for laminin subunit beta 1 (LAMB1), beta 3 (LAMB3), LAMC1, and gamma 2 (LAMC2) were higher in DecPDL fresh tissues, whereas transcripts for LAMB2 were increased in PermPDL (p<0.05). In conclusion, the differential expression of laminin chains in DecPDL and PermPDL suggests an involvement of laminin-dependent pathways in the control of physiological differences between them. PMID:27149379

  14. Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains

    PubMed Central

    Giovani, Priscila A.; Salmon, Cristiane R.; Martins, Luciane; Paes Leme, Adriana F.; Rebouças, Pedro; Puppin Rontani, Regina M.; Mofatto, Luciana S.; Sallum, Enilson A.; Nociti, Francisco H.; Kantovitz, Kamila R.

    2016-01-01

    It has been suggested that there are histological and functional distinctions between the periodontal ligament (PDL) of deciduous (DecPDL) and permanent (PermPDL) teeth. Thus, we hypothesized that DecPDL and PermPDL display differences in the constitutive expression of genes/proteins involved with PDL homeostasis. Primary PDL cell cultures were obtained for DecPDL (n = 3) and PermPDL (n = 3) to allow us to perform label-free quantitative secretome analysis. Although a highly similar profile was found between DecPDL and PermPDL cells, comparative secretome analysis evidenced that one of the most stickling differences involved cell adhesion molecules, including laminin subunit gamma 1 (LAMC1) and beta 2 (LAMB2). Next, total RNA and protein extracts were obtained from fresh PDL tissues of deciduous (n = 6) and permanent (n = 6) teeth, and Western blotting and qPCR analysis were used to validate our in vitro findings. Western blot analysis confirmed that LAMC1 was increased in DecPDL fresh tissues (p<0.05). Furthermore, qPCR data analysis revealed that mRNA levels for laminin subunit beta 1 (LAMB1), beta 3 (LAMB3), LAMC1, and gamma 2 (LAMC2) were higher in DecPDL fresh tissues, whereas transcripts for LAMB2 were increased in PermPDL (p<0.05). In conclusion, the differential expression of laminin chains in DecPDL and PermPDL suggests an involvement of laminin-dependent pathways in the control of physiological differences between them. PMID:27149379

  15. The folded and disordered domains of human ribosomal protein SA have both idiosyncratic and shared functions as membrane receptors.

    PubMed

    Zidane, Nora; Ould-Abeih, Mohamed B; Petit-Topin, Isabelle; Bedouelle, Hugues

    2013-01-01

    The human RPSA [ribosomal protein SA; also known as LamR1(laminin receptor 1)] belongs to the ribosome but is also a membrane receptor for laminin, growth factors, prion, pathogens and the anticarcinogen EGCG (epigallocatechin-gallate). It contributes to the crossing of the blood-brain barrier by neurotropic viruses and bacteria, and is a biomarker of metastasis. RPSA includes an N-terminal domain, which is folded and homologous to the prokaryotic RPS2, and a C-terminal extension, which is intrinsically disordered and conserved in vertebrates. We used recombinant derivatives of RPSA and its N- and C-domains to quantify its interactions with ligands by in-vitro immunochemical and spectrofluorimetric methods. Both N- and C-domains bound laminin with K(D) (dissociation constants) of 300 nM. Heparin bound only to the N-domain and competed for binding to laminin with the negatively charged C-domain, which therefore mimicked heparin. EGCG bound only to the N-domain with a K(D) of 100 nM. Domain 3 of the envelope protein from yellow fever virus and serotypes-1 and -2 of dengue virus bound preferentially to the C-domain whereas that from West Nile virus bound only to the N-domain. Our quantitative in-vitro approach should help clarify the mechanisms of action of RPSA, and ultimately fight against cancer and infectious agents.

  16. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice

    PubMed Central

    Godin, Lindsay M.; Sandri, Brian J.; Wagner, Darcy E.; Meyer, Carolyn M.; Price, Andrew P.; Akinnola, Ifeolu; Weiss, Daniel J.; Panoskaltsis-Mortari, Angela

    2016-01-01

    The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases. PMID:26954258

  17. The effect of microstructured surfaces and laminin-derived peptide coatings on soft tissue interactions with titanium dental implants.

    PubMed

    Werner, Sandra; Huck, Olivier; Frisch, Benoît; Vautier, Dominique; Elkaim, René; Voegel, Jean-Claude; Brunel, Gérard; Tenenbaum, Henri

    2009-04-01

    In the present study, we investigated the dental implant protection from peri-implant inflammation by improving the soft tissue adhesion on the titanium surface. Porous titanium was used to create, at the level of the transmucosal part of the implants (the "neck"), a microstructured 3-dimensional surface that would tightly seal the interface between the implant and soft tissue. Cell-specific adhesion properties were induced via an adhesion peptide derived from laminin-5 coupled to native or cross-linked PLL/PGA multilayered polyelectrolyte films (MPFs), which are used for biomedical device coatings. Porous titanium exhibited good cell-adhesion properties, but the colonisation of the material was further improved by a coating with laminin-5 functionalised MPFs and especially with (PLL/PGA)(6,5)-PGA-peptide film. Focal contact formation was observed on cross-linked architectures, reflecting cell anchorage on these surfaces. In contrast, when seeded on laminin-5-functionalised native films, epithelial cells formed only very diffuse focal contacts, but adhered via hemidesmosome formation. In vivo experiments confirmed that the porous titanium was colonised by cells of soft tissue. Altogether, the results indicate that the microstructure of the implant neck combined with a specific bioactive coating could constitute efficient routes to improve the integration of soft tissue on titanium dental implants, which could significantly protect implants from peri-implant inflammation and enhance long-term implant stabilisation.

  18. Studies on the structure and biological function of the carbohydrate moiety of the extracellular matrix component laminin

    SciTech Connect

    Knibbs, R.N.

    1987-01-01

    Laminin from the EHS murine tumor was isolated as a noncovalent complex with entactin by lectin affinity chromatography using the ..cap alpha..-D-galactosyl binding lectin Griffonia simplicifolia I (GS I). Entactin was removed from this complex by passage over Sephacryl S-1000 in the presence of SDS. Laminin was found to contain 25-30% carbohydrate by weight. Free oligosaccharides were derived from the asparagine-linked glycans of affinity-purified laminin by hydrazinolysis, followed by re-N-acetylation and reduction with NaB(/sup 3/H)/sub 4/. The oligosaccharides were fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose. A single peak corresponding to 20% of the total radioactivity was adsorbed, and specifically eluted with a linear gradient of 0-30 mM methyl ..cap alpha..-D-glucopyranoside. No additional material was eluted by 100 mM methyl ..cap alpha..-D-mannopyranoside. Further fractionation of the Con A-reactive oligosaccharides on GS I-Sepharose demonstrated that 70% of these oligosaccharides possess at least one terminal nonreducing ..cap alpha..-D-galactopyranosyl unit.

  19. Human Mesenchymal Cells from Adipose Tissue Deposit Laminin and Promote Regeneration of Injured Spinal Cord in Rats

    PubMed Central

    Menezes, Karla; Nascimento, Marcos Assis; Gonçalves, Juliana Pena; Cruz, Aline Silva; Lopes, Daiana Vieira; Curzio, Bianca; Bonamino, Martin; de Menezes, João Ricardo Lacerda; Borojevic, Radovan; Rossi, Maria Isabel Doria; Coelho-Sampaio, Tatiana

    2014-01-01

    Cell therapy is a promising strategy to pursue the unmet need for treatment of spinal cord injury (SCI). Although several studies have shown that adult mesenchymal cells contribute to improve the outcomes of SCI, a descripton of the pro-regenerative events triggered by these cells is still lacking. Here we investigated the regenerative properties of human adipose tissue derived stromal cells (hADSCs) in a rat model of spinal cord compression. Cells were delivered directly into the spinal parenchyma immediately after injury. Human ADSCs promoted functional recovery, tissue preservation, and axonal regeneration. Analysis of the cord tissue showed an abundant deposition of laminin of human origin at the lesion site and spinal midline; the appearance of cell clusters composed of neural precursors in the areas of laminin deposition, and the appearance of blood vessels with separated basement membranes along the spinal axis. These effects were also observed after injection of hADSCs into non-injured spinal cord. Considering that laminin is a well-known inducer of axonal growth, as well a component of the extracellular matrix associated to neural progenitors, we propose that it can be the paracrine factor mediating the pro-regenerative effects of hADSCs in spinal cord injury. PMID:24830794

  20. Immunohistochemical Analysis of Collagen IV and Laminin Expression in Spontaneous Melanoma Regression in the Melanoma-Bearing Libechov Minipig.

    PubMed

    Planska, Daniela; Burocziova, Monika; Strnadel, Jan; Horak, Vratislav

    2015-01-01

    Spontaneous regression (SR) of human melanoma is a rare, well-documented phenomenon that is not still fully understood. Its detailed study cannot be performed in patients due to ethical reasons. Using the Melanoma-bearing Libechov Minipig (MeLiM) animals of various ages (from 3 weeks to 8 months) we implemented a long-term monitoring of melanoma growth and SR. We focused on immunohistochemical detection of two important extracellular matrix proteins, collagen IV and laminin, which are associated with cancer. We showed that SR of melanoma is a highly dynamic process. The expression of collagen IV and laminin correlated with changes in population of melanoma cells. Tumours of 3-week-old animals consisted primarily of melanoma cells with a granular expression of collagen IV and laminin around them. Thereafter, melanoma cells were gradually destroyed and tumour tissue was rebuilt into the connective tissue. Collagen IV expression slightly increased in tumours of 10-week-old pigs showing extracellular fibrous appearance. In tumours of older animals, areas lacking melanoma cells demonstrated a low expression and areas still containing melanoma cells a high expression of both proteins. We considered the age of 10 weeks as a turning point in the transition between tumour growth and SR of the MeLiM melanoma.

  1. Human mesenchymal cells from adipose tissue deposit laminin and promote regeneration of injured spinal cord in rats.

    PubMed

    Menezes, Karla; Nascimento, Marcos Assis; Gonçalves, Juliana Pena; Cruz, Aline Silva; Lopes, Daiana Vieira; Curzio, Bianca; Bonamino, Martin; de Menezes, João Ricardo Lacerda; Borojevic, Radovan; Rossi, Maria Isabel Doria; Coelho-Sampaio, Tatiana

    2014-01-01

    Cell therapy is a promising strategy to pursue the unmet need for treatment of spinal cord injury (SCI). Although several studies have shown that adult mesenchymal cells contribute to improve the outcomes of SCI, a description of the pro-regenerative events triggered by these cells is still lacking. Here we investigated the regenerative properties of human adipose tissue derived stromal cells (hADSCs) in a rat model of spinal cord compression. Cells were delivered directly into the spinal parenchyma immediately after injury. Human ADSCs promoted functional recovery, tissue preservation, and axonal regeneration. Analysis of the cord tissue showed an abundant deposition of laminin of human origin at the lesion site and spinal midline; the appearance of cell clusters composed of neural precursors in the areas of laminin deposition, and the appearance of blood vessels with separated basement membranes along the spinal axis. These effects were also observed after injection of hADSCs into non-injured spinal cord. Considering that laminin is a well-known inducer of axonal growth, as well a component of the extracellular matrix associated to neural progenitors, we propose that it can be the paracrine factor mediating the pro-regenerative effects of hADSCs in spinal cord injury.

  2. The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

    PubMed

    Miner, J H; Patton, B L; Lentz, S I; Gilbert, D J; Snider, W D; Jenkins, N A; Copeland, N G; Sanes, J R

    1997-05-01

    Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously

  3. Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation

    PubMed Central

    Balaoing, Liezl Rae; Post, Allison Davis; Lin, Adam Yuh; Tseng, Hubert; Moake, Joel L.; Grande-Allen, K. Jane

    2015-01-01

    Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion

  4. Promoting differentiation of cultured myoblasts using biomimetic surfaces that present alpha-laminin-2 peptides.

    PubMed

    Parker, Francine; White, Kathryn; Phillips, Siȏn; Peckham, Michelle

    2016-10-01

    Traditionally, muscle cell lines are cultured on glass coverslips and differentiated to investigate myoblast fusion and differentiation. Efficient differentiation of myoblasts produces a dense network of myotubes with the correct organisation for contraction. Here we have tested the ability of artificially generated, precisely controlled peptide surfaces to enhance the efficiency of myoblast differentiation. We focused on specific short peptides from α-laminin-2 (IKVSV, VQLRNGFPYFSY and GLLFYMARINHA) as well as residues 15-155 from FGF1. We tested if these peptides in isolation, and/or in combination promoted muscle differentiation in culture, by promoting fusion and/or by improving sarcomere organisation. The majority of these peptides promoted fusion and differentiation in two different mouse myogenic cell lines and in primary human myoblasts. The additive effects of all four peptides gave the best results for both mouse cell lines tested, while primary human cell cultures differentiated equally well on most peptide surfaces tested. These data show that a mixture of short biomimetic peptides can reliably promote differentiation in mouse and human myoblasts. PMID:27507643

  5. Suppression of laminin-5 expression leads to increased motility, tumorigenicity, and invasion

    SciTech Connect

    Yuen Hengwai; Ziober, Amy F.; Gopal, Pallavi; Nasrallah, Ilya; Falls, Erica M.; Meneguzzi, Guerrino; Ang, Hwee-Quan; Ziober, Barry L. . E-mail: bziober@mail.med.upenn.edu

    2005-09-10

    Laminin-5 (Ln-5) is expressed in several human carcinomas and hypothesized to contribute to tumor invasion. To understand the role of Ln-5 in human cancers, we stably delivered small interfering RNAs (siRNAs) directed against the Ln-5 {gamma}2 chain into JHU-022-SCC cells (022), a non-invasive oral squamous cell carcinoma (OSCC) cell line which secretes Ln-5. Lysates from {gamma}2 siRNA cells (022-si{gamma}2) had nearly undetectable levels of the {gamma}2 chain while the {alpha}3 and {beta}3 subunits of Ln-5 remained unchanged compared to parental and control. In conditioned medium from 022-si{gamma}2 cells, the {gamma}2 chain and the Ln-5 heterotrimer were barely detectable, similar to an invasive OSCC cell line. Conditioned medium from 022-si{gamma}2 cells contained less {alpha}3 and {beta}3 subunits than both parental and control. Although the proliferation and adhesive properties of the 022-si{gamma}2 cells remained similar to parental and control cells, 022-si{gamma}2 cells showed increased detachment and a fibroblastic morphology similar to invasive cells. Moreover, migration, in vitro invasion, and in vivo tumorigenicity were enhanced in 022-si{gamma}2 cells. Our results suggest that the Ln-5 {gamma}2 chain regulates the secretion of the {alpha}3 and {beta}3 subunits. More importantly, suppression of Ln-5 results in a phenotype that is representative of invasive tumor cells.

  6. Omigapil ameliorates the pathology of muscle dystrophy caused by laminin-alpha2 deficiency.

    PubMed

    Erb, Michael; Meinen, Sarina; Barzaghi, Patrizia; Sumanovski, Lazar T; Courdier-Früh, Isabelle; Rüegg, Markus A; Meier, Thomas

    2009-12-01

    Laminin alpha2-deficient congenital muscular dystrophy, called MDC1A, is a rare, devastating genetic disease characterized by severe neonatal hypotonia ("floppy infant syndrome"), peripheral neuropathy, inability to stand or walk, respiratory distress, and premature death in early life. Transgenic overexpression of the apoptosis inhibitor protein BCL-2, or deletion of the proapoptotic Bax gene in a mouse model for MDC1A prolongs survival and mitigates pathology, indicating that apoptotic events are involved in the pathology. Here we demonstrate that the proapoptotic glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-Siah1-CBP/p300-p53 pathway is activated in a mouse model for MDC1A. Moreover, we show that omigapil, which inhibits GAPDH-Siah1-mediated apoptosis, ameliorates several pathological hallmarks in the MDC1A mouse model. Specifically, we demonstrate that treatment with omigapil inhibits apoptosis in muscle, reduces body weight loss and skeletal deformation, increases locomotive activity, and protects from early mortality. These data qualify omigapil, which is in late phase of clinical development for human use, as a drug candidate for the treatment of MDC1A.

  7. Molecular characterization of a laminin-derived oligopeptide with implications in biomimetic applications.

    PubMed

    Makohliso, S A; Melchionna, S

    2001-02-15

    The molecular properties of the laminin-derived oligopeptide, H-CDPGYIGSR-NH2, have been investigated with the aid of a tandem computer simulation/experimental approach. The simulation studies placed a particular emphasis on studying the oligopeptide in aqueous media, as well as in a grafted or immobilized state. The simulations revealed the presence of a stable double hydrogen bond between arginine (R) and aspartic acid (D) residues. The mutation of the terminal arginine with lysine, another hydrophilic and positively charged amino acid, resulted in a drastic structural change, thus suggesting a major role of the terminal arginine residue in the overall oligopeptide's conformation and, hence, its bioactivity. In addition, the involvement of the aspartic acid residue in overall peptide structural stabilization also illustrates a previously undetermined role for this region (i.e. CDPG) of the oligopeptide. A subsequent in vitro experiment demonstrated a significant loss of bioactivity upon mutating the terminal residue from arginine to lysine, thereby corroborating the overall findings of the computational model. PMID:11254207

  8. Identification of a fibronectin receptor specific for rat liver endothelial cells

    SciTech Connect

    Johansson, S.; Gustafson, S.; Pertoft, H. )

    1987-10-01

    Antibodies raised against the fibronectin receptor of rat hepatocytes recognized one protein in immunoblotting of solubilized rat liver endothelial cells (LEC). The antibodies specifically precipitated a 200-kDa protein together with the 135-kDa component from {sup 125}I-labeled LEC. Spreading of LEC on fibronectin, but not on laminin or collagen, was inhibited by monovalent Fab fragments of the antibodies, implicating that the 135/200-kDa complex is a specific fibronectin receptor. The results indicate that LEC, hepatocytes, and fibroblasts of rat carry different fibronectin receptors, suggesting that the interaction of fibronectin with these cells may have different functional roles.

  9. Basic components of connective tissues and extracellular matrix: elastin, fibrillin, fibulins, fibrinogen, fibronectin, laminin, tenascins and thrombospondins.

    PubMed

    Halper, Jaroslava; Kjaer, Michael

    2014-01-01

    Collagens are the most abundant components of the extracellular matrix and many types of soft tissues. Elastin is another major component of certain soft tissues, such as arterial walls and ligaments. Many other molecules, though lower in quantity, function as essential components of the extracellular matrix in soft tissues. Some of these are reviewed in this chapter. Besides their basic structure, biochemistry and physiology, their roles in disorders of soft tissues are discussed only briefly as most chapters in this volume deal with relevant individual compounds. Fibronectin with its muldomain structure plays a role of "master organizer" in matrix assembly as it forms a bridge between cell surface receptors, e.g., integrins, and compounds such collagen, proteoglycans and other focal adhesion molecules. It also plays an essential role in the assembly of fibrillin-1 into a structured network. Laminins contribute to the structure of the extracellular matrix (ECM) and modulate cellular functions such as adhesion, differentiation, migration, stability of phenotype, and resistance towards apoptosis. Though the primary role of fibrinogen is in clot formation, after conversion to fibrin by thrombin, it also binds to a variety of compounds, particularly to various growth factors, and as such fibrinogen is a player in cardiovascular and extracellular matrix physiology. Elastin, an insoluble polymer of the monomeric soluble precursor tropoelastin, is the main component of elastic fibers in matrix tissue where it provides elastic recoil and resilience to a variety of connective tissues, e.g., aorta and ligaments. Elastic fibers regulate activity of TGFβs through their association with fibrillin microfibrils. Elastin also plays a role in cell adhesion, cell migration, and has the ability to participate in cell signaling. Mutations in the elastin gene lead to cutis laxa. Fibrillins represent the predominant core of the microfibrils in elastic as well as non

  10. Novel mutations in the LAMC2 gene in non-Herlitz junctional epidermolysis bullosa: effects on laminin-5 assembly, secretion, and deposition.

    PubMed

    Castiglia, D; Posteraro, P; Spirito, F; Pinola, M; Angelo, C; Puddu, P; Meneguzzi, G; Zambruno, G

    2001-09-01

    Laminin-5 is the major adhesion ligand of epithelial cells. Mutations in the three genes (LAMA3, LAMB3, LAMC2) encoding the laminin-5 chains cause junctional epidermolysis bullosa, a clinically and genetically heterogeneous blistering skin disease. Here, we describe a non-Herlitz junctional epidermolysis bullosa patient, compound heterozygote for two novel mutations affecting the LAMC2 gene. The mutation in the paternal allele is a de novo splice site mutation (522-1G-->A) that results in in-frame skipping of exon 4 and synthesis of a mutated gamma2 polypeptide (gamma2Delta4) carrying a 33 amino acid deletion within the N-terminal domain V. The maternal mutation is a one base pair insertion (3511insA) in the 3' terminal exon of LAMC2 resulting in a frameshift and a premature termination codon. Mutation 3511insA is predicted to lead to the synthesis of a gamma2 polypeptide (gamma2t) disrupted in its alpha-helical C-terminal structure and truncated of the last 25 amino acids. Keratinocytes isolated from the patient's skin showed a markedly decreased level of gamma2 chain mRNA and secreted scant amounts of laminin-5, which undergoes physiologic proteolytic processing. To investigate the biologic function of the laminin-5 molecules synthesized by the patient, mutant gamma2 cDNAs were transiently expressed in gamma2-null keratinocytes. Transfection of the gamma2Delta4 cDNA resulted in restoration of laminin-5 deposition onto the culture substrate, which demonstrates that the gamma2 polypeptides carrying a deletion in domain V, upstream of the gamma2 proteolytic cleavage site, are assembled into native laminin-5 that is secreted and extracellularly processed. In contrast, transfection of a mutant cDNA expressing the gamma2t chain failed to restore laminin-5 immunoreactivity, which indicates that integrity of the gamma2 C-terminal amino acid sequences is required for laminin-5 assembly. These results correlate for the first time a functional alteration in a laminin-5

  11. Bortezomib Does Not Reduce Muscular Dystrophy in the dy2J/dy2J Mouse Model of Laminin α2 Chain-Deficient Muscular Dystrophy.

    PubMed

    Körner, Zandra; Durbeej, Madeleine

    2016-01-01

    Congenital muscular dystrophy with laminin α2 chain-deficiency, also known as MDC1A, is a severe neuromuscular disorder for which there is no cure. Patients with complete laminin α2 chain-deficiency typically have an early onset disease with a more severe muscle phenotype while patients with residual laminin α2 chain expression usually have a milder disease course. Similar genotype-phenotype correlations can be seen in the dy3K/dy3K and dy2J/dy2J mouse models of MDC1A, respectively, with dy3K/dy3K mice presenting the more severe phenotype. Recently, we demonstrated that the proteasome inhibitor bortezomib partially improves muscle morphology and increases lifespan in dy3K/dy3K mice. Here, we explore the use of bortezomib in dy2J/dy2J animals. However, bortezomib neither improved histological hallmarks of disease nor increased muscle strength and locomotive activity in dy2J/dy2J mice. Altogether our data suggest that proteasome inhibition does not mitigate muscle dysfunction caused by partial laminin α2 chain-deficiency. Still, it is possible that proteasome inhibition could be useful as a supportive therapy in patients with complete absence of laminin α2 chain.

  12. Laminin-binding integrin gene copy number alterations in distinct epithelial-type cancers

    PubMed Central

    Harryman, William L; Pond, Erika; Singh, Parminder; Little, Andrew S; Eschbacher, Jennifer M; Nagle, Raymond B; Cress, Anne E

    2016-01-01

    Background: The laminin-binding integrin (LBI) family are cell adhesion molecules that are essential for invasion and metastasis of human epithelial cancers and cell adhesion mediated drug resistance. We investigated whether copy number alteration (CNA) or mutations of a five-gene signature (ITGB4, ITGA3, LAMB3, PLEC, and SYNE3), representing essential genes for LBI adhesion, would correlate with patient outcomes within human epithelial-type tumor data sets currently available in an open access format. Methods: We investigated the relative alteration frequency of an LBI signature panel (integrin β4 (ITGB4), integrin α3 (ITGA3), laminin β3 chain (LAMB3), plectin (PLEC), and nesprin 3 (SYNE3)), independent of the epithelial cancer type, within publically available and published data using cBioPortal and Oncomine software. We rank ordered the results using a 20% alteration frequency cut-off and limited the analysis to studies containing at least 100 samples. Kaplan-Meier survival curves were analyzed to determine if alterations in the LBI signature correlated with patient survival. The Oncomine data mining tool was used to compare the heat map expression of the LBI signature without SYNE3 (as this was not included in the Oncomine database) to drug resistance patterns. Results: Twelve different cancer types, representing 5,647 samples, contained at least a 20% alteration frequency of the five-gene LBI signature. The frequency of alteration ranged from 38.3% to 19.8%. Within the LBI signature, PLEC was the most commonly altered followed by LAMB3, ITGB4, ITGA3, and SYNE3 across all twelve cancer types. Within cancer types, there was little overlap of the individual amplified genes from each sample, suggesting different specific amplicons may alter the LBI adhesion structures. Of the twelve cancer types, overall survival was altered by CNA presence in bladder urothelial carcinoma (p=0.0143*) and cervical squamous cell carcinoma and endocervical adenocarcinoma (p=0

  13. Tumour necrosis factor-alpha interacts with laminin and functions as a pro-adhesive cytokine.

    PubMed

    Hershkoviz, R; Goldkorn, I; Lider, O

    1995-05-01

    Certain cytokines, chemokines and growth factors interact with components of the extracellular matrix (ECM) and, in particular, sulphated polysaccharides and proteoglycans. Recently, we demonstrated that tumour necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, can bind fibronectin (FN), a cell-adhesive glycoprotein of the ECM, and that TNF-alpha bound to FN enhances the binding of T cells to the glycoprotein. In the present study, we studied the interactions of TNF-alpha and laminin (LN), another glycoprotein present in basement membranes and extracellular matrices. 125I-labelled TNF-alpha was found to bind to immobilized LN, and more avidly to the E1 and P1 fragments of LN, which contain its integrin- and non-integrin-dependent cell-adhesive sites, suggesting that cryptic TNF-alpha-binding sites are exposed upon proteolytic fragmentation of LN by enzymes such as elastase or pepsin. The bound cytokine did not dissociate from the LN and its fragments during a 24-hr period, indicating that in vivo LN can serve to restrict TNF-alpha adjacent to inflammatory sites. The LN-associated TNF-alpha retained at least some of its biological activities, since both diffusible and, to a greater extent, LN-bound TNF-alpha elevated the beta 1-integrin-dependent adhesion to LN of phorbol ester-activated human CD4+ T cells. Thus, LN and TNF-alpha may act in concert to transmit synergistic activating signals to infiltrating leucocytes, and thereby regulate immune cell reactions in extravascular inflammatory tissue. PMID:7635514

  14. Epoxy Cross-Linked Collagen and Collagen-Laminin Peptide Hydrogels as Corneal Substitutes

    PubMed Central

    Koh, Li Buay; Islam, Mohammad Mirazul; Mitra, Debbie; Noel, Christopher W.; Merrett, Kimberley; Odorcic, Silvia; Fagerholm, Per; Jackson, William. Bruce; Liedberg, Bo; Phopase, Jaywant; Griffith, May

    2013-01-01

    A bi-functional epoxy-based cross-linker, 1,4-Butanediol diglycidyl ether (BDDGE), was investigated in the fabrication of collagen based corneal substitutes. Two synthetic strategies were explored in the preparation of the cross-linked collagen scaffolds. The lysine residues of Type 1 porcine collagen were directly cross-linked using l,4-Butanediol diglycidyl ether (BDDGE) under basic conditions at pH 11. Alternatively, under conventional methodology, using both BDDGE and 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) as cross-linkers, hydrogels were fabricated under acidic conditions. In this latter strategy, Cu(BF4)2·XH2O was used to catalyze the formation of secondary amine bonds. To date, we have demonstrated that both methods of chemical cross-linking improved the elasticity and tensile strength of the collagen implants. Differential scanning calorimetry and biocompatibility studies indicate comparable, and in some cases, enhanced properties compared to that of the EDC/NHS controls. In vitro studies showed that human corneal epithelial cells and neuronal progenitor cell lines proliferated on these hydrogels. In addition, improvement of cell proliferation on the surfaces of the materials was observed when neurite promoting laminin epitope, IKVAV, and adhesion peptide, YIGSR, were incorporated. However, the elasticity decreased with peptide incorporation and will require further optimization. Nevertheless, we have shown that epoxy cross-linkers should be further explored in the fabrication of collagen-based hydrogels, as alternatives to or in conjunction with carbodiimide cross-linkers. PMID:24956085

  15. Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

    PubMed

    Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2010-01-01

    Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis.

  16. Nanoscale laminin coating modulates cortical scarring response around implanted silicon microelectrode arrays

    NASA Astrophysics Data System (ADS)

    He, Wei; McConnell, George C.; Bellamkonda, Ravi V.

    2006-12-01

    Neural electrodes could significantly enhance the quality of life for patients with sensory and/or motor deficits as well as improve our understanding of brain functions. However, long-term electrical connectivity between neural tissue and recording sites is compromised by the development of astroglial scar around the recording probes. In this study we investigate the effect of a nanoscale laminin (LN) coating on Si-based neural probes on chronic cortical tissue reaction in a rat model. Tissue reaction was evaluated after 1 day, 1 week, and 4 weeks post-implant for coated and uncoated probes using immunohistochemical techniques to evaluate activated microglia/macrophages (ED-1), astrocytes (GFAP) and neurons (NeuN). The coating did not have an observable effect on neuronal density or proximity to the electrode surface. However, the response of microglia/macrophages and astrocytes was altered by the coating. One day post-implant, we observed an ~60% increase in ED-1 expression near LN-coated probe sites compared with control uncoated probe sites. Four weeks post-implant, we observed an ~20% reduction in ED-1 expression along with an ~50% reduction in GFAP expression at coated relative to uncoated probe sites. These results suggest that LN has a stimulatory effect on early microglia activation, accelerating the phagocytic function of these cells. This hypothesis is further supported by the increased mRNA expression of several pro-inflammatory cytokines (TNF-α, IL-1 and IL-6) in cultured microglia on LN-bound Si substrates. LN immunostaining of coated probes immediately after insertion and retrieval demonstrates that the coating integrity is not compromised by the shear force during insertion. We speculate, based on these encouraging results, that LN coating of Si neural probes could potentially improve chronic neural recordings through dispersion of the astroglial scar.

  17. Ubiquitin-like epitopes associated with Candida albicans cell surface receptors.

    PubMed Central

    Sepulveda, P; Lopez-Ribot, J L; Gozalbo, D; Cervera, A; Martinez, J P; Chaffin, W L

    1996-01-01

    We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures. PMID:8926122

  18. A receptor tyrosine kinase, UFO/Axl, and other genes isolated by a modified differential display PCR are overexpressed in metastatic prostatic carcinoma cell line DU145.

    PubMed

    Jacob, A N; Kalapurakal, J; Davidson, W R; Kandpal, G; Dunson, N; Prashar, Y; Kandpal, R P

    1999-01-01

    We have used a modified differential display PCR protocol for isolating 3' restriction fragments of cDNAs specifically expressed or overexpressed in metastatic prostate carcinoma cell line DU145. Several cDNA fragments were identified that matched to milk fat globule protein, UFO/Axl, a receptor tyrosine kinase, human homologue of a Xenopus maternal transcript, laminin and laminin receptor, human carcinoma-associated antigen, and some expressed sequence tags. The transcript for milk fat globule protein, a marker protein shown to be overexpressed in breast tumors, was elevated in DU145 cells. The expression of UFO/Axl, a receptor tyrosine kinase, was considerably higher in DU145 cells as compared to normal prostate cells and prostatic carcinoma cell line PC-3. The overexpression of UFO oncogene in DU145 cells is discussed in the context of prostate cancer metastasis.

  19. Biosensor studies of collagen and laminin binding with immobilized Escherichia coli O157:H7 and inhibition with naturally occurring food additives

    NASA Astrophysics Data System (ADS)

    Medina, Marjorie B.

    1999-01-01

    Escherichia coli O157:H7 outbreaks were mostly due to consumption of undercooked contaminated beef which resulted in severe illness and several fatalities. Recalls of contaminated meat are costly for the meat industry. Our research attempts to understand the mechanisms of bacterial adhesion on animal carcass in order to eliminate or reduce pathogens in foods. We have reported the interactions of immobilized E. coli O157:H7 cells with extracellular matrix (ECM) components using a surface plasmon resonance biosensor (BIAcore). These studies showed that immobilized bacterial cells allowed the study of real-time binding interactions of bacterial surface with the ECM compounds, collagen I, laminin and fibronectin. Collagen I and laminin bound to the E. coli sensor surface with dissociation and association rates ranging from 106 to 109. Binding of collagen I and laminin mixture resulted in synergistic binding signals. An inhibition model was derived using collagen-laminin as the ligand which binds with E. coli sensor. A select group of naturally occurring food additives was evaluated by determining their effectivity in inhibiting the collagen-laminin binding to the bacterial sensor. Bound collagen-laminin was detached from the E. coli sensor surface with the aid of an organic acid. The biosensor results were verified with cell aggregation assays which were observed with optical and electron microscopes. These biosensor studies provided understanding of bacterial adhesion to connective tissue macromolecules. It also provided a model system for the rapid assessment of potential inhibitors that can be used in carcass treatment to inhibit or reduce bacterial contamination.

  20. A High Throughput Screen Identifies Chemical Modulators of the Laminin-Induced Clustering of Dystroglycan and Aquaporin-4 in Primary Astrocytes

    PubMed Central

    Noël, Geoffroy; Stevenson, Sarah; Moukhles, Hakima

    2011-01-01

    Background Aquaporin-4 (AQP4) constitutes the principal water channel in the brain and is clusteredat the perivascular astrocyte endfeet. This specific distribution of AQP4 plays a major role in maintaining water homeostasis in the brain. A growing body of evidence points to a role ofthe dystroglycan complex and its interaction with perivascular laminin in the clusteringof AQP4 atperivascular astrocyte endfeet. Indeed, mice lacking components of this complex or in which laminin-dystroglycan interaction is disrupted show a delayed onset of brain edema due to a redistribution of AQP4 away from astrocyte endfeet. It is therefore important to identify inhibitory drugs of laminin-dependent AQP4 clustering which may prevent or reduce brain edema. Methodolgy/Principal Findings In the present study we used primary rat astrocyte cultures toscreen a library of >3,500 chemicals and identified 6 drugs that inhibit the laminin-induced clustering of dystroglycan and AQP4. Detailed analysis of the inhibitory drug, chloranil, revealed that its inhibition of the clustering is due to the metalloproteinase-2-mediated ß-dystroglycan shedding and subsequent loss of laminin interaction with dystroglycan. Furthermore, chemical variants of chloranil induced a similar effect on ß-dystroglycan and this was prevented by the antioxidant N-acetylcysteine. Conclusion/Significance These findings reveal the mechanism of action of chloranil in preventing the laminin-induced clustering of dystroglycan and AQP4 and validate the use of high-throughput screening as a tool to identify drugs that modulate AQP4 clustering and that could be tested in models of brain edema. PMID:21408176

  1. Inhibition of hair follicle growth by a laminin-1 G-domain peptide, RKRLQVQLSIRT, in an organ culture of isolated vibrissa rudiment.

    PubMed

    Hayashi, Kazuhiro; Mochizuki, Mayumi; Nomizu, Motoyoshi; Uchinuma, Eijyu; Yamashina, Shohei; Kadoya, Yuichi

    2002-04-01

    We established a serum-free organ culture system of isolated single vibrissa rudiments taken from embryonic day 13 mice. This system allowed us to test more than 30 laminin-derived cell adhesive peptides to determine their roles on the growth and differentiation of vibrissa hair follicles. We found that the RKRLQVQLSIRT sequence (designated AG-73), which mapped to the LG-4 module of the laminin-alpha1 chain carboxyl-terminal G domain, perturbed the growth of hair follicles in vitro. AG-73 is one of the cell-binding peptides identified from more than 600 systematically synthesized 12 amino acid peptides covering the whole amino acid sequence of the laminin-alpha1, -beta1, and -gamma1 chains, by cell adhesion assay. Other cell-adhesive laminin peptides and a control scrambled peptide, LQQRRSVLRTKI, however, failed to show any significant effects on the growth of hair follicles. The AG-73 peptide binds to syndecan-1, a transmembrane heparan-sulfate proteoglycan. Syndecan-1 was expressed in both the mesenchymal condensation and the epithelial hair peg of developing vibrissa, suggesting that AG-73 binding to the cell surface syndecan-1 perturbed the epithelial-mesenchymal interactions of developing vibrissa. The formation of hair bulbs was aberrant in the explants treated with AG-73. In addition, impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in the AG-73-treated-hair matrix epithelium, indicating that AG-73 binding perturbs various steps of epithelial morphogenesis, including the basement membrane remodeling. We also found a region-specific loss of the laminin-alpha1 chain in the basement membrane at the distal region of the invading hair follicle epithelium, indicating that laminins play a part in hair morphogenesis.

  2. Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices: Role of astrocytes and extracellular matrix proteins.

    PubMed

    Giordano, Gennaro; Guizzetti, Marina; Dao, Khoi; Mattison, Hayley A; Costa, Lucio G

    2011-12-01

    In an in vitro co-culture system of astrocytes and neurons, stimulation of cholinergic muscarinic receptors in astrocytes had been shown to cause neuritogenesis in hippocampal neurons, and this effect was inhibited by ethanol. The present study sought to confirm these earlier findings in a more complex system, in vitro rat hippocampal slices in culture. Exposure of hippocampal slices to the cholinergic agonist carbachol (1mM for 24h) induced neurite outgrowth in hippocampal pyramidal neurons, which was mediated by activation of muscarinic M3 receptors. Specifically, carbachol induced a >4-fold increase in the length of the longest neurite, and a 4-fold increase in the length of minor neurites and in the number of branches. Co-incubation of carbachol with ethanol (50mM) resulted in significant inhibition of the effects induced by carbachol on all parameters measured. Neurite outgrowth in CNS neurons is dependent on various permissive factors that are produced and released by glial cells. In hippocampal slices carbachol increased the levels of two extracellular matrix protein, fibronectin and laminin-1, by 1.6-fold, as measured by Western blot. Co-incubation of carbachol with ethanol significantly inhibited these increases. Carbachol-induced increases in levels of extracellular matrix proteins were antagonized by a M3 muscarinic receptor antagonist. Furthermore, function-blocking fibronectin or laminin-1 antibodies antagonized the effect of carbachol on neurite outgrowth. These results indicate that in hippocampal slices stimulation of muscarinic M3 receptors induces neurite outgrowth, which is mediated by fibronectin and laminin-1, two extracellular matrix proteins released by astrocytes. By decreasing fibronectin and laminin levels ethanol prevents carbachol-induced neuritogenesis. These findings highlight the importance of glial-neuronal interactions as important targets in the developmental neurotoxicity of alcohol.

  3. Alpha-galactosidase stimulates acetylcholine receptor aggregation in skeletal muscle cells via PNA-binding carbohydrates.

    PubMed

    Parkhomovskiy, N; Martin, P T

    2000-04-21

    Aggregation of nicotinic acetylcholine receptors (AChRs) in skeletal muscle is an essential step in the formation of the mammalian neuromuscular junction. While proteins that bind to myotube receptors such as agrin and laminin can stimulate AChR aggregation in cultured myotubes, removal of cell surface sialic acids stimulates aggregation in a ligand-independent manner. Here, we show that removal of cell surface alpha-galactosides also stimulates AChR aggregation in the absence of added laminin or agrin. AChR aggregation stimulated by alpha-galactosidase was blocked by peanut agglutinin (PNA), which binds to lactosamine-containing disaccharides, but not by the GalNAc-binding lectin Vicia villosa agglutinin (VVA-B4). AChR aggregation stimulated by alpha-galactosidase potentiated AChR clustering induced by either neural agrin or laminin-1 and could be inhibited by muscle agrin. These data suggest that capping of cell surface lactosamines or N-acetyllactosamines with alpha-galactose affects AChR aggregation much as capping with sialic acids does.

  4. Detection of sequence variants in the gene encoding the beta 3 chain of laminin 5 (LAMB3).

    PubMed

    Pulkkinen, L; McGrath, J A; Christiano, A M; Uitto, J

    1995-01-01

    Laminin 5, a candidate gene/protein system for mutations in the junctional forms of epidermolysis bullosa (JEB), consists of three polypeptides encoded by the LAMA3, LAMB3, and LAMC2 genes. In this study, primer pairs for the amplification of the complete cDNA as well as 22 exons of the LAMB3 gene encoding the entire beta 3 chain of laminin 5, were established. The primers for amplification of individual exons from genomic DNA were placed at least 50 bp away from the exon-intron borders in the flanking intronic sequences. For amplification of cDNA generated by RT-PCR, eight primer pairs covering overlapping segments of mRNA were used. The amplified sequences were used to study sequence variations of the LAMB3 gene in patients with JEB and unrelated individuals using heteroduplex analysis. Nine out of 13 JEB patients examined showed heteroduplexes in at least one of the PCR products, indicating the existence of two variable alleles in their DNA. Sequence analyses revealed putative pathogenetic mutations in seven of the JEB patients, while four of the heteroduplexes resulted from polymorphisms, reflecting a single basepair substitution. The results demonstrate that this method is useful in the detection of JEB mutations, as well as polymorphisms in the LAMB3 gene. PMID:7550237

  5. Cloning of the beta 3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa.

    PubMed

    Pulkkinen, L; Gerecke, D R; Christiano, A M; Wagman, D W; Burgeson, R E; Uitto, J

    1995-01-01

    Laminin 5 consists of three polypeptides, alpha 3, beta 3, and gamma 2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. In this study, we have elucidated the exon-intron organization of the human LAMB3 gene. Characterization of five overlapping lambda phage DNA clones revealed that the gene was approximately 29 kb in size. Subsequent sequence data revealed that the gene consisted of 23 exons that varied from 64 to 379 bp in size, accounting for the full-length cDNA with an open reading frame of 3516 bp encoding 1172 amino acids. Comparison of the LAMB3 gene structure with the previously characterized LAMB1 gene revealed that LAMB3 was considerably more compact. Knowledge of the exon-intron organization of the LAMB3 gene will facilitate elucidation of mutations in patients with the junctional forms of epidermolysis bullosa, some of which have been associated with mutations in the laminin 5 genes. PMID:7774918

  6. Cloning of the {beta}3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa

    SciTech Connect

    Pulkkinen, L.; Christiano, A.M.; Uitto, J.

    1995-01-01

    Laminin 5 consists of three polypeptides, {alpha}3, {beta}3, and {gamma}2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. In this study, we have elucidated the exon-intron organization of the human LAMB3 gene. Characterization of five overlapping {lambda} phage DNA clones revealed that the gene was approximately 29 kb in size. Subsequent sequence data revealed that the gene consisted of 23 exons that varied from 64 to 379 bp in size, accounting for the full-length cDNA with an open reading frame of 3516 hp encoding 1172 amino acids. Comparison of the LAMB3 gene structure with the previously characterized LAMB1 gene revealed that LAMB3 was considerably more compact. Knowledge of the exon-intron organization of the LAMB3 gene will facilitate elucidation of mutations in patients with the junctional forms of epidermolysis bullosa, some of which have been associated with mutations in the laminin 5 genes. 33 refs., 3 figs., 2 tabs.

  7. Structural Study of Cell Attachment Peptide Derived from Laminin by Molecular Dynamics Simulation

    PubMed Central

    Yamada, Hironao; Mori, Sakiko; Miyakawa, Takeshi; Morikawa, Ryota; Katagiri, Fumihiko; Hozumi, Kentaro; Kikkawa, Yamato; Nomizu, Motoyoshi; Takasu, Masako

    2016-01-01

    Peptides with cell attachment activity are beneficial component of biomaterials for tissue engineering. Conformational structure is one of the important factors for the biological activities. The EF1 peptide (DYATLQLQEGRLHFMFDLG) derived from laminin promotes cell spreading and cell attachment activity mediated by α2β1 integrin. Although the sequence of the EF2 peptide (DFATVQLRNGFPYFSYDLG) is homologous sequence to that of EF1, EF2 does not promote cell attachment activity. To determine whether there are structural differences between EF1 and EF2, we performed replica exchange molecular dynamics (REMD) simulations and conventional molecular dynamics (MD) simulations. We found that EF1 and EF2 had β-sheet structure as a secondary structure around the global minimum. However, EF2 had variety of structures around the global minimum compared with EF1 and has easily escaped from the bottom of free energy. The structural fluctuation of the EF1 is smaller than that of the EF2. The structural variation of EF2 is related to these differences in the structural fluctuation and the number of the hydrogen bonds (H-bonds). From the analysis of H-bonds in the β-sheet, the number of H-bonds in EF1 is larger than that in EF2 in the time scale of the conventional MD simulation, suggesting that the formation of H-bonds is related to the differences in the structural fluctuation between EF1 and EF2. From the analysis of other non-covalent interactions in the amino acid sequences of EF1 and EF2, EF1 has three pairs of residues with hydrophobic interaction, and EF2 has two pairs. These results indicate that several non-covalent interactions are important for structural stabilization. Consequently, the structure of EF1 is stabilized by H-bonds and pairs of hydrophobic amino acids in the terminals. Hence, we propose that non-covalent interactions around N-terminal and C-terminal of the peptides are crucial for maintaining the β-sheet structure of the peptides. PMID:26889829

  8. Structural Study of Cell Attachment Peptide Derived from Laminin by Molecular Dynamics Simulation.

    PubMed

    Yamada, Hironao; Mori, Sakiko; Miyakawa, Takeshi; Morikawa, Ryota; Katagiri, Fumihiko; Hozumi, Kentaro; Kikkawa, Yamato; Nomizu, Motoyoshi; Takasu, Masako

    2016-01-01

    Peptides with cell attachment activity are beneficial component of biomaterials for tissue engineering. Conformational structure is one of the important factors for the biological activities. The EF1 peptide (DYATLQLQEGRLHFMFDLG) derived from laminin promotes cell spreading and cell attachment activity mediated by α2β1 integrin. Although the sequence of the EF2 peptide (DFATVQLRNGFPYFSYDLG) is homologous sequence to that of EF1, EF2 does not promote cell attachment activity. To determine whether there are structural differences between EF1 and EF2, we performed replica exchange molecular dynamics (REMD) simulations and conventional molecular dynamics (MD) simulations. We found that EF1 and EF2 had β-sheet structure as a secondary structure around the global minimum. However, EF2 had variety of structures around the global minimum compared with EF1 and has easily escaped from the bottom of free energy. The structural fluctuation of the EF1 is smaller than that of the EF2. The structural variation of EF2 is related to these differences in the structural fluctuation and the number of the hydrogen bonds (H-bonds). From the analysis of H-bonds in the β-sheet, the number of H-bonds in EF1 is larger than that in EF2 in the time scale of the conventional MD simulation, suggesting that the formation of H-bonds is related to the differences in the structural fluctuation between EF1 and EF2. From the analysis of other non-covalent interactions in the amino acid sequences of EF1 and EF2, EF1 has three pairs of residues with hydrophobic interaction, and EF2 has two pairs. These results indicate that several non-covalent interactions are important for structural stabilization. Consequently, the structure of EF1 is stabilized by H-bonds and pairs of hydrophobic amino acids in the terminals. Hence, we propose that non-covalent interactions around N-terminal and C-terminal of the peptides are crucial for maintaining the β-sheet structure of the peptides.

  9. The 67-kD elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of beta-galactosidase.

    PubMed Central

    Hinek, A; Rabinovitch, M; Keeley, F; Okamura-Oho, Y; Callahan, J

    1993-01-01

    We and others have previously shown that a 67-kD cell surface elastin/laminin-binding protein (EBP) is responsible for cell adhesion to elastin and laminin and for mediating the process of elastin fiber assembly, but the nature of this protein was unknown. In this report we provide evidence that a 67-kD catalytically inactive form of beta-galactosidase produced by alternative splicing demonstrates immunological and functional similarity and sequence homology to the 67-kD EBP, suggesting that the two might be the same. Antibody prepared to a synthetic peptide, N-Ac-GSPSAQDEASPL, corresponding to a frame-shift-generated sequence unique to the alternatively spliced form of human beta-galactosidase, also recognized sheep EBP both on Western blotting and in aortic tissue. Furthermore, this synthetic peptide (S-GAL) binds to elastin and laminin, but not to fibronectin, collagen I, or collagen III. Moreover, both tropoelastin and laminin which bind to S-GAL peptide affinity columns can be specifically eluted from them with an excess of free S-GAL peptides. In addition, sequence homology among this splice variant of human beta-galactosidase, sheep EBP, and NH2-terminal sequences of some elastases suggests that these proteins share a common ligand-binding motif that has not been previously recognized. Images PMID:8383699

  10. An unusual N-terminal deletion of the laminin alpha3a isoform leads to the chronic granulation tissue disorder laryngo-onycho-cutaneous syndrome.

    PubMed

    McLean, W H Irwin; Irvine, Alan D; Hamill, Kevin J; Whittock, Neil V; Coleman-Campbell, Carrie M; Mellerio, Jemima E; Ashton, Gabrielle S; Dopping-Hepenstal, Patricia J H; Eady, Robin A J; Jamil, Tanvir; Phillips, Roderic J; Shabbir, S Ghulam; Haroon, Tahir S; Khurshid, Khawar; Moore, Jonathan E; Page, Brian; Darling, Jonathan; Atherton, David J; Van Steensel, Maurice A M; Munro, Colin S; Smith, Frances J D; McGrath, John A; Phillips, Rodney J

    2003-09-15

    Laryngo-onycho-cutaneous (LOC or Shabbir) syndrome (OMIM 245660) is an autosomal recessive epithelial disorder confined to the Punjabi Muslim population. The condition is characterized by cutaneous erosions, nail dystrophy and exuberant vascular granulation tissue in certain epithelia, especially conjunctiva and larynx. Genome-wide homozygosity mapping localized the gene to a 2 Mb region on chromosome 18q11.2 with an LOD score of 19.8 at theta=0. This region includes the laminin alpha3 gene (LAMA3), in which loss-of-expression mutations cause the lethal skin blistering disorder Herlitz junctional epidermolysis bullosa. Detailed investigation showed that this gene possesses a further 38 exons (76 exons in total) spanning 318 kb of genomic DNA, and encodes three distinct proteins, designated laminin alpha3a, alpha3b1 and alpha3b2. The causative mutation in 15 families was a frameshift mutation 151insG predicting a stop codon 7 bp downstream in an exon that is specific to laminin alpha3a. This protein is secreted only by the basal keratinocytes of stratified epithelia, implying that LOC is caused by dysfunction of keratinocyte-mesenchymal communication. Surprisingly, the 151insG mutation does not result in nonsense-mediated mRNA decay due to rescue of the transcript by an alternative translation start site 6 exons downstream. The resultant N-terminal deletion of laminin alpha3a was confirmed by immunoprecipitation of secreted proteins from LOC keratinocytes. These studies show that the laminin alpha3a N-terminal domain is a key regulator of the granulation tissue response, with important implications not only in LOC but in a range of other clinical conditions associated with abnormal wound healing. PMID:12915477

  11. Behaviour of Human Induced Pluripotent Stem Cell-Derived Neural Progenitors on Collagen Scaffolds Varied in Freezing Temperature and Laminin Concentration

    PubMed Central

    Khayyatan, Fahimeh; Nemati, Shiva; Kiani, Sahar; Hojjati Emami, Shahriar; Baharvand, Hossein

    2014-01-01

    Objective: Biomaterial technology, when combined with emerging human induced pluripotent stem cell (hiPSC) technology, provides a promising strategy for patient-specific tissue engineering. In this study, we have evaluated the physical effects of collagen scaffolds fabricated at various freezing temperatures on the behavior of hiPSC-derived neural progenitors (hiPSC-NPs). In addition, the coating of scaffolds using different concentrations of laminin was examined on the cells. Materials and Methods: Initially, in this experimental study, the collagen scaffolds fabricated from different collagen concentrations and freezing temperatures were characterized by determining the pore size, porosity, swelling ratio, and mechanical properties. Effects of cross-linking on free amine groups, volume shrinkage and mass retention was also assessed. Then, hiPSC-NPs were seeded onto the most stable three-dimensional collagen scaffolds and we evaluated the effect of pore structure. Additionally, the different concentrations of laminin coating of the scaffolds on hiPSC-NPs behavior were assessed. Results: Scanning electron micrographs of the scaffolds showed a pore diameter in the range of 23-232 μm for the scaffolds prepared with different fabrication parameters. Also porosity of all scaffolds was >98% with more than 94% swelling ratio. hiPSC-NPs were subsequently seeded onto the scaffolds that were made by different freezing temperatures in order to assess for physical effects of the scaffolds. We observed similar proliferation, but more cell infiltration in scaffolds prepared at lower freezing temperatures. The laminin coating of the scaffolds improved NPs proliferation and infiltration in a dose-dependent manner. Immunofluorescence staining and scanning electron microscopy confirmed the compatibility of undifferentiated and differentiated hiPSC-NPs on these scaffolds. Conclusion: The results have suggested that the pore structure and laminin coating of collagen scaffolds

  12. Bidirectional signalling between EphA2 and ephrinA1 increases tubular cell attachment, laminin secretion and modulates erythropoietin expression after renal hypoxic injury.

    PubMed

    Rodriguez, Stéphane; Rudloff, Stefan; Koenig, Katrin Franziska; Karthik, Swapna; Hoogewijs, David; Huynh-Do, Uyen

    2016-08-01

    Acute kidney injury (AKI) is common in hospitalized patients and has a poor prognosis, the severity of AKI being linked to progression to chronic kidney disease. This stresses the need to search for protective mechanisms during the acute phase. We investigated kidney repair after hypoxic injury using a rat model of renal artery branch ligation, which led to an oxygen gradient vertical to the corticomedullary axis. Three distinct zones were observed: tubular necrosis, infarction border zone and preserved normal tissue. EphA2 is a receptor tyrosine kinase with pivotal roles in cell architecture, migration and survival, upon juxtacrine contact with its membrane-bound ligand EphrinA1. Following hypoxia, EphA2 was up-regulated in cortical and medullary tubular cells, while EphrinA1 was up-regulated in interstitial cells adjacent to peritubular capillaries. Moreover, erythropoietin (EPO) messenger RNA (mRNA) was strongly expressed in the border zone of infarcted kidney within the first 6 h. To gain more insight into the biological impact of EphA2 and EphrinA1 up-regulation, we activated the signalling pathways in vitro using recombinant EphrinA1/Fc or EphA2/Fc proteins. Stimulation of EphA2 forward signalling in the proximal tubular cell line HK2 increased cell attachment and laminin secretion at the baso-lateral side. Conversely, activation of reverse signalling through EphrinA1 expressed by Hep3B cells promoted EPO production at both the transcriptional and protein level. Strikingly, in co-culture experiments, juxtacrine contact between EphA2 expressing MDCK and EphrinA1 expressing Hep3B was sufficient to induce a significant up-regulation of EPO mRNA production in the latter cells, even in the absence of hypoxic conditions. The synergistic effects of EphA2 and hypoxia led to a 15-20-fold increase of EPO expression. Collectively, our results suggest an important role of EphA2/EphrinA1 signalling in kidney repair after hypoxic injury through stimulation of (i) tubular

  13. RhoA-dependent Switch between α2β1 and α3β1 Integrins Is Induced by Laminin-5 during Early Stage of HT-29 Cell Differentiation

    PubMed Central

    Gout, Stéphanie P.; Jacquier-Sarlin, Muriel R.; Rouard-Talbot, Laurence; Rousselle, Patricia; Block, Marc R.

    2001-01-01

    Integrin-mediated interactions between the basement membrane and epithelial cells control the differentiation of epithelia. We characterized the modulation of adhesive behaviors to basement membrane proteins and of integrin function in the human colon adenocarcinoma HT-29 cell line, which differentiates into enterocytes after the substitution of galactose for glucose in the medium. We demonstrate an increased capability of these cells to adhere to collagen type IV during the early stage of differentiation. This effect occurs without any changes in integrin cell surface expression but rather results from an α2β1/α3β1 integrin switch, α3β1 integrin becoming the major collagen receptor. The increase in laminin-5 secretion and deposit on the matrix is a key factor in the mechanism regulating cell adhesion, because it is responsible for the activation of α3β1 integrin. Furthermore, down-regulation of RhoA GTPase activity occurs during HT-29 cell differentiation and correlates with the activation of the integrin α3β1. Indeed, C3 transferase, a RhoA GTPase inhibitor, induces a similar α2β1/α3β1 switch in undifferentiated HT-29 cells. These results indicate that the decrease in RhoA activation is the biochemical mechanism underlying this integrin switch observed during cell differentiation. The physiological relevance of such modulation of integrin activity in the functioning of the crypt-villus axis is discussed. PMID:11598208

  14. Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors.

    PubMed

    Guizzetti, Marina; Moore, Nadia H; Giordano, Gennaro; VanDeMark, Kathryn L; Costa, Lucio G

    2010-09-01

    In utero alcohol exposure can lead to fetal alcohol spectrum disorders, characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. We have recently shown that stimulation of M(3) muscarinic receptors in astrocytes increases the synthesis and release of fibronectin, laminin, and plasminogen activator inhibitor-1, causing neurite outgrowth in hippocampal neurons. As M(3) muscarinic receptor signaling in astroglial cells is strongly inhibited by ethanol, we hypothesized that ethanol may also inhibit neuritogenesis in hippocampal neurons induced by carbachol-stimulated astrocytes. In the present study, we report that the effect of carbachol-stimulated astrocytes on hippocampal neuron neurite outgrowth was inhibited in a concentration-dependent manner (25-100 mM) by ethanol. This effect was because of the inhibition of the release of fibronectin, laminin, and plasminogen activator inhibitor-1. Similar effects on neuritogenesis and on the release of astrocyte extracellular proteins were observed after the incubation of astrocytes with carbachol in the presence of 1-butanol, another short-chain alcohol, which like ethanol is a competitive substrate for phospholipase D, but not by tert-butanol, its analog that is not a substrate for this enzyme. This study identifies a potential novel mechanism involved in the developmental effects of ethanol mediated by the interaction of ethanol with cell signaling in astrocytes, leading to an impairment in neuron-astrocyte communication.

  15. Prognostic significance of circulating laminin gamma2 for early-stage non-small-cell lung cancer

    PubMed Central

    Teng, Yu; Wang, Zitong; Ma, Li; Zhang, Lina; Guo, Yinan; Gu, Meng; Wang, Ziyu; Wang, Yue; Yue, Wentao

    2016-01-01

    Background Laminin gamma2 (Ln-γ2) chain, a distinctive subunit of heterotrimeric laminin-332, is frequently upregulated in carcinomas and is of great importance in cell migration and invasion. Despite this, the status of circulating Ln-γ2 in lung cancer patients is still uncertain. Patients and methods In this retrospective study, serum samples from 538 all-stage (stages I–IV) patients with non-small-cell lung cancer (NSCLC) and 94 age-matched healthy volunteers were investigated by enzyme-linked immunosorbent assay. Data were statistically analyzed in combination with clinicopathological information. Results Circulating Ln-γ2 was markedly increased in NSCLC, even in stage I cases (P<0.01), reflecting the progression of lung cancer. Survival analysis on 370 eligible patients indicated that serum Ln-γ2-negative patients survived much longer compared with Ln-γ2-positive individuals (P=0.028), and it was especially the case for stage I (P<0.001), stage T1 (P=0.001), and stage N0 patients (P=0.038), all of whom represented early-stage cases. For the advanced patients, however, overall survivals were not significantly different among stages II–IV (P=0.830), stages T2–T4 (P=0.575), stages N1–N3 (P=0.669), and stage M1 (P=0.849). Cox analysis subsequently defined serum Ln-γ2 as an independent prognostic indicator of NSCLC, particularly for early-stage patients. Furthermore, we demonstrated the association of serum Ln-γ2 with smoking behavior, but its association with tumor progression and early prognostic significance were not altered in the nonsmoking cohort. Conclusion Our study demonstrated that elevation of circulating Ln-γ2 was an early-emerging event in NSCLC and was significantly associated with poor prognosis in NSCLC, especially for early-stage cases. PMID:27462170

  16. Laminin-332 alters connexin profile, dye coupling and intercellular Ca2+ waves in ciliated tracheal epithelial cells

    PubMed Central

    Isakson, Brant E; Olsen, Colin E; Boitano, Scott

    2006-01-01

    Background Tracheal epithelial cells are anchored to a dynamic basement membrane that contains a variety of extracellular matrix proteins including collagens and laminins. During development, wound repair and disease of the airway epithelium, significant changes in extracellular matrix proteins may directly affect cell migration, differentiation and events mediated by intercellular communication. We hypothesized that alterations in cell matrix, specifically type I collagen and laminin α3β3γ2 (LM-332) proteins within the matrix, directly affect intercellular communication in ciliated rabbit tracheal epithelial cells (RTEC). Methods Functional coupling of RTEC was monitored by microinjection of the negatively charged fluorescent dyes, Lucifer Yellow and Alexa 350, into ciliated RTEC grown on either a LM-332/collagen or collagen matrix. Coupling of physiologically significant molecules was evaluated by the mechanism and extent of propagated intercellular Ca2+ waves. Expression of connexin (Cx) mRNA and proteins were assayed by reverse transcriptase – polymerase chain reaction and immunocytochemistry, respectively. Results When compared to RTEC grown on collagen alone, RTEC grown on LM-332/collagen displayed a significant increase in dye transfer. Although mechanical stimulation of RTEC grown on either LM-332/collagen or collagen alone resulted in intercellular Ca2+ waves, the mechanism of transfer was dependent on matrix: RTEC grown on LM-332/collagen propagated Ca2+waves via extracellular purinergic signaling whereas RTEC grown on collagen used gap junctions. Comparison of RTEC grown on collagen or LM-332/collagen matrices revealed a reorganization of Cx26, Cx43 and Cx46 proteins. Conclusion Alterations in airway basement membrane proteins such as LM-332 can induce connexin reorganizations and result in altered cellular communication mechanisms that could contribute to airway tissue function. PMID:16884540

  17. Androgen receptor non-nuclear regulation of prostate cancer cell invasion mediated by Src and matriptase.

    PubMed

    Zarif, Jelani C; Lamb, Laura E; Schulz, Veronique V; Nollet, Eric A; Miranti, Cindy K

    2015-03-30

    Castration-resistant prostate cancers still depend on nuclear androgen receptor (AR) function despite their lack of dependence on exogenous androgen. Second generation anti-androgen therapies are more efficient at blocking nuclear AR; however resistant tumors still develop. Recent studies indicate Src is highly active in these resistant tumors. By manipulating AR activity in several different prostate cancer cell lines through RNAi, drug treatment, and the use of a nuclear-deficient AR mutant, we demonstrate that androgen acting on cytoplasmic AR rapidly stimulates Src tyrosine kinase via a non-genomic mechanism. Cytoplasmic AR, acting through Src enhances laminin integrin-dependent invasion. Active Matriptase, which cleaves laminin, is elevated within minutes after androgen stimulation, and is subsequently shed into the medium. Matriptase activation and shedding induced by cytoplasmic AR is dependent on Src. Concomitantly, CDCP1/gp140, a Matriptase and Src substrate that controls integrin-based migration, is activated. However, only inhibition of Matriptase, but not CDCP1, suppresses the AR/Src-dependent increase in invasion. Matriptase, present in conditioned medium from AR-stimulated cells, is sufficient to enhance invasion in the absence of androgen. Thus, invasion is stimulated by a rapid but sustained increase in Src activity, mediated non-genomically by cytoplasmic AR, leading to rapid activation and shedding of the laminin protease Matriptase.

  18. Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila

    PubMed Central

    2014-01-01

    Background The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Results Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis. Conclusions Assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational

  19. Abnormal expression of laminin suggests disturbance of sarcolemma-extracellular matrix interaction in Japanese patients with autosomal recessive muscular dystrophy deficient in adhalin.

    PubMed Central

    Higuchi, I; Yamada, H; Fukunaga, H; Iwaki, H; Okubo, R; Nakagawa, M; Osame, M; Roberds, S L; Shimizu, T; Campbell, K P

    1994-01-01

    Dystrophin is associated with several novel sarcolemmal proteins, including a laminin-binding extracellular glycoprotein of 156 kD (alpha-dystroglycan) and a transmembrane glycoprotein of 50 kD (adhalin). Deficiency of adhalin characterizes a severe autosomal recessive muscular dystrophy prevalent in Arabs. Here we report for the first time two mongoloid (Japanese) patients with autosomal recessive muscular dystrophy deficient in adhalin. Interestingly, adhalin was not completely absent and was faintly detectable in a patchy distribution along the sarcolemma in our patients. Although the M and B2 subunits of laminin were preserved, the B1 subunit was greatly reduced in the basal lamina surrounding muscle fibers. Our results raise a possibility that the deficiency of adhalin may be associated with the disturbance of sarcolemma-extracellular matrix interaction leading to sarcolemmal instability. Images PMID:8040315

  20. Tumor suppressive microRNA-218 inhibits cancer cell migration and invasion through targeting laminin-332 in head and neck squamous cell carcinoma.

    PubMed

    Kinoshita, Takashi; Hanazawa, Toyoyuki; Nohata, Nijiro; Kikkawa, Naoko; Enokida, Hideki; Yoshino, Hirofumi; Yamasaki, Takeshi; Hidaka, Hideo; Nakagawa, Masayuki; Okamoto, Yoshitaka; Seki, Naohiko

    2012-11-01

    Recent our microRNA (miRNA) expression signature revealed that expression of microRNA-218 (miR-218) was reduced in cancer tissues, suggesting a candidate of tumor suppressor in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the functional significance of miR-218 and its mediated moleculer pathways in HNSCC. Restoration of miR-218 in cancer cells led to significant inhibition of cell migration and invasion activities in HNSCC cell lines (FaDu and SAS). Genome-wide gene expression analysis of miR-218 transfectants and in silico database analysis showed that focal adhesion pathway was a promising candidate of miR-218 target pathways. The laminins are an important and biologically active part of the basal lamina, the function of that are various such as influencing cell differentiation, migration and adhesion as well as proliferation and cell survival. Interestingly, all components of laminin-332 (LAMA3, LAMB3 and LAMC2) are listed on the candidate genes in focal adhesion pathway. Furthermore, we focused on LAMB3 which has a miR-218 target site and gene expression studies and luciferase reporter assays showed that LAMB3 was directly regulated by miR-218. Silencing study of LAMB3 demonstrated significant inhibition of cell migration and invasion. In clinical specimens with HNSCC, the expression levels of laminin-332 were significantly upregulated in cancer tissues compared to adjacent non-cancerous tissues. Our analysis data showed that tumor suppressive miR-218 contributes to cancer cell migration and invasion through regulating focal adhesion pathway, especially laminin-332. Tumor suppressive miRNA-mediated novel cancer pathways provide new insights into the potential mechanisms of HNSCC oncogenesis. PMID:23159910

  1. A comprehensive survey of the laminins and collagens type IV expressed in mouse Leydig cells and their regulation by LH/hCG.

    PubMed

    Mazaud Guittot, Séverine; Vérot, Adélie; Odet, Fanny; Chauvin, Marie-Agnès; le Magueresse-Battistoni, Brigitte

    2008-04-01

    Extracellular matrix (ECM) proteins have been shown to alter Leydig cell steroidogenesis in vitro, substantiating the hypothesis that Leydig cell steroidogenic activity and matrix environment are interdependent events. However, the nature of the ECM components synthesized by Leydig cells and their regulation by LH/human chorionic gonadotropin (hCG) remain unknown. Here, we examine the occurrence of the 11 laminin subunits and the 6 alpha chains of collagen IV (COL4A1-6) by RT-PCR in Leydig cells cultured with or without LH/hCG. Leydig cells were a tumor Leydig cell line (mLTC-1) or 8-week-old mice Leydig cells. Based on PCR data, it is suggested that normal Leydig cells may synthesize a maximum of 11 laminin heterotrimers and the 6 alpha chains of collagen IV. They also may synthesize various proteases and inhibitors of the metzincin family. The mLTC-1 cells have a limited repertoire as compared with normal Leydig cells. Interestingly, none of the ten proteases and inhibitors monitored is under LH-hCG regulation whereas every protease and inhibitor of the serine protease family yet identified in Leydig cells is under gonadotropin regulation. In addition, a few laminin and collagen subunit genes are regulated by LH/hCG. These are laminins alpha3 and gamma3 (Lama3 and Lamc3), Col4a3, and Col4a6, which are negatively regulated by LH/hCG in both Leydig cell types, and Col4a4, which was downregulated in primary cultures but not in mLTC-1 cells. Collectively, the present study suggests that Leydig cells modulate in a selective fashion their matrix environment in response to their trophic hormone. This may alter the steroidogenic outcome of Leydig cells. PMID:18367508

  2. Laminin alpha 5, a major transcript of normal and malignant rat liver epithelial cells, is differentially expressed in developing and adult liver.

    PubMed

    Seebacher, T; Medina, J L; Bade, E G

    1997-11-25

    The laminin family of extracellular matrix glycoproteins plays a major role in cell migration and differentiation and in tumor cell invasion. As previously shown, the laminin deposited by normal and malignant rat liver epithelial cells in their extracellular matrix (ECM) and into their ECM migration tracks does not contain a typical (EHS-like) alpha 1 heavy chain. By RT-PCR screening we have now identified two alpha chains among a total of five additional laminin chains produced by these cells. Three of the newly identified chains were not previously known for the rat. Their sequences have been deposited in the EMBL nucleotide sequence data bank. The alpha 5 chain now identified is expressed at comparably high levels by both the normal and the malignant liver epithelial cells. The chain is also expressed in fetal liver together with the alpha 2 and beta 2 chains, but it is only vestigially expressed in the mature organ as shown by RT-PCR. These results suggest for alpha 5 a role in development and production of the chain by only a small subset of cells in adult liver. At the level of detection used, no changes were observed in regenerating liver after partial hepatectomy. In addition to the alpha 5 chain, the cultured cells express the beta 1 and beta 2 light chains, indicating the expression of more than one laminin isoform by the same cell line. The expression of the alpha 5 chain and of the other new non-EHS isoform chains was also analyzed in various tissues. The malignant liver epithelial cells, but not their nontumorigenic parental cells, also express, in addition to the alpha 5 chain the alpha 2 chain, which is expressed at high level by the NBT II bladder carcinoma cell line, suggesting a relationship with malignancy. PMID:9417868

  3. A recurrent laminin 5 mutation in British patients with lethal (Herlitz) junctional epidermolysis bullosa: evidence for a mutational hotspot rather than propagation of an ancestral allele.

    PubMed

    Ashton, G H; Mellerio, J E; Dunnill, M G; Pulkkinen, L; Christiano, A M; Uitto, J; Eady, R A; McGrath, J A

    1997-05-01

    The three genes (LAMA3, LAB3 and LAMC2) that encode the anchoring filament protein, laminin 5, may all harbour pathogenetic mutations in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa (JEB). Recently, one particular mutation, R635X in the LAMB3 gene, has been found to account for approximately 40% of all JEB laminin 5 mutations (Kivirikko et al., Hum Mol Genet 1996; 5: 231-7). In this study, we assessed the frequency of this mutation in 12 British patients with lethal (Herlitz) JEB using PCR amplification of genomic DNA and restriction endonuclease digestion. The mutation R635X was fond in seven of 24 (29%) mutant alleles, confirming its relative frequency within the British gene pool. In addition, haplotype analysis using intragenic polymorphisms showed that the mutation arose on at least four different haplotype backgrounds, suggesting it represents a mutational hotspot rather than propagation of a common British ancestral allele. These findings support the hypermutable nature of this CpG dinucleotide and have implications in screening for laminin 5 gene mutations in British and other patients with JEB. PMID:9205497

  4. Laminin 5 genes and Herlitz junctional epidermolysis bullosa: novel mutations and polymorphisms in the LAMB3 and LAMC2 genes. Mutations in brief no. 190. Online.

    PubMed

    Kon, A; Pulkkinen, L; Hara, M; Tamai, K; Tagami, H; Hashimoto, I; Uitto, J

    1998-01-01

    Herlitz junctional epidermolysis bullosa (H-JEB; OMIM #226700) is a lethal, autosomal recessive blistering disorder characterized by fragility of the skin and other specialized epithelia. Previously, mutations in the laminin 5 genes (LAMA3, LAMB3, and LAMC2) have been disclosed, most of them in LAMB3. In this study, we have examined the genetic basis of H-JEB in three families utilizing heteroduplex analysis and automated nucleotide sequencing. In one family, the proband was compound heterozygote for previously unpublished LAMB3 mutations, 1482delC and W95X. In two other families, the probands were found to be homozygous for novel nonsense mutations C553X and K822X in the LAMC2 gene. These mutations result in premature termination codons and predict truncation of the corresponding polypeptides. Also, during the search of laminin 5 mutations, 18 LAMB3 and LAMC2 polymorphisms were discovered, 9 of them being previously undescribed. Delineation of novel homozygous nonsense mutations in the LAMB3 and LAMC2 genes, with previous demonstrations of LAMA3 mutations, re-emphasizes the concept that stop codon mutations in both alleles of any of the three laminin 5 genes result in the severe H-JEB phenotype. PMID:10660342

  5. A homozygous nonsense mutation in the alpha 3 chain gene of laminin 5 (LAMA3) in lethal (Herlitz) junctional epidermolysis bullosa.

    PubMed

    Kivirikko, S; McGrath, J A; Baudoin, C; Aberdam, D; Ciatti, S; Dunnill, M G; McMillan, J R; Eady, R A; Ortonne, J P; Meneguzzi, G

    1995-05-01

    The inherited mechanobullous disorder, junctional epidermolysis bullosa (JEB), is characterized by extensive blistering and erosions of the skin and mucous membranes. The diagnostic hallmarks of JEB include ultrastructural abnormalities in the hemidesmosomes of the cutaneous basement membrane zone, as well as an absence of staining with antibodies against the anchoring filament protein, laminin 5. Therefore, the three genes encoding alpha 3, beta 3 and gamma 2 chains of laminin 5, known as LAMA3, LAMB3 and LAMC2, are candidate genes for JEB. We have previously demonstrated mutations in the LAMB3 and LAMC2 genes in several families with JEB. We initiated mutation analysis from an affected child by PCR amplification of individual LAMA3 exons, followed by heteroduplex analysis. Nucleotide sequencing of heteroduplexes identified a homozygous nonsense mutation within domain I/II of the alpha 3 chain. These findings provide the first evidence that nonsense mutations within the LAMA3 gene are also involved in the pathogenesis of JEB, and indicate that mutations of all three genes of laminin 5 can result in the JEB phenotype. PMID:7633458

  6. Quantitative proteomic analysis reveals metabolic alterations, calcium dysregulation, and increased expression of extracellular matrix proteins in laminin α2 chain-deficient muscle.

    PubMed

    de Oliveira, Bruno Menezes; Matsumura, Cintia Y; Fontes-Oliveira, Cibely C; Gawlik, Kinga I; Acosta, Helena; Wernhoff, Patrik; Durbeej, Madeleine

    2014-11-01

    Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

  7. Adsorption of HSA, IgG and laminin-1 on model titania surfaces--effects of glow discharge treatment on competitively adsorbed film composition.

    PubMed

    Santos, Olga; Svendsen, Ida E; Lindh, Liselott; Arnebrant, Thomas

    2011-10-01

    This study investigated the effect of glow discharge treatment of titania surfaces on plasma protein adsorption, by means of ellipsometry and mechanically assisted SDS elution. The adsorption and film elution of three plasma proteins, viz. human serum albumin (HSA), human immunoglobulin G (IgG) and laminin-1, as well as competitive adsorption from a mixture of the three proteins, showed that the adsorbed amount of the individual proteins after 1 h increased in the order HSA laminin-1 ≤ protein mixture. Film elutability showed that 30 min of SDS interaction resulted in almost complete removal of adsorbed films. No difference in the total adsorbed amounts of individual proteins, or from the mixture, was observed between untreated and glow discharge treated titania surfaces. However, the composition of the adsorbed films from the mixture differed between the untreated and glow discharge treated substrata. On glow discharge-treated titania the fraction of HSA increased, the fraction of laminin-1 decreased and the fraction of IgG was unchanged compared to the adsorption on the untreated titania, which was attributed to protein-protein interactions and competitive/associative adsorption behaviour.

  8. Long-term self-renewal of human ES/iPS-derived hepatoblast-like cells on human laminin 111-coated dishes.

    PubMed

    Takayama, Kazuo; Nagamoto, Yasuhito; Mimura, Natsumi; Tashiro, Katsuhisa; Sakurai, Fuminori; Tachibana, Masashi; Hayakawa, Takao; Kawabata, Kenji; Mizuguchi, Hiroyuki

    2013-01-01

    The establishment of self-renewing hepatoblast-like cells (HBCs) from human pluripotent stem cells (PSCs) would realize a stable supply of hepatocyte-like cells for medical applications. However, the functional characterization of human PSC-derived HBCs was not enough. To purify and expand human PSC-derived HBCs, human PSC-derived HBCs were cultured on dishes coated with various types of human recombinant laminins (LN). Human PSC-derived HBCs attached to human laminin-111 (LN111)-coated dish via integrin alpha 6 and beta 1 and were purified and expanded by culturing on the LN111-coated dish, but not by culturing on dishes coated with other laminin isoforms. By culturing on the LN111-coated dish, human PSC-derived HBCs were maintained for more than 3 months and had the ability to differentiate into both hepatocyte-like cells and cholangiocyte-like cells. These expandable human PSC-derived HBCs would be manageable tools for drug screening, experimental platforms to elucidate mechanisms of hepatoblasts, and cell sources for hepatic regenerative therapy. PMID:24319667

  9. A stop-gain in the laminin, alpha 3 gene causes recessive junctional epidermolysis bullosa in Belgian Blue cattle.

    PubMed

    Sartelet, Arnaud; Harland, Chad; Tamma, Nico; Karim, Latifa; Bayrou, Calixte; Li, Wanbo; Ahariz, Naima; Coppieters, Wouter; Georges, Michel; Charlier, Carole

    2015-10-01

    Four newborn purebred Belgian Blue calves presenting a severe form of epidermolysis bullosa were recently referred to our heredo-surveillance platform. SNP array genotyping followed by autozygosity mapping located the causative gene in a 8.3-Mb interval on bovine chromosome 24. Combining information from (i) whole-genome sequencing of an affected calf, (ii) transcriptomic data from a panel of tissues and (iii) a list of functionally ranked positional candidates pinpointed a private G to A nucleotide substitution in the LAMA3 gene that creates a premature stop codon (p.Arg2609*) in exon 60, truncating 22% of the corresponding protein. The LAMA3 gene encodes the alpha 3 subunit of the heterotrimeric laminin-332, a key constituent of the lamina lucida that is part of the skin basement membrane connecting epidermis and dermis layers. Homozygous loss-of-function mutations in this gene are known to cause severe junctional epidermolysis bullosa in human, mice, horse, sheep and dog. Overall, our data strongly support the causality of the identified gene and mutation. PMID:26370913

  10. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy.

    PubMed

    Li, Mei; Arner, Anders

    2015-01-01

    Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of extended immobilization or muscle relaxation in vivo in different dystrophy models. We have examined effects of immobilization in larvae from two zebrafish strains with muscular dystrophy, the Sapje dystrophin-deficient and the Candyfloss laminin α2-chain-deficient strains. Larvae (4 days post fertilization, dpf) of both mutants have significantly lower active force in vitro, alterations in the muscle structure with gaps between muscle fibers and altered birefringence patterns compared to their normal siblings. Complete immobilization (18 hrs to 4 dpf) was achieved using a small molecular inhibitor of actin-myosin interaction (BTS, 50 μM). This treatment resulted in a significantly weaker active contraction at 4 dpf in both mutated larvae and normal siblings, most likely reflecting a general effect of immobilization on myofibrillogenesis. The immobilization also significantly reduced the structural damage in the mutated strains, showing that muscle activity is an important pathological mechanism. Following one-day washout of BTS, muscle tension partly recovered in the Candyfloss siblings and caused structural damage in these mutants, indicating activity-induced muscle recovery and damage, respectively. PMID:26536238

  11. Degradation of extracellular matrix proteins (fibronectin, vitronectin and laminin) by serine-proteinases isolated from Lonomia achelous caterpillar hemolymph.

    PubMed

    Lucena, Sara; Guerrero, Belsy; Salazar, Ana M; Gil, Amparo; Arocha-Piñango, Carmen L

    2006-09-01

    Lonomia achelous is a caterpillar distributed in southern Venezuela and in northern Brazil that causes an acute hemorrhagic syndrome in people who have contact with its bristles. The effect of the crude hemolymph and its chromatographic fractions (FDII, Lonomin V and Lonomin V-2) on extracellular matrix proteins was studied. The chromatographic fractions show activities similar to plasmin and urokinase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both lonomins appear as a protein band of 25 kDa under reduced conditions. By exclusion chromatography, the molecular weights of Lonomin V and Lonomin V-2 were 26.5 and 24.5 kDa, respectively. Fibronectin, laminin and vitronectin were degraded by all venom components. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reduced conditions, shows that lonomins degrade fibronectin in four main fragments of 116, 60, 50 and 30 kDa. Molecular exclusion chromatography in native conditions shows that the molecular masses of these fragments are > or = 300, 62 and 27 kDa. The proteolytic effect of lonomins was abolished by benzamidine/HCl, iodoacetic acid and aprotinin. The extracellular matrix protein degradation together with the fibrino(geno)lytic activity of hemolymph and its fractions could explain, in part, the hemorrhagic syndrome, and the wound dehiscence in persons who have had contact with the L. achelous caterpillar.

  12. Aggregation dynamics of laminin-1 in a physiological solution: A time-resolved static light scattering study

    NASA Astrophysics Data System (ADS)

    Onuma, Kazuo; Kanzaki, Noriko

    2005-11-01

    The aggregation of laminin-1 in a physiological solution was observed using time-resolved static light scattering. In a solution containing 150 mM of NaCl and 1 mM of CaCl 2, with a pH of 7.2 buffered by 50 mM Tris, and maintained at 25 °C, the weight-averaged mass (molecular weight) Mw, and radius of gyration Rg, of the aggregate were measured at 10 s intervals. The aggregation kinetics changed from reaction-limited cluster aggregation (RLCA) to diffusion-limited cluster aggregation (DLCA) over time. The fractal dimension estimated in the DLCA regime was 1.71 from the M˜Rgd relationship, which is consistent with the df of 1.75 obtained from an R˜t plot. Direct calculation of df using the scattering intensity and scattering vector revealed that df gradually increased over time in the DLCA regime, suggesting that spontaneous restructuring of the aggregate had occurred. This restructuring would have been caused by hydrophobic contact in the aggregate. The form factor of the aggregate was well fitted by a linear random coil model and not by a simple spherical model.

  13. Galaptin Mediates the Effect of Hypergravity on Vascular Smooth Muscle cell (SMC) Adhesion to Laminin Containing Matrices

    NASA Technical Reports Server (NTRS)

    Enahora, Fatisha T.; Bosah, Francis N.; Harris-Hooker, Sandra; Sanford, Gary L.

    1997-01-01

    Galaptin, an endogenous beta-galactoside specific lectin, has been reported to bind to laminin and subsequently decrease the binding of SMC. Cellular function depend on cell:matrix interactions. Hypergravity (HGrav) affect a number of cellular functions, yet little is known about its affect on cell adhesion. We examined the possibility that galaptin mediates the effects of hypergravity on SMC adherence. Confluent primate aorta SMC cultures were subjected to Hgrav (centrifuged at 6G) for 24 and 48 hr. Cells were non-enzymatically dispersed, pretreated with antisense (AS-oligo) or control sense (SS-oligo) oligonucleotides to galaptin mRNA (0.01 micro g/ml), then seeded in uncoated or ECL-matrix coated plates. Adhesion of cells were monitored after 6 hr. HGrav increased adhesion by 100-300% compared to controls. AS-oligo decreased adhesion for both HGrav and control cells. SS-oligo did not affect adhesion for either HGrav or control cells. These studies show that HGrav affects cell adhesion and that galaptin expression is required for this effect.

  14. Regeneration of adult rat spinal cord is promoted by the soluble KDI domain of gamma1 laminin.

    PubMed

    Wiksten, Markus; Väänänen, Antti J; Liebkind, Ron; Liesi, Päivi

    2004-11-01

    Regeneration in the central nervous system (CNS) of adult mammals is hampered by formation of a glial scar and by proteins released from the myelin sheaths of injured neuronal pathways. Our recent data indicate that the KDI (Lys-Asp-Ile) domain of gamma1 laminin neutralizes both glial- and myelin-derived inhibitory signals and promotes survival and neurite outgrowth of cultured human spinal cord neurons. We show that after complete transection of the adult rat spinal cord, animals receiving onsite infusion of the KDI domain via osmotic mini-pumps recover and are able to sustain their body weights and walk with their hindlimbs. Animals treated with placebo suffer from irreversible hindlimb paralysis. Microscopic and molecular analyses of the spinal cords indicate that the KDI domain reduces tissue damage at the lesion site and enables neurite outgrowth through the injured area to effect functional recovery of the initially paralyzed animals. That the KDI domain enhances regeneration of acute spinal cord injuries in the adult rat suggests that it may be used to promote regeneration of spinal cord injuries in humans.

  15. Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells

    PubMed Central

    Månsson-Broberg, Agneta; Rodin, Sergey; Bulatovic, Ivana; Ibarra, Cristián; Löfling, Marie; Genead, Rami; Wärdell, Eva; Felldin, Ulrika; Granath, Carl; Alici, Evren; Le Blanc, Katarina; Smith, C.I. Edvard; Salašová, Alena; Westgren, Magnus; Sundström, Erik; Uhlén, Per; Arenas, Ernest; Sylvén, Christer; Tryggvason, Karl; Corbascio, Matthias; Simonson, Oscar E.; Österholm, Cecilia; Grinnemo, Karl-Henrik

    2016-01-01

    Summary The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/β-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo. PMID:27052314

  16. A laminin-2-derived peptide promotes early-stage peripheral nerve regeneration in a dual-component artificial nerve graft.

    PubMed

    Seo, S Y; Min, S-K; Bae, H K; Roh, D; Kang, H K; Roh, S; Lee, S; Chun, G-S; Chung, D-J; Min, B-M

    2013-10-01

    The DLTIDDSYWYRI motif (Ln2-P3) of human laminin-2 has been reported to promote PC12 cell attachment through syndecan-1; however, the in vivo effects of Ln2-P3 have not been studied. In Schwann cells differentiated from skin-derived precursors, the peptide was effective in promoting cell attachment and spreading in vitro. To examine the effects of Ln2-P3 in peripheral nerve regeneration in vivo, we developed a dual-component poly(p-dioxanone) (PPD)/poly(lactic-co-glycolic acid) (PLGA) artificial nerve graft. The novel graft was coated with scrambled peptide or Ln2-P3 and used to bridge a 10 mm defect in rat sciatic nerves. The dual-component nerve grafts provided tensile strength comparable to that of a real rat nerve trunk. The Ln2-P3-treated grafts promoted early-stage peripheral nerve regeneration by enhancing the nerve regeneration rate and significantly increased the myelinated fibre density compared with scrambled peptide-treated controls. These findings indicate that Ln2-P3, combined with tissue-engineering scaffolds, has potential biomedical applications in peripheral nerve injury repair. PMID:22438104

  17. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy

    PubMed Central

    Li, Mei; Arner, Anders

    2015-01-01

    Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of extended immobilization or muscle relaxation in vivo in different dystrophy models. We have examined effects of immobilization in larvae from two zebrafish strains with muscular dystrophy, the Sapje dystrophin-deficient and the Candyfloss laminin α2-chain-deficient strains. Larvae (4 days post fertilization, dpf) of both mutants have significantly lower active force in vitro, alterations in the muscle structure with gaps between muscle fibers and altered birefringence patterns compared to their normal siblings. Complete immobilization (18 hrs to 4 dpf) was achieved using a small molecular inhibitor of actin-myosin interaction (BTS, 50 μM). This treatment resulted in a significantly weaker active contraction at 4 dpf in both mutated larvae and normal siblings, most likely reflecting a general effect of immobilization on myofibrillogenesis. The immobilization also significantly reduced the structural damage in the mutated strains, showing that muscle activity is an important pathological mechanism. Following one-day washout of BTS, muscle tension partly recovered in the Candyfloss siblings and caused structural damage in these mutants, indicating activity-induced muscle recovery and damage, respectively. PMID:26536238

  18. Microfabrication procedure of PDMS microbeam array using photolithography for laminin printing and piconewton force transduction on axons.

    PubMed

    Sasoglu, F Mert; Bohl, Andrew J; Layton, Bradley E

    2006-01-01

    The purpose of this paper is to introduce our design for transducing forces on the order of tens of piconewtons by optically measuring deflection of a microfabricated beam tip as it pulls on an array of flexible structures such as axons in an array of laminin-printed neurons. To achieve this we have designed polymeric beams with spring constants on the order of 10 pN/microm. We have fabricated circular microbeams with Sylgard polydimethylsiloxane (PDMS). The elastic modulus of PDMS was determined experimentally using a microscale and a micrometer at different concentrations of curing agent and base agent and found to be on the order of 100 kPa. The designed geometry is a 100x100 tapered microcone array with each beam having a length of 100 microm, and a base diameter of 10 microm. A SU-8 negative photoresist is etched using photolithography and used as a mold for PDMS soft lithography. PDMS was injected into the mold and the array peeled from the mold.

  19. Anti-La (SS-B) but not anti-Ro52 (SS-A) antibodies cross-react with laminin--a role in the pathogenesis of congenital heart block?

    PubMed Central

    Li, J M; Horsfall, A C; Maini, R N

    1995-01-01

    Cross-reactions between maternally derived autoantibodies and fetal cardiac antigens have been postulated to play a role in the pathogenesis of congenital heart block (CHB). We have explored the cross-reactivity of autoantibodies to the small ribonuclear autoantigens, La/SS-B and Ro/SS-A, with laminin, the major component of cardiac sarcolemmal membrane using affinity-purified antibodies from patients with Sjögren's syndrome (SS). Anti-La antibodies purified from eight of 10 patients cross-reacted significantly with mouse laminin by ELISA. In contrast, purified antibodies to Ro52 from the same 10 patients showed little or no binding to laminin. Laminin inhibited up to 70% binding of anti-La antibodies to La antigen, and La inhibited up to 65% binding of anti-La antibodies to laminin. The cross-reaction was further examined on cryosections of 10 human fetal hearts aged from 8.7 to 14.9 weeks of gestation, two normal adult hearts, and one pathological adult heart with a diagnosis of dilated cardiomyopathy. Anti-Ro52 antibodies did not bind to the surface of cardiac cells. However, anti-La antibodies from seven of 10 patients tested bound to the surface of fetal myocytes from hearts aged 9.4 to 14.9 weeks of gestation, and also to the myocytes from the pathological adult heart but not to normal adult hearts. Preincubation with La antigen abolished the binding of anti-La antibodies to the surface of adult heart myocytes with dilated cardiomyopathy, and pre-incubation with mouse laminin could partially block this binding. These results suggest that molecular mimicry between laminin and La, but not Ro52, may act as a target for specific maternal autoantibodies, and contribute to the pathogenesis of CHB at a critical stage during fetal cardiac development. Images Fig. 2 Fig. 3 Fig. 4 PMID:7882552

  20. Anti-La (SS-B) but not anti-Ro52 (SS-A) antibodies cross-react with laminin--a role in the pathogenesis of congenital heart block?

    PubMed

    Li, J M; Horsfall, A C; Maini, R N

    1995-03-01

    Cross-reactions between maternally derived autoantibodies and fetal cardiac antigens have been postulated to play a role in the pathogenesis of congenital heart block (CHB). We have explored the cross-reactivity of autoantibodies to the small ribonuclear autoantigens, La/SS-B and Ro/SS-A, with laminin, the major component of cardiac sarcolemmal membrane using affinity-purified antibodies from patients with Sjögren's syndrome (SS). Anti-La antibodies purified from eight of 10 patients cross-reacted significantly with mouse laminin by ELISA. In contrast, purified antibodies to Ro52 from the same 10 patients showed little or no binding to laminin. Laminin inhibited up to 70% binding of anti-La antibodies to La antigen, and La inhibited up to 65% binding of anti-La antibodies to laminin. The cross-reaction was further examined on cryosections of 10 human fetal hearts aged from 8.7 to 14.9 weeks of gestation, two normal adult hearts, and one pathological adult heart with a diagnosis of dilated cardiomyopathy. Anti-Ro52 antibodies did not bind to the surface of cardiac cells. However, anti-La antibodies from seven of 10 patients tested bound to the surface of fetal myocytes from hearts aged 9.4 to 14.9 weeks of gestation, and also to the myocytes from the pathological adult heart but not to normal adult hearts. Preincubation with La antigen abolished the binding of anti-La antibodies to the surface of adult heart myocytes with dilated cardiomyopathy, and pre-incubation with mouse laminin could partially block this binding. These results suggest that molecular mimicry between laminin and La, but not Ro52, may act as a target for specific maternal autoantibodies, and contribute to the pathogenesis of CHB at a critical stage during fetal cardiac development. PMID:7882552

  1. The C-terminus of the {gamma}2 chain but not of the {beta}3 chain of laminin-332 is indirectly but indispensably necessary for integrin-mediated cell reactions

    SciTech Connect

    Navdaev, Alexei; Heitmann, Vanessa; Santana Evangelista, Karla de; Moergelin, Matthias; Wegener, Joachim; Eble, Johannes A.

    2008-02-01

    Using a recombinant mini-laminin-332, we showed that truncation of the three C-terminal amino acids of the {gamma}2 chain, but not of the C-terminal amino acid of the {beta}3 chain, completely abolished {alpha}3{beta}1 integrin binding and its cellular functions, such as attachment and spreading. However, a synthetic peptide mimicking the {gamma}2 chain C-terminus did not interfere with {alpha}3{beta}1 integrin binding or cell adhesion and spreading on laminin-332 as measured by protein interaction assays and electric cell-substrate impedance sensing. Nor was the soluble peptide able to restore the loss of integrin-mediated cell adhesiveness to mini-laminin-332 after deletion of the {gamma}2 chain C-terminus. These findings spoke against the hypothesis that the {gamma}2 chain C-terminus of laminin-332 is a part of the {alpha}3{beta}1 integrin interaction site. In addition, structural studies with electron microscopy showed that truncation of the {gamma}2 chain C-terminus opened up the compact supradomain structure of LG1-3 domains. Thus, by inducing or stabilizing an integrin binding-competent conformation or array of the LG1-3 domains, the {gamma}2 chain C-terminus plays an indirect but essential role in laminin-332 recognition by {alpha}3{beta}1 integrin and, hence, its cellular functions.

  2. A Tc-99m labeled laminin derived peptide, Tc-99m-YIGSR for thrombus specific imaging

    SciTech Connect

    Wang, G.J.; Oster, Z.H.; Som, P.

    1994-05-01

    Laminin derived adhesive peptides were studied as potential agents for thrombus specific imaging. Using a novel peptide Tc-99m labeling method studies were performed in vitro using human whole blood clots and platelets, and in vivo scintigraphy in animals with experimental thrombi. Aliquots of 0.1 ml human blood were placed in inclined Petri dishes until clot was well formed. Clots were rinsed 3x with phosphate buffer and 10 {mu}Ci Tc-99m YIGSR II was added. After incubation at room temperature for 1 hr, clots were again washed 3x. Residual activity was measured. Platelets were harvested using routine methods and incubated with Tc-99m YIGSR II, washed and assayed. Blocking experiments using cold YIGSR II showed that the Tc-99m labeled peptide preparation YIGSR II binds specifically and selectively to clot and platelets as compared to control experiments using nonspecific human Tc-99m IgG. Tissue distribution studies showed rapid blood clearance, urinary excretion and to a lesser degree GI tract excretion. Tc-99m YIGSR II was lower in all organs except kidneys compared to Tc-99m 50 H.19, Tc-99m IgG and Tc-99m YIGSR I. Tc-99m-YIGSR II consistently visualized thrombi within 30 min p.i. In vivo scintigraphic (thrombus/contralateral side) ratio was 3:1 and ex vivo direct counting (thrombosed to nonthrombosed vessel segment) was 5.4: 1. Compared to monoclonal antibodies peptide preparations are non- or minimally immunogenic, preparation is probably less expensive and there is also less danger of viral DNA contamination. These considerations and our data indicate that the Tc-99m-YIGSR II peptide has significant potential as a thrombus imaging agent.

  3. Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction.

    PubMed

    Somajo, Sofia; Ahnström, Josefin; Fernandez-Recio, Juan; Gierula, Magdalena; Villoutreix, Bruno O; Dahlbäck, Björn

    2015-05-01

    Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50-60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LG-domains.

  4. A laminin 511 matrix is regulated by TAZ and functions as the ligand for the α6Bβ1 integrin to sustain breast cancer stem cells.

    PubMed

    Chang, Cheng; Goel, Hira Lal; Gao, Huijie; Pursell, Bryan; Shultz, Leonard D; Greiner, Dale L; Ingerpuu, Sulev; Patarroyo, Manuel; Cao, Shiliang; Lim, Elgene; Mao, Junhao; McKee, Karen Kulju; Yurchenco, Peter D; Mercurio, Arthur M

    2015-01-01

    Understanding how the extracellular matrix impacts the function of cancer stem cells (CSCs) is a significant but poorly understood problem. We report that breast CSCs produce a laminin (LM) 511 matrix that promotes self-renewal and tumor initiation by engaging the α6Bβ1 integrin and activating the Hippo transducer TAZ. Although TAZ is important for the function of breast CSCs, the mechanism is unknown. We observed that TAZ regulates the transcription of the α5 subunit of LM511 and the formation of a LM511 matrix. These data establish a positive feedback loop involving TAZ and LM511 that contributes to stemness in breast cancer.

  5. Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction.

    PubMed

    Singhal, Neha; Martin, Paul T

    2011-11-01

    The vertebrate neuromuscular junction (NMJ) remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning, and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. This is also the case at the NMJ, where the motor nerve contributes additional factors. Before most of the current molecular components were known, it was clear that the synaptic ECM of adult skeletal muscles was unique in composition and contained factors sufficient to induce the differentiation of both pre- and postsynaptic membranes. Biochemical, genetic, and microscopy studies have confirmed that agrin, laminin (221, 421, and 521), collagen IV (α3-α6), collagen XIII, perlecan, and the ColQ-bound form of acetylcholinesterase are all synaptic ECM proteins with important roles in neuromuscular development. The roles of their many potential receptors and/or binding proteins have been more difficult to assess at the genetic level due to the complexity of membrane interactions with these large proteins, but roles for MuSK-LRP4 in agrin signaling and for integrins, dystroglycan, and voltage-gated calcium channels in laminin-dependent phenotypes have been identified. Synaptic ECM proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability, and transmission. PMID:21766463

  6. The PPFLMLLKGSTR motif in globular domain 3 of the human laminin-5 {alpha}3 chain is crucial for integrin {alpha}3{beta}1 binding and cell adhesion

    SciTech Connect

    Kim, Jin-Man; Park, Won Ho; Min, Byung-Moo . E-mail: bmmin@snu.ac.kr

    2005-03-10

    Laminin-5 regulates various cellular functions, including cell adhesion, spreading, and motility. Here, we expressed the five human laminin {alpha}3 chain globular (LG) domains as monomeric, soluble fusion proteins, and examined their biological functions and signaling. Recombinant LG3 (rLG3) protein, unlike rLG1, rLG2, rLG4, and rLG5, played roles in cell adhesion, spreading, and integrin {alpha}3{beta}1 binding. More significantly, we identified a novel motif (PPFLMLLKGSTR) in the LG3 domain that is crucial for these responses. Studies with the synthetic peptides delineated the PPFLMLLKGSTR peptide within LG3 domain as a major site for both integrin {alpha}3{beta}1 binding and cell adhesion. Substitution mutation experiments suggest that the Arg residue is important for these activities. rLG3 protein- and PPFLMLLKGSTR peptide-induced keratinocyte adhesion triggered cell signaling through FAK phosphorylation at tyrosine-397 and -577. To our knowledge, this is the first report demonstrating that the PPFLMLLKGSTR peptide within the LG3 domain is a novel motif that is capable of supporting integrin {alpha}3{beta}1-dependent cell adhesion and spreading.

  7. Three-layer microfibrous peripheral nerve guide conduit composed of elastin-laminin mimetic artificial protein and poly(L-lactic acid)

    NASA Astrophysics Data System (ADS)

    Kakinoki, Sachiro; Nakayama, Midori; Moritan, Toshiyuki; Yamaoka, Tetsuji

    2014-07-01

    We developed a microfibrous poly(L-lactic acid) (PLLA) nerve conduit with a three-layered structure to simultaneously enhance nerve regeneration and prevent adhesion of surrounding tissue. The inner layer was composed of PLLA microfiber containing 25% elastin-laminin mimetic protein (AG73-(VPGIG)30) that promotes neurite outgrowth. The thickest middle layer was constructed of pure PLLA microfibers that impart the large mechanical stremgth to the conduit. A 10% poly(ethylene glycol) was added to the outer layer to prevent the adhesion with the surrounding tissue. The AG73-(VPGIG)30 composisting of an elastin-like repetitive sequence (VPGIG)30 and a laminin-derived sequence (RKRLQVQLSIRT: AG73) was biosynthesized using Escherichia coli. The PLLA microfibrous conduits were fabricated using an electrospinning procedure. AG73-(VPGIG)30 was successfully mixed in the PLLA microfibers, and the PLLA/AG73-(VPGIG)30 microfibers were stable under physiological conditions. The PLLA/AG73-(VPGIG)30 microfibers enhanced adhesion and neurite outgrowth of PC12 cells. The electrospun microfibrous conduit with a three-layered structure was implanted for bridging a 2.0-cm gap in the tibial nerve of a rabbit. Two months after implantation, no adhesion of surrounding tissue was observed, and the action potential was slightly improved in the nerve conduit with the PLLA/AG73-(VPGIG)30 inner layer.

  8. Decreased collagen types I and IV, laminin, CK-19 and α-SMA expression after bone marrow cell transplantation in rats with liver fibrosis.

    PubMed

    Carvalho, S N; Lira, D C; Oliveira, G P; Thole, A A; Stumbo, A C; Caetano, C E; Marques, R G; Carvalho, L

    2010-11-01

    Bone marrow cells have frequently been tested in animal models of liver fibrosis to assess their role in hepatic regeneration. The mononuclear fraction of bone marrow cells is of particular interest, as many studies show that these cells may be beneficial to treat hepatic fibrosis. In this study, we used the bile duct ligation model to induce hepatic fibrosis in an irreversible manner, and rats were treated with bone marrow mononuclear (BMMN) cells after fibrosis was established. Analysis of collagen types I and IV, laminin and α-SMA showed a decreased expression of these proteins in fibrotic livers after 7 days of BMMN cell injection. Moreover, cytokeratin-19 analysis showed a reduction in bile ducts in the BMMN cell-treated group. These results were accompanied by ameliorated levels of hepatic enzymes GPT (Glutamic-pyruvic transaminase), GOT (glutamic-oxaloacetic transaminase) and alkaline phosphatase (AP). Therefore, we showed that BMMN cells decrease hepatic fibrosis by significantly reducing myofibroblast numbers and through reduction of the collagen and laminin-rich extracellular matrix of fibrotic septa and hepatic sinusoids.

  9. Three-layer microfibrous peripheral nerve guide conduit composed of elastin-laminin mimetic artificial protein and poly(L-lactic acid)

    PubMed Central

    Kakinoki, Sachiro; Nakayama, Midori; Moritan, Toshiyuki; Yamaoka, Tetsuji

    2014-01-01

    We developed a microfibrous poly(L-lactic acid) (PLLA) nerve conduit with a three-layered structure to simultaneously enhance nerve regeneration and prevent adhesion of surrounding tissue. The inner layer was composed of PLLA microfiber containing 25% elastin-laminin mimetic protein (AG73-(VPGIG)30) that promotes neurite outgrowth. The thickest middle layer was constructed of pure PLLA microfibers that impart the large mechanical strength to the conduit. A 10% poly(ethylene glycol) was added to the outer layer to prevent the adhesion with the surrounding tissue. The AG73-(VPGIG)30 compositing of an elastin-like repetitive sequence (VPGIG)30 and a laminin-derived sequence (RKRLQVQLSIRT: AG73) was biosynthesized using Escherichia coli. The PLLA microfibrous conduits were fabricated using an electrospinning procedure. AG73-(VPGIG)30 was successfully mixed in the PLLA microfibers, and the PLLA/AG73-(VPGIG)30 microfibers were stable under physiological conditions. The PLLA/AG73-(VPGIG)30 microfibers enhanced adhesion and neurite outgrowth of PC12 cells. The electrospun microfibrous conduit with a three-layered structure was implanted for bridging a 2.0-cm gap in the tibial nerve of a rabbit. Two months after implantation, no adhesion of surrounding tissue was observed, and the action potential was slightly improved in the nerve conduit with the PLLA/AG73-(VPGIG)30 inner layer. PMID:25101261

  10. Neuronal degeneration and a decrease in laminin-like immunoreactivity is associated with elevated tissue-type plasminogen activator in the rat hippocampus after kainic acid injection.

    PubMed

    Nagai, N; Urano, T; Endo, A; Takahashi, H; Takada, Y; Takada, A

    1999-02-01

    Tissue-type plasminogen activator (tPA) is a serine protease that converts the inactive precursor plasminogen to the active protease plasmin. In the central nervous system, tPA has been suggested to participate in plasticity, memory and the neuronal degeneration caused by excitotoxins, but its precise functions during these processes are still unclear. We show in this report that tPA antigen level and extracellular tPA activity increased in the hippocampus during the early stages of neuronal degeneration in the CA3 region following the injection of kainic acid (KA) into the lateral cerebral ventricles. The increase in tPA antigen level was transient and its peak was at 4 h after the injection. tPA activity was also increased 4 h after the injection, but it remained at a high level for more than 8 h. Histological zymography showed that the increase in tPA activity was mainly localized in the CA3 region. In the same region, the disappearance of interneuronal laminin-like immunoreactivity and atrophic changes in pyramidal neurons were observed 4 h after the injection of KA. These results suggested that such focal and transient increases in tPA synthesis and release, which result in the proteolysis of laminin through plasminogen activation, could be involved in the neuronal degeneration in the CA3 region after the injection of KA.

  11. The formation of complex acetylcholine receptor clusters requires MuSK kinase activity and structural information from the MuSK extracellular domain

    PubMed Central

    Mazhar, Sania; Herbst, Ruth

    2012-01-01

    Efficient synaptic transmission at the neuromuscular junction (NMJ) requires the topological maturation of the postsynaptic apparatus from an oval acetylcholine receptor (AChR)-rich plaque into a complex pretzel-shaped array of branches. However, compared to NMJ formation very little is known about the mechanisms that regulate NMJ maturation. Recently the process of in vivo transformation from plaque into pretzel has been reproduced in vitro by culturing myotubes aneurally on laminin-coated substrate. It was proposed that the formation of complex AChR clusters is regulated by a MuSK-dependent muscle intrinsic program. To elucidate the structure–function role of MuSK in the aneural maturation of AChR pretzels, we used muscle cell lines expressing MuSK mutant and chimeric proteins. Here we report, that besides its role during agrin-induced AChR clustering, MuSK kinase activity is also necessary for substrate-dependent cluster formation. Constitutive-active MuSK induces larger AChR clusters, a faster cluster maturation on laminin and increases the anchorage of AChRs to the cytoskeleton compared to MuSK wild-type. In addition, we find that the juxtamembrane region of MuSK, which has previously been shown to regulate agrin-induced AChR clustering, is unable to induce complex AChR clusters on laminin substrate. Most interestingly, MuSK kinase activity is not sufficient for laminin-dependent AChR cluster formation since the MuSK ectodomain is also required suggesting a so far undiscovered instructive role for the extracellular domain of MuSK. PMID:22210232

  12. Fibroblast growth factor receptor-1 signaling in pancreatic islet beta-cells is modulated by the extracellular matrix.

    PubMed

    Kilkenny, Dawn M; Rocheleau, Jonathan V

    2008-01-01

    Maintenance of pancreatic beta-cell mass depends on extracellular stimuli that promote survival and proliferation. In the islet, these stimuli come from the beta-cell microenvironment and include extracellular matrix deposited by associated vascular endothelial cells. Fibroblast growth factor receptor-1 (FGFR1) has recently been implicated as a signaling pathway that is important for normal beta-cell function. We would like to understand how extracellular matrix and FGFR1 signaling interact to promote beta-cell survival and proliferation. To examine beta-cell-specific receptor responses, we created lentiviral vectors with rat insulin promoter-driven expression of Venus fluorescent protein-tagged full-length (R1betav) and kinase-deficient (KDR1betav) FGFR1. Significant FGF-1-dependent activation of ERK1/2 was observed in betaTC3 cells, dispersed beta-cells, and beta-cells in intact islets. This response was enhanced by R1betav expression and reduced by KDR1betav expression. Plating-dispersed beta-cells on collagen type IV resulted in enhanced expression of endogenous FGFR1 that was associated with sustained activation of ERK1/2. Conversely, plating cells on laminin reduced expression of FGFR1, and this reduction was associated with transient activation of ERK1/2. Addition of neutralizing antibodies to inhibit beta-cell attachment to laminin via alpha(6)-integrin increased high-affinity FGF-1-binding at the plasma membrane and resulted in sustained ERK1/2 activity similar to cells plated on collagen type IV. These data show that the FGF-stimulated beta-cell response is negatively affected by alpha(6)-integrin binding to laminin and suggest regulation associated with vascular endothelial cell remodeling. PMID:17916654

  13. Overexpression of β3 Chains of Laminin-332 is Associated With Clinicopathologic Features and Decreased Survival in Patients With Pancreatic Adenocarcinoma

    PubMed Central

    Chen, Jun; Wang, Weilin; Wei, Jianfeng; Zhou, Dongkai; Zhao, Xinyi; Song, Wei; Sun, Qiang; Huang, Pengfei

    2015-01-01

    Background: Laminin-332 (LM-332, formerly termed laminin-5) is a heterotrimeric glycoprotein that promotes cellular adhesion and migration. The heterotrimer consists of an α3, a β3, and a γ2 chain. The aim of this investigation was to clarify the clinicopathologic significance of laminin-332β3 (LNβ3) chain expression and determine its influence on survival in pancreatic ductal adenocarcinoma. Materials and Methods: Quantitative real-time polymerase chain reaction was used to validate and detect the expression of LNβ3 mRNA in 37 pancreatic carcinoma tissue specimens and non-neoplastic pancreatic tissue samples. In addition, the protein expression of LNβ3 was detected by immunohistochemistry methods in 96 pancreatic carcinoma specimens and 90 non-neoplastic pancreatic tissues. We analyzed the association between immunohistochemically detected LNβ3 expression in pancreatic ductal adenocarcinoma and clinicopathologic characteristics. Survival curves were completed using the Kaplan-Meier method and compared using log-rank analysis. Results: Quantitative real-time polymerase chain reaction indicated that the relative value of LNβ3 mRNA was 1.427±1.554 and 1.423±1.439 by 2−Δ(ΔCt) in pancreatic carcinoma and non-neoplastic pancreatic tissues, respectively, values that were not statistically associated (P=0.991). Immunostaining for LNβ3 was expressed in all patients with pancreatic ductal adenocarcinoma. LNβ3 expression was related to differentiation (P=0.000) and advanced stage (P=0.034). Tumors with low expression of LNβ3 had a survival advantage compared with tumors that had high expression of LNβ3 (P=0.016). Multivariate analysis indicated that location is an independent predictor of overall survival, whereas other clinicopathologic characteristics such as tumor size, duodenal invasion, differentiation, extent of invasion, hepatic metastasis, and expression of LNβ3 were not. Conclusion: Our results suggest that LNβ3 expression may play a key role in

  14. A homozygous nonsense mutation in the alpha 3 chain gene of laminin 5 (LAMA3) in Herlitz junctional epidermolysis bullosa: prenatal exclusion in a fetus at risk.

    PubMed

    McGrath, J A; Kivirikko, S; Ciatti, S; Moss, C; Dunnill, G S; Eady, R A; Rodeck, C H; Christiano, A M; Uitto, J

    1995-09-01

    Mutations in the three genes (LAMA3, LAMB3, and LAMC2) that encode the three chains (alpha 3, beta 3, and gamma 2, respectively) of laminin 5, a protein involved in epidermal-dermal adhesion, have been established as the genetic basis for the inherited blistering skin disorder, Herlitz junctional epidermolysis bullosa (H-JEB). In this study, we performed mutational analysis on genomic DNA from a child with H-JEB and identified a nonsense mutation in the alpha 3 chain gene (LAMA3) consisting of a homozygous C-to-T transition resulting in a premature termination codon (CGA-->TGA) on both alleles. The parents were shown to be heterozygous carriers of the same mutation. Direct mutation analysis was used to perform DNA-based prenatal diagnosis from a chorionic villus biopsy at 10 weeks' gestation in a subsequent pregnancy. The fetus was predicted to be genotypically normal with respect to the LAMA3 mutation. PMID:8530087

  15. Somatostatin receptors.

    PubMed

    Srikant, C B; Patel, Y C

    1985-01-01

    It is now well established that the biological actions of tetradecapeptide somatostatin (somatostatin-14, S-14) are receptor-mediated. These receptors were first quantified in GH4C pituitary tumor cells using [125I-Tyr1] S-14 as radioligand which was found to exhibit high non-specific binding to membrane receptor preparations from normal tissues. Our studies have shown that [125I-Tyr11] S-14 in which the radiolabel is situated away from the N-terminus exhibits significantly lower non-specific binding and therefore is more suitable for S-14 receptor studies. In the CNS, highest concentration of S-14 receptors was found in the cerebral cortex, followed by thalamus, hypothalamus, striatum, amygdala and hippocampus while medulla-pons, cerebellum and spinal cord exhibited negligible binding. Outside the CNS membrane receptors for S-14 have been characterized in pituitary, adrenal cortex and pancreatic acini. In all these tissues a single class of high affinity binding sites for S-14 were present, the receptors in pancreatic acinar cells exhibiting significantly greater affinity for binding S-14 than in other tissues.

  16. Murine laminin alpha3A and alpha3B isoform chains are generated by usage of two promoters and alternative splicing.

    PubMed

    Ferrigno, O; Virolle, T; Galliano, M F; Chauvin, N; Ortonne, J P; Meneguzzi, G; Aberdam, D

    1997-08-15

    We already identified two distinct laminin alpha3A and alpha3B chain isoforms which differ in their amino-terminal ends and display different tissue-specific expression patterns. In this study we have investigated whether these two different isoforms are products of the same laminin alpha3 (lama3) gene and transcribed from one or two separate promoters. Genomic clones were isolated that encompass the sequences upstream to the 5' ends of both the alpha3A and the alpha3B cDNAs. Sequence analysis of the region upstream to the alpha3A open reading frame revealed the presence of a TATA box and potential binding sites for responsive elements. By primer extension analysis, the transcription start site of the alpha3B mRNA isoform was defined. The sequences upstream to the alpha3B mRNA transcription start site do not contain a TATA box near the transcription initiation sites, but AP-1, AP-2, and Sp1 consensus binding site sequences were identified. The genomic regions located immediately upstream of the alpha3A and alpha3B transcription start sites were shown to possess promoter activities in transfection experiments. In the promoter regions, response elements for the acute phase reactant signal and NF-interleukin 6 were found, and their possible relevance in the context of inflammation and wound healing is discussed. Our results demonstrate that the lama3 gene produces the two polypeptides by alternative splicing and contains two promoters, which regulate the production of the two isoforms alpha3A and alpha3B. PMID:9252362

  17. Novel and recurrent mutations in the laminin-5 genes causing lethal junctional epidermolysis bullosa: molecular basis and clinical course of Herlitz disease.

    PubMed

    Mühle, Christiane; Jiang, Qiu-Jie; Charlesworth, Alexandra; Bruckner-Tuderman, Leena; Meneguzzi, Guerrino; Schneider, Holm

    2005-01-01

    Herlitz disease (H-JEB), the lethal form of junctional epidermolysis bullosa, is a rare genodermatosis presenting from birth with widespread erosions and blistering of skin and mucosae because of tissue cleavage within the epidermal basement membrane. Mutations in any of the three genes encoding the alpha3, beta3 and gamma2 chains of laminin-5 underlie this recessively inherited disorder. Here, we report the molecular basis and clinical course of H-JEB in 12 patients. Two novel nonsense mutations in the gene LAMA3 (E281X and K1299X) and a novel frame-shift mutation in the gene LAMB3 (1628insG) leading to a premature termination codon were identified by DNA sequencing and confirmed by restriction fragment length polymorphism analysis. In the four patients affected, neither the resulting truncated polypeptide chains nor assembled laminin-5 protein were detectable by immunofluorescence. Three patients were found to be heterozygous for the known hotspot mutation R635X and the recurrent mutations Q373X or 29insC in the gene LAMB3, whereas five others were homozygous for R635X. Significant variations in the disease progression and survival times between 1 and 30 months in this group of H-JEB patients emphasised the impact of modifying factors and the importance of immunostaining or mRNA assessment as parallel diagnostic methods. Interestingly, the only patients who survived for longer than 6 months were four females carrying the mutation R635X homozygously. In one of them, the clinical course may have been improved by treatment with artificial skin equivalents. These data may stimulate further investigation of genotype-phenotype correlations and facilitate mutation analysis and genetic counselling of affected families. PMID:15538630

  18. The role of a recombinant fragment of laminin-332 in integrin α3β1-dependent cell binding, spreading and migration

    PubMed Central

    Yamashita, Hironobu; Tripathi, Manisha; Harris, Mark P.; Liu, Shanshan; Weidow, Brandy; Zent, Roy; Quaranta, Vito

    2010-01-01

    The extracellular matrix (ECM) is thought to be an essential component of tissue scaffolding and engineering because it fulfills fundamental functions related to cell adhesion, migration, and three-dimensional organization. Natural ECM preparations, however, are challenging to work with because they are comprised of macromolecules that are large and insoluble in their functional state. Functional fragments of ECM macromolecules are a viable answer to this challenge, as demonstrated by the RGD-based engineered scaffolds, where the tri-peptide, Arg– Gly–Asp (RGD), represents the minimal functional unit of fibronectin and related ECM. Laminins (Ln) are main components of epithelial tissues, since they enter into the composition of basement membranes (BM). Application of Ln to epithelial tissue engineering would be desirable, since they could help mimic ideal functional conditions for both lining and glandular epithelial tissues. However, functional fragments of Ln that could be used in artificial settings have not been characterized in detail. In this paper, we describe the production and application of the recombinant LG4 (rLG4) fragment of laminin-332 (Ln-332), and show that it mimics three fundamental functional properties of Ln-332: integrin mediated cell adhesion, spreading, and migration. Adhesive structures formed by cells on rLG4 closely resemble those formed on Ln-332, as judged by microscopy-based analyses of their molecular composition. As on Ln-332, focal adhesion kinase (FAK) is phosphorylated in cells adhering to rLG4, and colocalized with other focal adhesion components. We conclude that rLG4 could be a useful substitute to recapitulate, in vitro, the tissue scaffolding properties of Ln-332. PMID:20347131

  19. Trypanosoma cruzi Binds to Cytokeratin through Conserved Peptide Motifs Found in the Laminin-G-Like Domain of the gp85/Trans-sialidase Proteins

    PubMed Central

    Teixeira, Andre Azevedo Reis; de Vasconcelos, Veronica de Cássia Sardinha; Colli, Walter; Alves, Maria Júlia Manso; Giordano, Ricardo José

    2015-01-01

    Background Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a disease that affects millions of people most of them living in South and Central Americas. There are few treatment options for individuals with Chagas' disease making it important to understand the molecular details of parasite infection, so novel therapeutic alternatives may be developed for these patients. Here, we investigate the interaction between host cell intermediate filament proteins and the T. cruzi gp85 glycoprotein superfamily with hundreds of members that have long been implicated in parasite cell invasion. Methodology/Principal Findings An in silico analysis was utilized to identify peptide motifs shared by the gp85 T. cruzi proteins and, using phage display, these selected peptide motifs were screened for their ability to bind to cells. One peptide, named TS9, showed significant cell binding capacity and was selected for further studies. Affinity chromatography, phage display and invasion assays revealed that peptide TS9 binds to cytokeratins and vimentin, and prevents T. cruzi cell infection. Interestingly, peptide TS9 and a previously identified binding site for intermediate filament proteins are disposed in an antiparallel β-sheet fold, present in a conserved laminin-G-like domain shared by all members of the family. Moreover, peptide TS9 overlaps with an immunodominant T cell epitope. Conclusions/Significance Taken together, the present study reinforces previous results from our group implicating the gp85 superfamily of glycoproteins and the intermediate filament proteins cytokeratin and vimentin in the parasite infection process. It also suggests an important role in parasite biology for the conserved laminin-G-like domain, present in all members of this large family of cell surface proteins. PMID:26398185

  20. EGF-receptor and extracellular matrix changes in mouse pulmonary carcinogenesis

    SciTech Connect

    Smith, G.J.; Morris, C.; Leigh, D.; Rhodes, G.C.; Wong, A. )

    1991-03-01

    Malignant Balb/c mouse lung cell clones related to alveologenic carcinoma exhibited low levels of epidermal growth factor (EGF) receptor activity compared to nonmalignant cell clones. Immunoprecipitation of cell homogenates and immunohistochemistry on urethane-induced lung tumors suggest that the absence of activity reflects decreased amounts of EGF receptor protein. Low levels of EGF receptor alone cannot cause neoplastic transformation, since a nonneoplastic cell cone, B5D3, exhibited low levels of EGF receptor despite its nontransformed phenotype. The reduced levels of EGF receptor in malignant clones have been mimicked by long-term (12 h) treatment of a nontransformed cell clone with 200 nM phorbol dibutyrate. The detection of mutated ras oncogene in the transformed cell lines, taken together with the EGF receptor findings, suggests that more than one alteration in the signal transduction pathway may be necessary for transformation in alveologenic adenoma and carcinoma cell systems. A further phenotypic feature of transformation, reduced expression of the extracellular matrix proteins fibronectin and laminin, may be mediated at the transcriptional level.

  1. Somatostatin receptors.

    PubMed

    Patel, Y C; Srikant, C B

    1997-12-01

    The diverse biological effects of somatostatin (SRIF) are mediated by a family of G protein-coupled receptors (termed sst) that are encoded by five nonallelic genes located on separate chromosomes. The receptors can be further divided into two subfamilies: sst(2,3,5) react with octapeptide and hexapeptide SRIF analogues and belong to one subclass; sst(1,4) react poorly with these compounds and fall into another subclass. This review focuses on the molecular pharmacology and function of these receptors, with particular emphasis on the ligand-binding domain, subtype-selective analogues, agonist-dependent receptor regulation and desensitization responses, subtype-specific effector coupling, and signal transduction pathways responsible for inhibiting cell secretion and cell growth or induction of apoptosis.

  2. Lipoxin receptors.

    PubMed

    Romano, Mario; Recchia, Irene; Recchiuti, Antonio

    2007-01-01

    Lipoxins (LXs) represent a class of arachidonic acid (AA) metabolites that carry potent immunoregulatory and anti-inflammatory properties, LXA4 and LXB4 being the main components of this series. LXs are generated by cooperation between 5-lipoxygenase (LO) and 12- or 15-LO during cell-cell interactions or by single cell types. LX epimers at carbon 15, the 15-epi-LXs, are formed by aspirin-acetylated cyclooxygenase-2 (COX-2) in cooperation with 5-LO. 15-epi-LXA4 is also termed aspirin-triggered LX (ATL). In vivo studies with stable LX and ATL analogs have established that these eicosanoids possess potent anti-inflammatory activities. A LXA4 receptor has been cloned. It belongs to the family of chemotactic receptors and clusters with formyl peptide receptors on chromosome 19. Therefore, it was initially denominated formyl peptide receptor like 1 (FPRL1). This receptor binds with high affinity and stereoselectivity LXA4 and ATL. It also recognizes a variety of peptides, synthetic, endogenously generated, or disease associated, but with lower affinity compared to LXA4. For this reason, this receptor has been renamed ALX. This review summarizes the current knowledge on ALX expression, signaling, and potential pathophysiological role. The involvement of additional recognition sites in LX bioactions is also discussed. PMID:17767357

  3. Matricryptins Network with Matricellular Receptors at the Surface of Endothelial and Tumor Cells

    PubMed Central

    Ricard-Blum, Sylvie; Vallet, Sylvain D.

    2016-01-01

    The extracellular matrix (ECM) is a source of bioactive fragments called matricryptins or matrikines resulting from the proteolytic cleavage of extracellular proteins (e.g., collagens, elastin, and laminins) and proteoglycans (e.g., perlecan). Matrix metalloproteinases (MMPs), cathepsins, and bone-morphogenetic protein-1 release fragments, which regulate physiopathological processes including tumor growth, metastasis, and angiogenesis, a pre-requisite for tumor growth. A number of matricryptins, and/or synthetic peptides derived from them, are currently investigated as potential anti-cancer drugs both in vitro and in animal models. Modifications aiming at improving their efficiency and their delivery to their target cells are studied. However, their use as drugs is not straightforward. The biological activities of these fragments are mediated by several receptor families. Several matricryptins may bind to the same matricellular receptor, and a single matricryptin may bind to two different receptors belonging or not to the same family such as integrins and growth factor receptors. Furthermore, some matricryptins interact with each other, integrins and growth factor receptors crosstalk and a signaling pathway may be regulated by several matricryptins. This forms an intricate 3D interaction network at the surface of tumor and endothelial cells, which is tightly associated with other cell-surface associated molecules such as heparan sulfate, caveolin, and nucleolin. Deciphering the molecular mechanisms underlying the behavior of this network is required in order to optimize the development of matricryptins as anti-cancer agents. PMID:26869928

  4. Matricryptins Network with Matricellular Receptors at the Surface of Endothelial and Tumor Cells.

    PubMed

    Ricard-Blum, Sylvie; Vallet, Sylvain D

    2016-01-01

    The extracellular matrix (ECM) is a source of bioactive fragments called matricryptins or matrikines resulting from the proteolytic cleavage of extracellular proteins (e.g., collagens, elastin, and laminins) and proteoglycans (e.g., perlecan). Matrix metalloproteinases (MMPs), cathepsins, and bone-morphogenetic protein-1 release fragments, which regulate physiopathological processes including tumor growth, metastasis, and angiogenesis, a pre-requisite for tumor growth. A number of matricryptins, and/or synthetic peptides derived from them, are currently investigated as potential anti-cancer drugs both in vitro and in animal models. Modifications aiming at improving their efficiency and their delivery to their target cells are studied. However, their use as drugs is not straightforward. The biological activities of these fragments are mediated by several receptor families. Several matricryptins may bind to the same matricellular receptor, and a single matricryptin may bind to two different receptors belonging or not to the same family such as integrins and growth factor receptors. Furthermore, some matricryptins interact with each other, integrins and growth factor receptors crosstalk and a signaling pathway may be regulated by several matricryptins. This forms an intricate 3D interaction network at the surface of tumor and endothelial cells, which is tightly associated with other cell-surface associated molecules such as heparan sulfate, caveolin, and nucleolin. Deciphering the molecular mechanisms underlying the behavior of this network is required in order to optimize the development of matricryptins as anti-cancer agents.

  5. alpha6beta1-Integrin, a major cell surface carrier of beta1-6-branched oligosaccharides, mediates migration of EJ-ras-transformed fibroblasts on laminin-1 independently of its glycosylation state.

    PubMed

    Jasiulionis, M G; Chammas, R; Ventura, A M; Travassos, L R; Brentani, R R

    1996-04-01

    EJ-ras oncogene-induced malignant transformation is characterized by a series of changes in cell surface carbohydrates and cell-cell and cell-matrix interactions. Here, we show that EJ-ras-transformed NIH-3T3 fibroblasts acquired a migratory phenotype on laminin-1 surfaces. Such a phenotype was accompanied by overexpression of: (a) functional alpha6beta1, but not other laminin binding beta1-integrins; and (b) glycoconjugates on the cell surface bearing large oligosaccharides recognized by leukoagglutinin from Phaseolus vulgaris (L-PHA). The internal pool of pre-beta1-integrins was differently regulated in EJ-ras-transformed cells compared with nontransfected fibroblasts. Conversion of pre-beta1- into mature beta1-integrins was faster in EJ-ras-transformed cells, a process associated with the overexpression of the alpha6-chain. Overexpression of L-PHA-reactive oligosaccharides is dependent on the activity of N-acetylglucosaminyltransferase V, which is increased in transformed cells [J. W. Dennis et al., Science (Washington DC), 236: 582-585, 1987]. We show that beta1-integrins were the major carriers of L-PHA-reactive oligosaccharides on the cell surface. This glycosylation pattern, however, was not necessary for either the cell surface expression of beta1-integrins or their functional activity in the migratory response to laminin-1. Moreover, EJ-ras-transformed fibroblasts aggregated spontaneously. These effects were not observed in c-jun-transfected fibroblasts, which were unable to migrate on laminin, did not overexpress either beta1-integrins or L-PHA-reactive oligosaccharides, and did not self-aggregate.

  6. Differential cytokine modulation of the genes LAMA3, LAMB3, and LAMC2, encoding the constitutive polypeptides, alpha 3, beta 3, and gamma 2, of human laminin 5 in epidermal keratinocytes.

    PubMed

    Korang, K; Christiano, A M; Uitto, J; Mauviel, A

    1995-07-24

    Laminin 5, an anchoring filament protein previously known as nicein/kalinin/epiligrin, consists of three polypeptide chains, alpha 3, beta 3, and gamma 2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. The expression of laminin 5 was detected by Northern hybridization with specific cDNA probes in various epidermal keratinocyte cultures, whereas no expression of any of the three genes could be detected in foreskin fibroblast cultures. Transforming growth factor-beta (TGF-beta) enhanced LAMA3, LAMB3, and LAMC2 gene expression in human epidermal keratinocytes, as well as in HaCaT and Balb/K cells in culture, although the extent of enhancement was greater for LAMA3 and LAMC2 genes than for LAMB3. Interestingly, tumor necrosis factor-alpha, (TNF-alpha) alone did not alter the expression of LAMB3 and LAMC2 genes in human epidermal keratinocytes, whereas it inhibited the expression of LAMA3. These results suggest that the expression of the three genes encoding the laminin 5 subunits is not coordinately regulated by the cytokines tested. PMID:7635220

  7. Vascular Delivery of rAAVrh74.MCK.GALGT2 to the Gastrocnemius Muscle of the Rhesus Macaque Stimulates the Expression of Dystrophin and Laminin α2 Surrogates

    PubMed Central

    Chicoine, Louis G.; Rodino-Klapac, Louise R.; Shao, Guohong; Xu, Rui; Bremer, William G.; Camboni, Marybeth; Golden, Bethannie; Montgomery, Chrystal L.; Shontz, Kimberly; Heller, Kristin N.; Griffin, Danielle A.; Lewis, Sarah; Coley, Brian D.; Walker, Christopher M.; Clark, K. Reed; Sahenk, Zarife; Mendell, Jerry R.; Martin, Paul T.

    2014-01-01

    Overexpression of GALGT2 in skeletal muscle can stimulate the glycosylation of α dystroglycan and the upregulation of normally synaptic dystroglycan-binding proteins, some of which are dystrophin and laminin α2 surrogates known to be therapeutic for several forms of muscular dystrophy. This article describes the vascular delivery of GALGT2 gene therapy in a large animal model, the rhesus macaque. Recombinant adeno-associated virus, rhesus serotype 74 (rAAVrh74), was used to deliver GALGT2 via the femoral artery to the gastrocnemius muscle using an isolated focal limb perfusion method. GALGT2 expression averaged 44 ± 4% of myofibers after treatment in macaques with low preexisting anti-rAAVrh74 serum antibodies, and expression was reduced to 9 ± 4% of myofibers in macaques with high preexisting rAAVrh74 immunity (P < 0.001; n = 12 per group). This was the case regardless of the addition of immunosuppressants, including prednisolone, tacrolimus, and mycophenolate mofetil. GALGT2-treated macaque muscles showed increased glycosylation of α dystroglycan and increased expression of dystrophin and laminin α2 surrogate proteins, including utrophin, plectin1, agrin, and laminin α5. These experiments demonstrate successful transduction of rhesus macaque muscle with rAAVrh74.MCK.GALGT2 after vascular delivery and induction of molecular changes thought to be therapeutic in several forms of muscular dystrophy. PMID:24145553

  8. Membrane glucocorticoid receptors are localised in the extracellular matrix and signal through the MAPK pathway in mammalian skeletal muscle fibres

    PubMed Central

    Boncompagni, Simona; Arthurton, Lewis; Akujuru, Eugene; Pearson, Timothy; Steverding, Dietmar; Protasi, Feliciano; Mutungi, Gabriel

    2015-01-01

    A number of studies have previously proposed the existence of glucocorticoid receptors on the plasma membrane of many cell types, including skeletal muscle fibres. However, their exact localisation and the cellular signalling pathway(s) they utilise to communicate with the rest of the cell are still poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the non-genomic physiological functions of these receptors in mouse skeletal muscle cells. The results show that the receptors were localised in the cytoplasm in myoblasts, in the nucleus in myotubes, in the extracellular matrix, in satellite cells and in the proximity of mitochondria in adult muscle fibres. Also, they bound laminin in a glucocorticoid-dependent manner. Treating small skeletal muscle fibre bundles with the synthetic glucocorticoid beclomethasone dipropionate increased the phosphorylation (= activation) of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. This occurred within 5 min and depended on the fibre type and the duration of the treatment. It was also abolished by the glucocorticoid receptor inhibitor, mifepristone, and a monoclonal antibody against the receptor. From these results we conclude that the non-genomic/non-canonical physiological functions of glucocorticoids, in adult skeletal muscle fibres, are mediated by a glucocorticoid receptor localised in the extracellular matrix, in satellite cells and close to mitochondria, and involve activation of the mitogen-activated protein kinase pathway. PMID:25846902

  9. A dystonia-like movement disorder with brain and spinal neuronal defects is caused by mutation of the mouse laminin β1 subunit, Lamb1

    PubMed Central

    Liu, Yi Bessie; Tewari, Ambika; Salameh, Johnny; Arystarkhova, Elena; Hampton, Thomas G; Brashear, Allison; Ozelius, Laurie J; Khodakhah, Kamran; Sweadner, Kathleen J

    2015-01-01

    A new mutant mouse (lamb1t) exhibits intermittent dystonic hindlimb movements and postures when awake, and hyperextension when asleep. Experiments showed co-contraction of opposing muscle groups, and indicated that symptoms depended on the interaction of brain and spinal cord. SNP mapping and exome sequencing identified the dominant causative mutation in the Lamb1 gene. Laminins are extracellular matrix proteins, widely expressed but also known to be important in synapse structure and plasticity. In accordance, awake recording in the cerebellum detected abnormal output from a circuit of two Lamb1-expressing neurons, Purkinje cells and their deep cerebellar nucleus targets, during abnormal postures. We propose that dystonia-like symptoms result from lapses in descending inhibition, exposing excess activity in intrinsic spinal circuits that coordinate muscles. The mouse is a new model for testing how dysfunction in the CNS causes specific abnormal movements and postures. DOI: http://dx.doi.org/10.7554/eLife.11102.001 PMID:26705335

  10. Molecular detection of noninvasive and invasive bladder tumor tissues and exfoliated cells by aberrant promoter methylation of laminin-5 encoding genes.

    PubMed

    Sathyanarayana, Ubaradka G; Maruyama, Riichiroh; Padar, Asha; Suzuki, Makoto; Bondaruk, Jolanta; Sagalowsky, Arthur; Minna, John D; Frenkel, Eugene P; Grossman, H Barton; Czerniak, Bogdan; Gazdar, Adi F

    2004-02-15

    Laminin-5 (LN5) anchors epithelial cells to the underlying basement membrane, and it is encoded by three distinct genes: LAMA3, LAMB3, and LAMC2. To metastasize and grow, cancer cells must invade and destroy the basement membrane. Our previous work has shown that epigenetic inactivation is a major mechanism of silencing LN5 genes in lung cancers. We extended our methylation studies to resected bladder tumors (n = 128) and exfoliated cell samples (bladder washes and voided urine; n = 71) and correlated the data with clinicopathologic findings. Nonmalignant urothelium had uniform expression of LN5 genes and lacked methylation. The methylation frequencies for LN5 genes in tumors were 21-45%, and there was excellent concordance between methylation in tumors and corresponding exfoliated cells. Methylation of LAMA3 and LAMB3 and the methylation index were correlated significantly with several parameters of poor prognosis (tumor grade, growth pattern, muscle invasion, tumor stage, and ploidy pattern), whereas methylation of LAMC2 and methylation index were associated with shortened patient survival. Of particular interest, methylation frequencies of LAMA3 helped to distinguish invasive (72%) from noninvasive (12%) tumors. These results suggest that methylation of LN5 genes has potential clinical applications in bladder cancers. PMID:14973053

  11. Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

    PubMed

    Rodin, Sergey; Antonsson, Liselotte; Hovatta, Outi; Tryggvason, Karl

    2014-10-01

    A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

  12. Self-enhanced N-(aminobutyl)-N-(ethylisoluminol) derivative-based electrochemiluminescence immunosensor for sensitive laminin detection using PdIr cubes as a mimic peroxidase

    NASA Astrophysics Data System (ADS)

    Jiang, Xinya; Wang, Huijun; Wang, Haijun; Zhuo, Ying; Yuan, Ruo; Chai, Yaqin

    2016-04-01

    Herein, a self-enhanced N-(aminobutyl)-N-(ethylisoluminol) (ABEI) derivative-based electrochemiluminescence (ECL) immunosensor was constructed for the determination of laminin (LN) using PdIr cubes as a mimic peroxidase for signal amplification. Initially, PdIr cubes with efficient peroxidase mimicking properties, large specific surface areas, and good stability and uniformity were synthesized. Then, l-cysteine (l-Cys) and ABEI were immobilized on the PdIr cubes to form the self-enhanced ECL nanocomplex (PdIr-l-Cys-ABEI). In this nanocomplex, PdIr cubes, whose catalytic constant is higher than that of horseradish peroxidase (HRP), could effectively catalyze H2O2 decomposition and thus enhance the ECL intensity of ABEI. Moreover, PdIr cubes can be easily modified with functional groups, which make them adaptable to desired supported platforms. On the other hand, l-Cys as a coreactant of ABEI could effectively enhance the luminous efficiency due to the intramolecular ECL reaction which could reduce the energy loss between l-Cys and ABEI by giving a shorter electron transfer distance. The developed strategy combined an ABEI derivative as a self-enhanced ECL luminophore and PdIr cubes as a mimic peroxidase, resulting in a significantly enhanced ECL signal output. Also, the strategy showed high sensitivity and selectivity for LN, which suggested that our new approach could be potentially applied in monitoring different proteins.

  13. Self-enhanced N-(aminobutyl)-N-(ethylisoluminol) derivative-based electrochemiluminescence immunosensor for sensitive laminin detection using PdIr cubes as a mimic peroxidase.

    PubMed

    Jiang, Xinya; Wang, Huijun; Wang, Haijun; Zhuo, Ying; Yuan, Ruo; Chai, Yaqin

    2016-04-21

    Herein, a self-enhanced N-(aminobutyl)-N-(ethylisoluminol) (ABEI) derivative-based electrochemiluminescence (ECL) immunosensor was constructed for the determination of laminin (LN) using PdIr cubes as a mimic peroxidase for signal amplification. Initially, PdIr cubes with efficient peroxidase mimicking properties, large specific surface areas, and good stability and uniformity were synthesized. Then, L-cysteine (L-Cys) and ABEI were immobilized on the PdIr cubes to form the self-enhanced ECL nanocomplex (PdIr-L-Cys-ABEI). In this nanocomplex, PdIr cubes, whose catalytic constant is higher than that of horseradish peroxidase (HRP), could effectively catalyze H2O2 decomposition and thus enhance the ECL intensity of ABEI. Moreover, PdIr cubes can be easily modified with functional groups, which make them adaptable to desired supported platforms. On the other hand, L-Cys as a coreactant of ABEI could effectively enhance the luminous efficiency due to the intramolecular ECL reaction which could reduce the energy loss between L-Cys and ABEI by giving a shorter electron transfer distance. The developed strategy combined an ABEI derivative as a self-enhanced ECL luminophore and PdIr cubes as a mimic peroxidase, resulting in a significantly enhanced ECL signal output. Also, the strategy showed high sensitivity and selectivity for LN, which suggested that our new approach could be potentially applied in monitoring different proteins.

  14. A homozygous nonsense mutation in the {alpha}3 chain gene of laminin 5 (LAMA3) in Herlitz junctional epidermolysis bullosa: Prenatal exclusion in a fetus at risk

    SciTech Connect

    McGrath, J.A. |; Ciatti, S.; Christiano, A.M.

    1995-09-01

    Mutations in the three genes (LAMA3, LAMB3, and LAMC2) that encode the three chains ({alpha}3, {Beta}3, and {gamma}2, respectively) of laminin 5, a protein involved in epidermal-dermal adhesion, have been established as the genetic basis for the inherited blistering skin disorder, Herlitz junctional epidermolysis bullosa (H-JEB). In this study, we performed mutational analysis on genomic DNA from a child with H-JEB and identified a nonsense mutation in the {alpha}3 chain gene (LAMA3) consisting of a homozygous C-to-T transition resulting in a premature termination codon (CGA {r_arrow} TGA) on both alleles. The parents were shown to be heterozygous carriers of the same mutation. Direct mutation analysis was used to perform DNA-based prenatal diagnosis from a chorionic villus biopsy at 10 weeks` gestation in a subsequent pregnancy. The fetus was predicted to be genotypically normal with respect to the LAMA3 mutation. 15 refs., 1 fig.

  15. The laminin binding protein p40 is involved in inducing limb abnormality of mouse fetuses as the effects of methoxyacetic acid treatment.

    PubMed

    Ruyani, Aceng; Sudarwati, Sri; Sutasurya, Lien A; Sumarsono, Sony H; Gloe, Torsten

    2003-09-01

    This study is intended to characterize a protein that is linked with mouse limb teratogenicity as the effects of methoxyacetic acid (MAA) treatment. A single dose of MAA (10 mmol/kg body weight) was given by gavage on gestation day (GD) 11, whereas the control group were administered vehicle only. The pregnant mice were killed at 4 h after MAA treatment, and forelimb buds were isolated from both the control and treated group embryos. Proteins from forelimb buds GD 11 + 4 h, which were precipitated out using 40-60% ammonium sulfate, then were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) technique. The 2-D gels reveal one protein with 41.6 kDa and pI 6.4, which expression was downregulated after MAA treatment. Tentative protein identification via peptide mass database search and definitive protein identification via a primary sequence database search indicate that the protein matches exactly to 34/67 kDa laminin binding protein (LBP; P14206, SwissProt), which is encoded by p40 gene (MGI:105381). The identity was further verified by Western blotting with an antibody against the 67 kDa LBP. The results suggest that MAA treatment to pregnant mice downregulates the LBP-p40 in the forelimb buds.

  16. A modular, plasmin-sensitive, clickable poly(ethylene glycol)-heparin-laminin microsphere system for establishing growth factor gradients in nerve guidance conduits.

    PubMed

    Roam, Jacob L; Yan, Ying; Nguyen, Peter K; Kinstlinger, Ian S; Leuchter, Michael K; Hunter, Daniel A; Wood, Matthew D; Elbert, Donald L

    2015-12-01

    Peripheral nerve regeneration is a complex problem that, despite many advancements and innovations, still has sub-optimal outcomes. Compared to biologically derived acellular nerve grafts and autografts, completely synthetic nerve guidance conduits (NGC), which allow for precise engineering of their properties, are promising but still far from optimal. We have developed an almost entirely synthetic NGC that allows control of soluble growth factor delivery kinetics, cell-initiated degradability and cell attachment. We have focused on the spatial patterning of glial-cell derived human neurotrophic factor (GDNF), which promotes motor axon extension. The base scaffolds consisted of heparin-containing poly(ethylene glycol) (PEG) microspheres. The modular microsphere format greatly simplifies the formation of concentration gradients of reversibly bound GDNF. To facilitate axon extension, we engineered the microspheres with tunable plasmin degradability. 'Click' cross-linking chemistries were also added to allow scaffold formation without risk of covalently coupling the growth factor to the scaffold. Cell adhesion was promoted by covalently bound laminin. GDNF that was released from these microspheres was confirmed to retain its activity. Graded scaffolds were formed inside silicone conduits using 3D-printed holders. The fully formed NGC's contained plasmin-degradable PEG/heparin scaffolds that developed linear gradients in reversibly bound GDNF. The NGC's were implanted into rats with severed sciatic nerves to confirm in vivo degradability and lack of a major foreign body response. The NGC's also promoted robust axonal regeneration into the conduit.

  17. A modular, plasmin-sensitive, clickable poly(ethylene glycol)-heparin-laminin microsphere system for establishing growth factor gradients in nerve guidance conduits.

    PubMed

    Roam, Jacob L; Yan, Ying; Nguyen, Peter K; Kinstlinger, Ian S; Leuchter, Michael K; Hunter, Daniel A; Wood, Matthew D; Elbert, Donald L

    2015-12-01

    Peripheral nerve regeneration is a complex problem that, despite many advancements and innovations, still has sub-optimal outcomes. Compared to biologically derived acellular nerve grafts and autografts, completely synthetic nerve guidance conduits (NGC), which allow for precise engineering of their properties, are promising but still far from optimal. We have developed an almost entirely synthetic NGC that allows control of soluble growth factor delivery kinetics, cell-initiated degradability and cell attachment. We have focused on the spatial patterning of glial-cell derived human neurotrophic factor (GDNF), which promotes motor axon extension. The base scaffolds consisted of heparin-containing poly(ethylene glycol) (PEG) microspheres. The modular microsphere format greatly simplifies the formation of concentration gradients of reversibly bound GDNF. To facilitate axon extension, we engineered the microspheres with tunable plasmin degradability. 'Click' cross-linking chemistries were also added to allow scaffold formation without risk of covalently coupling the growth factor to the scaffold. Cell adhesion was promoted by covalently bound laminin. GDNF that was released from these microspheres was confirmed to retain its activity. Graded scaffolds were formed inside silicone conduits using 3D-printed holders. The fully formed NGC's contained plasmin-degradable PEG/heparin scaffolds that developed linear gradients in reversibly bound GDNF. The NGC's were implanted into rats with severed sciatic nerves to confirm in vivo degradability and lack of a major foreign body response. The NGC's also promoted robust axonal regeneration into the conduit. PMID:26352518

  18. Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

    PubMed

    Rodin, Sergey; Antonsson, Liselotte; Hovatta, Outi; Tryggvason, Karl

    2014-10-01

    A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities. PMID:25211513

  19. 3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) disrupts blood-brain barrier integrity through a mechanism involving P2X7 receptors.

    PubMed

    Rubio-Araiz, Ana; Perez-Hernandez, Mercedes; Urrutia, Andrés; Porcu, Francesca; Borcel, Erika; Gutierrez-Lopez, Maria Dolores; O'Shea, Esther; Colado, Maria Isabel

    2014-08-01

    The recreational drug 3,4-methylenedioxymethamphetamine (MDMA; 'ecstasy') produces a neuro-inflammatory response in rats characterized by an increase in microglial activation and IL-1β levels. The integrity of the blood-brain barrier (BBB) is important in preserving the homeostasis of the brain and has been shown to be affected by neuro-inflammatory processes. We aimed to study the effect of a single dose of MDMA on the activity of metalloproteinases (MMPs), expression of extracellular matrix proteins, BBB leakage and the role of the ionotropic purinergic receptor P2X7 (P2X7R) in the changes induced by the drug. Adult male Dark Agouti rats were treated with MDMA (10 mg/kg, i.p.) and killed at several time-points in order to evaluate MMP-9 and MMP-3 activity in the hippocampus and laminin and collagen-IV expression and IgG extravasation in the dentate gyrus. Microglial activation, P2X7R expression and localization were also determined in the dentate gyrus. Separate groups were treated with MDMA and the P2X7R antagonists Brilliant Blue G (BBG; 50 mg/kg, i.p.) or A-438079 (30 mg/kg, i.p.). MDMA increased MMP-3 and MMP-9 activity, reduced laminin and collagen-IV expression and increased IgG immunoreactivity. In addition, MDMA increased microglial activation and P2X7R immunoreactivity in these cells. BBG suppressed the increase in MMP-9 and MMP-3 activity, prevented basal lamina degradation and IgG extravasation into the brain parenchyma. A-438079 also prevented the MDMA-induced reduction in laminin and collagen-IV immunoreactivity. These results indicate that MDMA alters BBB permeability through an early P2X7R-mediated event, which in turn leads to enhancement of MMP-9 and MMP-3 activity and degradation of extracellular matrix.

  20. Results of a two-year pilot study of clinical outcome measures in collagen VI- and laminin alpha2-related congenital muscular dystrophies.

    PubMed

    Meilleur, Katherine G; Jain, Minal S; Hynan, Linda S; Shieh, Ching-Yi; Kim, Eunice; Waite, Melissa; McGuire, Michelle; Fiorini, Courtney; Glanzman, Allan M; Main, Marion; Rose, Kristy; Duong, Tina; Bendixen, Roxanna; Linton, Melody M; Arveson, Irene C; Nichols, Carmel; Yang, Kelly; Fischbeck, Kenneth H; Wagner, Kathryn R; North, Kathryn; Mankodi, Ami; Grunseich, Christopher; Hartnett, Elizabeth J; Smith, Michaele; Donkervoort, Sandra; Schindler, Alice; Kokkinis, Angela; Leach, Meganne; Foley, A Reghan; Collins, James; Muntoni, Francesco; Rutkowski, Anne; Bönnemann, Carsten G

    2015-01-01

    Potential therapies are currently under development for two congenital muscular dystrophy (CMD) subtypes: collagen VI-related muscular dystrophy (COL6-RD) and laminin alpha 2-related dystrophy (LAMA2-RD). However, appropriate clinical outcome measures to be used in clinical trials have not been validated in CMDs. We conducted a two-year pilot study to evaluate feasibility, reliability, and validity of various outcome measures, particularly the Motor Function Measure 32, in 33 subjects with COL6-RD and LAMA2-RD. In the first year, outcome measures tested included: Motor Function Measure 32 (MFM32), forced vital capacity (FVC) percent predicted sitting, myometry, goniometry, 10-meter walk, Egen Klassification 2, and PedsQL(TM) Generic and Neuromuscular Cores. In the second year, we added the North Star Ambulatory Assessment (NSAA), Hammersmith Functional Motor Scale (HFMS), timed functional tests, Measure of Activity Limitations (ACTIVLIM), Quality of Upper Extremity Skills Test (QUEST), and Patient-Reported Outcomes Measurement Information System (PROMIS) fatigue subscale. The MFM32 showed strong inter-rater (0.92) and internal consistency (0.96) reliabilities. Concurrent validity for the MFM32 was supported by large correlations (range 0.623-0.936) with the following: FVC, NSAA, HFMS, timed functional tests, ACTIVLIM, and QUEST. Significant correlations of the MFM32 were also found with select myometry measurements, mainly of the proximal extremities and domains of the PedsQL(TM) scales focusing on physical health and neuromuscular disease. Goniometry measurements were less reliable. The Motor Function Measure is reliable and valid in the two specific subtypes of CMD evaluated, COL6-RD and LAMA2-RD. The NSAA is useful as a complementary outcome measure in ambulatory individuals. Preliminary concurrent validity of several other clinical outcome measures was also demonstrated for these subtypes. PMID:25307854

  1. The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein.

    PubMed

    Miranda-Ozuna, Jesús F T; Hernández-García, Mar S; Brieba, Luis G; Benítez-Cardoza, Claudia G; Ortega-López, Jaime; González-Robles, Arturo; Arroyo, Rossana

    2016-10-01

    Triosephosphate isomerase of Trichomonas vaginalis (TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2, that participates in glucose metabolism. TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated TvTIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of TvTIM in T. vaginalis Affinity ligand assays demonstrated that the recombinant TvTIM1 and TvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r and TvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that TvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis. PMID:27481251

  2. Translating human embryonic stem cells from 2-dimensional to 3-dimensional cultures in a defined medium on laminin- and vitronectin-coated surfaces.

    PubMed

    Heng, Boon Chin; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul; Cool, Simon M; Birch, William R; Oh, Steve Kah-Weng

    2012-07-01

    While defining the environment for human embryonic stem cell (hESC) culture on 2-dimensional (2D) surfaces has made rapid progress, the industrial-scale implementation of this technology will benefit from translating this knowledge into a 3-dimensional (3D) system, thus enabling better control, automation, and volumetric scale-up in bioreactors. The current study describes a system with defined conditions that are capable of supporting the long-term 2D culture of hESCs and the transposing of these conditions to 3D microcarrier (MC) cultures. Vitronectin (VN) and laminin (LN) were chosen as matrices for the long-term propagation of hESCs in a defined culture medium (STEMPRO(®)) for conventional 2D culture. Adsorption of these proteins onto 2D tissue culture polystyrene (TCPS) indicated that surface density saturation of 510 and 850 ng/cm(2) for VN and LN, respectively, was attained above 20 μg/mL deposition solution concentration. Adsorption of these proteins onto spherical (97±10 μm), polystyrene MC followed a similar trend and coating surface densities of 450 and 650 ng/cm(2) for VN and LN, respectively, were used to support hESC propagation. The long-term expansion of hESCs was equally successful on TCPS and MC, with consistently high expression (>90%) of pluripotent markers (OCT-4, MAB-84, and TRA-1-60) over 20 passages and maintenance of karyotypic normality. The average fold increase in cell numbers on VN-coated MC per serial passage was 8.5±1.0, which was similar to LN-coated MC (8.5±0.9). Embryoid body differentiation assays and teratoma formation confirmed that hESCs retained the ability to differentiate into lineages of all 3 germ layers, thus demonstrating the first translation to a fully defined MC-based environment for the expansion of hESCs.

  3. Inhibition of vimentin or B1 integrin reverts morphology of prostate tumor cells grown in laminin-rich extracellular matrix gels and reduces tumor growth in vivo

    SciTech Connect

    Zhang, Xueping; Fournier, Marcia V; Ware, Joy L; Bissell, Mina J; Yacoub, Adly; Zehner, Zendra E

    2008-06-12

    Prostate epithelial cells grown embedded in laminin-rich extracellular matrix (lrECM) undergo morphologic changes that closely resemble their architecture in vivo. In this study, growth characteristics of three human prostate epithelial sublines derived from the same cellular lineage, but displaying different tumorigenic and metastatic properties in vivo, were assessed in three-dimensional lrECM gels. M12, a highly tumorigenic and metastatic subline, was derived from the immortalized, prostate epithelial P69 cell line by selection in athymic, nude mice and found to contain a deletion of 19p-q13.1. The stable reintroduction of an intact human chromosome 19 into M12 resulted in a poorly tumorigenic subline, designated F6. When embedded in lrECM gels, the parental, nontumorigenic P69 line produced acini with clearly defined lumena. Immunostaining with antibodies to {beta}-catenin, E-cadherin, or {alpha}6 and {beta}1 integrins showed polarization typical of glandular epithelium. In contrast, the metastatic M12 subline produced highly disorganized cells with no evidence of polarization. The F6 subline reverted to acini-like structures exhibiting basal polarity marked with integrins. Reducing either vimentin levels via small interfering RNA interference or the expression of {alpha}6 and {beta}1 integrins by the addition of blocking antibodies, reorganized the M12 subline into forming polarized acini. The loss of vimentin significantly reduced M12-Vim tumor growth when assessed by s.c. injection in athymic mice. Thus, tumorigenicity in vivo correlated with disorganized growth in three-dimensional lrECM gels. These studies suggest that the levels of vimentin and {beta}1 integrin play a key role in the homeostasis of the normal acinus in prostate and that their dysregulation may lead to tumorigenesis. [Mol Cancer Ther 2009;8(3):499-508].

  4. The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein.

    PubMed

    Miranda-Ozuna, Jesús F T; Hernández-García, Mar S; Brieba, Luis G; Benítez-Cardoza, Claudia G; Ortega-López, Jaime; González-Robles, Arturo; Arroyo, Rossana

    2016-10-01

    Triosephosphate isomerase of Trichomonas vaginalis (TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes, tvtim1 and tvtim2, that participates in glucose metabolism. TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associated TvTIM in T. vaginalis and to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that the tvtim genes were differentially expressed in response to glucose concentration. tvtim1 was overexpressed under glucose-restricted (GR) conditions, whereas tvtim2 was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization of TvTIM in T. vaginalis Affinity ligand assays demonstrated that the recombinant TvTIM1 and TvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r and TvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover, T. vaginalis was exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate that TvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor of T. vaginalis.

  5. Identification of Novel Laminin- and Fibronectin-binding Proteins by Far-Western Blot: Capturing the Adhesins of Streptococcus suis Type 2

    PubMed Central

    Li, Quan; Liu, Hanze; Du, Dechao; Yu, Yanfei; Ma, Caifeng; Jiao, Fangfang; Yao, Huochun; Lu, Chengping; Zhang, Wei

    2015-01-01

    Bacterial cell wall (CW) and extracellular (EC) proteins are often involved in interactions with extracellular matrix (ECM) proteins such as laminin (LN) and fibronectin (FN), which play important roles in adhesion and invasion. In this study, an efficient method combining proteomic analysis and Far-Western blotting assays was developed to screen directly for bacterial surface proteins with LN- and FN-binding capacity. With this approach, fifteen potential LN-binding proteins and five potential FN-binding proteins were identified from Streptococcus suis serotype 2 (SS2) CW and EC proteins. Nine newly identified proteins, including oligopeptide-binding protein OppA precursor (OppA), elongation factor Tu (EF-Tu), enolase, lactate dehydrogenase (LDH), fructose-bisphosphate aldolase (FBA), 3-ketoacyl-ACP reductase (KAR), Gly ceraldehyde-3-phosphate dehydrogenase (GAPDH), Inosine 5′-monophosphate dehydrogenase (IMPDH), and amino acid ABC transporter permease (ABC) were cloned, expressed, purified and further confirmed by Far-Western blotting and ELISA. Five proteins (OppA, EF-Tu, enolase, LDH, and FBA) exhibited specifically binding activity to both human LN and human FN. Furthermore, seven important recombinant proteins were selected and identified to have the ability to bind Hep-2 cells by the indirect immunofluorescent assay. In addition, four recombinant proteins, and their corresponding polyclonal antibodies, were observed to decrease SS2 adhesion to Hep-2 cells, which indicates that these proteins contribute to the adherence of SS2 to host cell surface. Collectively, these results show that the approach described here represents a useful tool for investigating the host-pathogen interactions. PMID:26636044

  6. Covalent co-immobilization of heparin/laminin complex that with different concentration ratio on titanium surface for selectively direction of platelets and vascular cells behavior

    NASA Astrophysics Data System (ADS)

    Wang, Jian; Chen, Yuan; Liu, Tao; Wang, Xue; Liu, Yang; Wang, Yuan; Chen, Junying; Huang, Nan

    2014-10-01

    Surface biofunctional modification of coronary artery stent to improve the hemocompatibility and selectively accelerate endothelium regeneration but prevent restenosis have been become a new hotspot. For this, a novel method was developed in this work by co-immobilization of Ln and heparin complex on poly-L-lysine modified Ti surface. Take the advantage of the specific interaction between Ln and heparin, Ln and heparin complexes with different concentration ratios were set up for creating different exposure density of these two types of biomolecules. According to biocompatibility evaluation results, the Hep/Ln complexes modified surface displayed less platelet adhesion and activation. Especially, on L(150)H and L(200)H surface, the AT III binding quantity, APTT value and anti-coagulation property of modified surface were significantly promoted. Furthermore, the adherent density and proliferation activity of ECs and EPCs were positively correlated with Ln concentration. Notably, the proliferation of both ECs and EPCs on L(100)H, L(150)H and L(200)H surface were greatly promoted. Another hand, the proliferation activity of SMCs was significantly inhibited on Hep/Ln modified surfaces, which was considered mainly due to the inhibitory effect of heparin to SMCs. According to the existing results, this study demonstrated that in a certain range of heparin and laminin concentration ratio, the biological behavior of platelets, ECs, EPCs and SMCs could be selectively directed. We suggested that this article provided a potential method to construct an adequate platform on a stent surface for accelerate endothelialization with low side effects.

  7. Tissue engineering of composite grafts: Cocultivation of human oral keratinocytes and human osteoblast-like cells on laminin-coated polycarbonate membranes and equine collagen membranes under different culture conditions.

    PubMed

    Glaum, R; Wiedmann-Al-Ahmad, M; Huebner, U; Schmelzeisen, R

    2010-05-01

    In complex craniomaxillofacial defects, the simultaneous reconstruction of hard and soft tissue is often necessary. Until now, oral keratinocytes and osteoblast-like cells have not been cocultivated on the same carrier. For the first time, the cocultivation of human oral keratinocytes and human osteoblast-like cells has been investigated in this study. Different carriers (laminin-coated polycarbonate and equine collagen membranes) and various culture conditions were examined. Human oral keratinocytes and human osteoblast-like cells from five patients were isolated from tissue samples, seeded on the opposite sides of the carriers and cultivated for 1 and 2 weeks under static conditions in an incubator and in a perfusion chamber. Proliferation and morphology of the cells were analyzed by EZ4U-tests, light microscopy, and scanning electron microscopy. Cocultivation of both cell-types seeded on one carrier was possible. Quantitative and qualitative growth was significantly better on collagen membranes when compared with laminin-coated polycarbonate membranes independent of the culture conditions. Using perfusion culture in comparison to static culture, the increase of cell proliferation after 2 weeks of cultivation when compared with the proliferation after 1 week was significantly lower, independent of the carriers used. In conclusion, the contemporaneous cultivation of human oral keratinocytes and human osteoblast-like cells on the same carrier is possible, a prerequisite for planned in vivo studies. As carrier collagen is superior to laminin-coated polycarbonate membranes. Regarding the development over time, the increase of proliferation rate is lower in perfusion culture. Examinations of cellular differentiation over time under various culture conditions will be subject of further investigations.

  8. Defect of Fc receptors and phenotypical changes in sinusoidal endothelial cells in human liver cirrhosis.

    PubMed Central

    Muro, H.; Shirasawa, H.; Kosugi, I.; Nakamura, S.

    1993-01-01

    To analyze the pathological changes occurring in Fc receptors (FcRs) in sinusoidal endothelial cells (SECs) in chronic liver diseases, we first characterized immunohistochemically the SEC FcRs by using monoclonal antibodies (MAbs) to FcRs and then investigated the distribution of the SEC FcRs by using peroxidase-antiperoxidase IgG complexes as a ligand on frozen sections. MAb 2E1 to FcRII reacted with SECs in a similar manner to peroxidase-antiperoxidase IgG and blocked the peroxidase-antiperoxidase IgG binding to SECs, whereas MAbs 3G8 and Leu-11b to FcRIII did not. FcRs in normal liver were found along the sinusoidal walls, except for those in the outer periportal zones, but FcRs in chronic active hepatitis and cirrhosis were intermittently or focally absent. The lengths of the FcR-positive portion of sinusoids in unit areas were respectively about 54% and 76% of the normal values in active and inactive cirrhosis. Where FcRs were absent, the MAbs CD36, CD31, and EN4 revealed the presence of sinusoids and, in active cirrhosis, frequently the thickening of liver cell plates. The FcR-negative SECs in the outer periportal zones of normal livers were different from the SECs of other sites in the presence of PAL-E antigen and a rich amount of EN4 antigen, though these sinusoids possessed Kupffer cells and no perisinusoidal deposition of laminin. The FcR-negative SECs in liver diseases occasionally presented the character of ordinary blood vessels, viz., PAL-E antigen, CD34 antigen, and a deficiency of Kupffer cells, regardless of perisinusoidal laminin deposition. However, they preserved the character of normally FcR-possessing SECs, viz., CD36 antigen, and a small amount of EN4 and CD31 antigens. These findings indicate that the outer-periportal SECs in normal livers are phenotypically different from other SECs and that the SECs in diseased livers frequently undergo phenotypical changes, including loss of FcRs, regardless of perisinusoidal laminin deposition, i

  9. The LDL receptor.

    PubMed

    Goldstein, Joseph L; Brown, Michael S

    2009-04-01

    In this article, the history of the LDL receptor is recounted by its codiscoverers. Their early work on the LDL receptor explained a genetic cause of heart attacks and led to new ways of thinking about cholesterol metabolism. The LDL receptor discovery also introduced three general concepts to cell biology: receptor-mediated endocytosis, receptor recycling, and feedback regulation of receptors. The latter concept provides the mechanism by which statins selectively lower plasma LDL, reducing heart attacks and prolonging life. PMID:19299327

  10. Endothelin A receptor activation on mesangial cells initiates Alport glomerular disease.

    PubMed

    Dufek, Brianna; Meehan, Daniel T; Delimont, Duane; Cheung, Linda; Gratton, Michael Anne; Phillips, Grady; Song, Wenping; Liu, Shiguang; Cosgrove, Dominic

    2016-08-01

    Recent work demonstrates that Alport glomerular disease is mediated through a biomechanical strain-sensitive activation of mesangial actin dynamics. This occurs through a Rac1/CDC42 cross-talk mechanism that results in the invasion of the subcapillary spaces by mesangial filopodia. The filopodia deposit mesangial matrix proteins in the glomerular basement membrane, including laminin 211, which activates focal adhesion kinase in podocytes culminating in the up-regulation of proinflammatory cytokines and metalloproteinases. These events drive the progression of glomerulonephritis. Here we test whether endothelial cell-derived endothelin-1 is up-regulated in Alport glomeruli and further elevated by hypertension. Treatment of cultured mesangial cells with endothelin-1 activates the formation of drebrin-positive actin microspikes. These microspikes do not form when cells are treated with the endothelin A receptor antagonist sitaxentan or under conditions of small, interfering RNA knockdown of endothelin A receptor mRNA. Treatment of Alport mice with sitaxentan results in delayed onset of proteinuria, normalized glomerular basement membrane morphology, inhibition of mesangial filopodial invasion of the glomerular capillaries, normalization of glomerular expression of metalloproteinases and proinflammatory cytokines, increased life span, and prevention of glomerulosclerosis and interstitial fibrosis. Thus endothelin A receptor activation on mesangial cells is a key event in initiation of Alport glomerular disease in this model.

  11. Molecular analysis of the human laminin alpha3a chain gene (LAMA3a): a strategy for mutation identification and DNA-based prenatal diagnosis in Herlitz junctional epidermolysis bullosa.

    PubMed

    Pulkkinen, L; Cserhalmi-Friedman, P B; Tang, M; Ryan, M C; Uitto, J; Christiano, A M

    1998-09-01

    Mutations in the genes (LAMA3, LAMB3, and LAMC2) encoding the subunit polypeptides of the cutaneous basement membrane zone protein laminin 5 have been reported in different forms of junctional epidermolysis bullosa (JEB), an inherited blistering skin disease. In this study, we present the complete exon-intron organization of the "a" transcript of the laminin alpha3 chain gene, LAMA3a, which is expressed primarily in the skin. We have performed fine-resolution mapping of this gene on chromosome 18q11.2 using a human-hamster radiation hybrid panel. We have also developed a mutation-detection strategy based on the exon-intron structure of LAMA3a. This strategy, based on PCR amplification of genomic sequences, followed by heteroduplex scanning and automated nucleotide sequencing, was used for successful mutation screening in a family with the lethal (Herlitz) type of JEB, and two novel LAMA3 mutations were identified in the proband. The mutations consisted of a single-base pair deletion in LAMA3a exon A11 on the paternal allele, designated 1239delC, and a two-base pair deletion in LAMA3a exon A23 on the maternal allele, designated 2959delGG. This information was also used for DNA-based prenatal testing in a subsequent pregnancy in this family. Collectively, these results attest to our expanding capability to elucidate the genetic basis of various forms of epidermolysis bullosa using molecular techniques. PMID:9759651

  12. A syndecan-4 binding peptide derived from laminin 5 uses a novel PKCε pathway to induce cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells.

    PubMed

    Filla, Mark S; Clark, Ross; Peters, Donna M

    2014-10-01

    In this study, we examined the role(s) of syndecan-4 in regulating the formation of an actin geodesic dome structure called a cross-linked actin network (CLAN) in which syndecan-4 has previously been localized. CLANs have been described in several different cell types, but they have been most widely studied in human trabecular meshwork (HTM) cells where they may play a key role in controlling intraocular pressure by regulating aqueous humor outflow from the eye. In this study we show that a loss of cell surface synedcan-4 significantly reduces CLAN formation in HTM cells. Analysis of HTM cultures treated with or without dexamethasone shows that laminin 5 deposition within the extracellular matrix is increased by glucocorticoid treatment and that a laminin 5-derived, syndecan-4-binding peptide (PEP75), induces CLAN formation in TM cells. This PEP75-induced CLAN formation was inhibited by heparin and the broad spectrum PKC inhibitor Ro-31-7549. In contrast, the more specific PKCα inhibitor Gö 6976 had no effect, thus excluding PKCα as a downstream effector of syndecan-4 signaling. Analysis of PKC isozyme expression showed that HTM cells also expressed both PKCγ and PKCε. Cells treated with a PKCε agonist formed CLANs while a PKCα/γ agonist had no effect. These data suggest that syndecan-4 is essential for CLAN formation in HTM cells and that a novel PKCε-mediated signaling pathway can regulate formation of this unique actin structure. PMID:25128150

  13. A syndecan-4 binding peptide derived from laminin 5 uses a novel PKCε pathway to induce cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells

    PubMed Central

    Filla, Mark S.; Clark, Ross; Peters, Donna M.

    2014-01-01

    In this study, we examined the role(s) of syndecan-4 in regulating the formation of an actin geodesic dome structure called a cross-linked actin network (CLAN) in which syndecan-4 has previously been localized. CLANs have been described in several different cell types, but they have been most widely studied in human trabecular meshwork (HTM) cells where they may play a key role in controlling intraocular pressure by regulating aqueous humor outflow from the eye. In this study we show that a loss of cell surface synedcan-4 significantly reduces CLAN formation in HTM cells. Analysis of HTM cultures treated with or without dexamethasone shows that laminin 5 deposition within the extracellular matrix is increased by glucocorticoid treatment and that a laminin 5-derived, syndecan-4-binding peptide (PEP75), induces CLAN formation in TM cells. This PEP75-induced CLAN formation was inhibited by heparin and the broad spectrum PKC inhibitor Ro-31-7549. In contrast, the more specific PKCα inhibitor Go 6976 had no effect, thus excluding PKCα as a downstream effector of syndecan-4 signaling. Analysis of PKC isozyme expression showed that HTM cells also expressed both PKCγ and PKCε. Cells treated with a PKCε agonist formed CLANs while a PKCα/γ agonist had no effect. These data suggest that syndecan-4 is essential for CLAN formation in HTM cells and that a novel PKCε-mediated signaling pathway can regulate formation of this unique actin structure. PMID:25128150

  14. Clearance Kinetics and Matrix Binding Partners of the Receptor for Advanced Glycation End Products

    PubMed Central

    Milutinovic, Pavle S.; Englert, Judson M.; Crum, Lauren T.; Mason, Neale S.; Ramsgaard, Lasse; Enghild, Jan J.; Sparvero, Louis J.; Lotze, Michael T.; Oury, Tim D.

    2014-01-01

    Elucidating the sites and mechanisms of sRAGE action in the healthy state is vital to better understand the biological importance of the receptor for advanced glycation end products (RAGE). Previous studies in animal models of disease have demonstrated that exogenous sRAGE has an anti-inflammatory effect, which has been reasoned to arise from sequestration of pro-inflammatory ligands away from membrane-bound RAGE isoforms. We show here that sRAGE exhibits in vitro binding with high affinity and reversibly to extracellular matrix components collagen I, collagen IV, and laminin. Soluble RAGE administered intratracheally, intravenously, or intraperitoneally, does not distribute in a specific fashion to any healthy mouse tissue, suggesting against the existence of accessible sRAGE sinks and receptors in the healthy mouse. Intratracheal administration is the only effective means of delivering exogenous sRAGE to the lung, the organ in which RAGE is most highly expressed; clearance of sRAGE from lung does not differ appreciably from that of albumin. PMID:24642901

  15. A fibronectin receptor on Candida albicans mediates adherence of the fungus to extracellular matrix

    SciTech Connect

    Klotz, S.A.; Smith, R.L. )

    1991-03-01

    Binding of fibronectin, an extracellular matrix (ECM) protein, to Candida albicans was measured, and adherence of the fungus to immobilized ECM proteins, fibronectin, laminin, types I and IV collagen, and subendothelial ECM was studied. 125I-labeled fibronectin was inhibited from binding to the fungus by unlabeled human plasma fibronectin and by Arg-Gly-Asp (RGD), Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), and Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP), but binding was not inhibited by Gly-Arg-Gly-Asp-Ser-Pro. Soluble fibronectin, RGD, GRGESP, and GRGDTP also inhibited fungal adherence to the individual immobilized ECM proteins in a complex pattern, but only soluble fibronectin (10(-7) M) inhibited fungal adherence to subendothelial ECM. Thus, C. albicans possesses at least one type of cell surface receptor for binding soluble fibronectin that can be inhibited with peptides. This receptor apparently is used to bind the fungus to immobilized ECM proteins and to subendothelial ECM and may play a role in the initiation of disseminated disease by bloodborne fungi by providing for adherence of the microorganisms to ECM proteins.

  16. Silencing Met receptor tyrosine kinase signaling decreased oral tumor growth and increased survival of nude mice

    PubMed Central

    Tao, X.; Hill, K.S.; Gaziova, I.; Sastry, S.K.; Qui, S.; Szaniszlo, P.; Fennewald, S.; Resto, V.A.; Elferink, L.A.

    2013-01-01

    SUMMARY Objectives The hepatocyte growth factor receptor (Met) is frequently overexpressed in Head and Neck Squamous Cell Carcinoma (HNSCC), correlating positively with high-grade tumors and shortened patient survival. As such, Met may represent an important therapeutic target. The purpose of this study was to explore the role of Met signaling for HNSCC growth and locoregional dissemination. Materials and methods Using a lentiviral system for RNA interference, we knocked down Met in established HNSCC cell lines that express high levels of the endogenous receptor. The effect of Met silencing on in vitro proliferation, cell survival and migration was examined using western analysis, immunohisto-chemistry and live cell imaging. In vivo tumor growth, dissemination and mouse survival was assessed using an orthotopic tongue mouse model for HNSCC. Results We show that Met knockdown (1) impaired activation of downstream MAPK signaling; (2) reduced cell viability and anchorage independent growth; (3) abrogated HGF-induced cell motility on laminin; (4) reduced In vivo tumor growth by increased cell apoptosis; (5) caused reduced incidence of tumor dissemination to regional lymph nodes and (6) increased the survival of nude mice with orthotopic xenografts. Conclusion Met signaling is important for HNSCC growth and locoregional dissemination In vivo and that targeting Met may be an important strategy for therapy. PMID:24268630

  17. Sulfur mustard disrupts human α3β1-integrin receptors in concert with α6β4-integrin receptors and collapse of the keratin K5/K14 cytoskeleton

    NASA Astrophysics Data System (ADS)

    Werrlein, Robert J.; Braue, Catherine R.

    2004-06-01

    Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a chemical warfare agent that produces persistent, incapacitating blisters of the skin. The lesions inducing vesication remain elusive, and there is no completely effective treatment. Using mulitphoton microscopy and immunofluorescent staining, we found that exposing human epidermal keratinocytes (HEK) and intact epidermis to SM (400 μm for 5 min) caused progressive collapse of the keratin (K5/K14) cytoskeleton and depletion of α6β integrins. We now report that SM causes concomitant disruption nad collapse of the basal cell's α3β1-integrin receptors. At 1 h postexposure, images of Alexa488-conjugated HEK/α3β1 integrins showed almost complete withdrawal and disappearance of retraction fibers and a progressive loss of polarized mobility. With stero imaging, in vitro expression of this SM effect was characterized by collapse and abutment of adjacent cell membranes. At 2 h postexposure, there was an average 13% dorso-ventral collapse of HEK membranes that paralleled progressive collapse of the K5/K14 cytoskeleton. α3β1 integrin, like α6β4 integrin, is a regulator of cytoskeletal assembly, a receptor for laminin 5 and a mediator of HEK attachment to the basement membrane. Our images indicate that SM disrupts these receptors. We suggest that the progressive disruption destabilizes and potentiates blistering of the epidermal-dermal junction.

  18. Historical overview of nuclear receptors.

    PubMed

    Gustafsson, Jan-Ake

    2016-03-01

    This review summarizes the birth of the field of nuclear receptors, from Jensen's discovery of estrogen receptor alpha, Gustafsson's discovery of the three-domain structure of the glucocorticoid receptor, the discovery of the glucocorticoid response element and the first partial cloning of the glucocorticoid receptor. Furthermore the discovery of the novel receptors called orphan receptors is described.

  19. Standardizing Scavenger Receptor Nomenclature

    PubMed Central

    PrabhuDas, Mercy; Bowdish, Dawn; Drickamer, Kurt; Febbraio, Maria; Herz, Joachim; Kobzik, Lester; Krieger, Monty; Loike, John; Means, Terry K.; Moestrup, Soren K.; Post, Steven; Sawamura, Tatsuya; Silverstein, Samuel; Wang, Xiang-Yang; El Khoury, Joseph

    2014-01-01

    Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a variety of ligands, including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the U.S. National Institute of Allergy and Infectious Diseases, National Institutes of Health to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of non-self or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. The discussion and nomenclature recommendations described in this report only refer to mammalian scavenger receptors. The purpose of this article is to describe the proposed mammalian nomenclature and classification developed at the workshop and to solicit additional feedback from the broader research community. PMID:24563502

  20. P2X receptors.

    PubMed

    North, R Alan

    2016-08-01

    Extracellular adenosine 5'-triphosphate (ATP) activates cell surface P2X and P2Y receptors. P2X receptors are membrane ion channels preferably permeable to sodium, potassium and calcium that open within milliseconds of the binding of ATP. In molecular architecture, they form a unique structural family. The receptor is a trimer, the binding of ATP between subunits causes them to flex together within the ectodomain and separate in the membrane-spanning region so as to open a central channel. P2X receptors have a widespread tissue distribution. On some smooth muscle cells, P2X receptors mediate the fast excitatory junction potential that leads to depolarization and contraction. In the central nervous system, activation of P2X receptors allows calcium to enter neurons and this can evoke slower neuromodulatory responses such as the trafficking of receptors for the neurotransmitter glutamate. In primary afferent nerves, P2X receptors are critical for the initiation of action potentials when they respond to ATP released from sensory cells such as taste buds, chemoreceptors or urothelium. In immune cells, activation of P2X receptors triggers the release of pro-inflammatory cytokines such as interleukin 1β. The development of selective blockers of different P2X receptors has led to clinical trials of their effectiveness in the management of cough, pain, inflammation and certain neurodegenerative diseases.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377721

  1. Signals and Receptors.

    PubMed

    Heldin, Carl-Henrik; Lu, Benson; Evans, Ron; Gutkind, J Silvio

    2016-04-01

    Communication between cells in a multicellular organism occurs by the production of ligands (proteins, peptides, fatty acids, steroids, gases, and other low-molecular-weight compounds) that are either secreted by cells or presented on their surface, and act on receptors on, or in, other target cells. Such signals control cell growth, migration, survival, and differentiation. Signaling receptors can be single-span plasma membrane receptors associated with tyrosine or serine/threonine kinase activities, proteins with seven transmembrane domains, or intracellular receptors. Ligand-activated receptors convey signals into the cell by activating signaling pathways that ultimately affect cytosolic machineries or nuclear transcriptional programs or by directly translocating to the nucleus to regulate transcription. PMID:27037414

  2. Signaling by Sensory Receptors

    PubMed Central

    Julius, David; Nathans, Jeremy

    2012-01-01

    Sensory systems detect small molecules, mechanical perturbations, or radiation via the activation of receptor proteins and downstream signaling cascades in specialized sensory cells. In vertebrates, the two principal categories of sensory receptors are ion channels, which mediate mechanosensation, thermosensation, and acid and salt taste; and G-protein-coupled receptors (GPCRs), which mediate vision, olfaction, and sweet, bitter, and umami tastes. GPCR-based signaling in rods and cones illustrates the fundamental principles of rapid activation and inactivation, signal amplification, and gain control. Channel-based sensory systems illustrate the integration of diverse modulatory signals at the receptor, as seen in the thermosensory/pain system, and the rapid response kinetics that are possible with direct mechanical gating of a channel. Comparisons of sensory receptor gene sequences reveal numerous examples in which gene duplication and sequence divergence have created novel sensory specificities. This is the evolutionary basis for the observed diversity in temperature- and ligand-dependent gating among thermosensory channels, spectral tuning among visual pigments, and odorant binding among olfactory receptors. The coding of complex external stimuli by a limited number of sensory receptor types has led to the evolution of modality-specific and species-specific patterns of retention or loss of sensory information, a filtering operation that selectively emphasizes features in the stimulus that enhance survival in a particular ecological niche. The many specialized anatomic structures, such as the eye and ear, that house primary sensory neurons further enhance the detection of relevant stimuli. PMID:22110046

  3. Estrogen and progesterone receptors have distinct roles in the establishment of the hyperplastic phenotype in PR-A transgenic mice

    SciTech Connect

    Simian, Marina; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Shyamala, Gopalan

    2009-05-11

    Expression of the A and B forms of progesterone receptor (PR) in an appropriate ratio is critical for mammary development. Mammary glands of PR-A transgenic mice, carrying an additional A form of PR as a transgene, exhibit morphological features associated with the development of mammary tumors. Our objective was to determine the roles of estrogen (E) and progesterone (P) in the genesis of mammary hyperplasias/preneoplasias in PR-A transgenics. We subjected PR-A mice to hormonal treatments and analyzed mammary glands for the presence of hyperplasias and used BrdU incorporation to measure proliferation. Quantitative image analysis was carried out to compare levels of latency-associated peptide and transforming growth factor beta 1 (TGF{beta}1) between PR-A and PR-B transgenics. Basement membrane disruption was examined by immunofluorescence and proteolytic activity by zymography. The hyperplastic phenotype of PR-A transgenics is inhibited by ovariectomy, and is reversed by treatment with E + P. Studies using the antiestrogen ICI 182,780 or antiprogestins RU486 or ZK 98,299 show that the increase in proliferation requires signaling through E/estrogen receptor alpha but is not sufficient to give rise to hyperplasias, whereas signaling through P/PR has little impact on proliferation but is essential for the manifestation of hyperplasias. Increased proliferation is correlated with decreased TGF{beta}1 activation in the PR-A transgenics. Analysis of basement membrane integrity showed loss of laminin-5, collagen III and collagen IV in mammary glands of PR-A mice, which is restored by ovariectomy. Examination of matrix metalloproteases (MMPs) showed that total levels of MMP-2 correlate with the steady-state levels of PR, and that areas of laminin-5 loss coincide with those of activation of MMP-2 in PR-A transgenics. Activation of MMP-2 is dependent on treatment with E and P in ovariectomized wild-type mice, but is achieved only by treatment with P in PR-A mice. These data

  4. Leukotriene receptor antagonist therapy

    PubMed Central

    Dempsey, O

    2000-01-01

    Leukotriene receptor antagonists (LTRA) are a new class of drugs for asthma treatment, available in tablet form. Their unique mechanism of action results in a combination of both bronchodilator and anti-inflammatory effects. While their optimal place in asthma management is still under review, LTRA represent an important advance in asthma pharmacotherapy.


Keywords: leukotriene receptor antagonist; asthma; montelukast; zafirlukast PMID:11085767

  5. Genetics of Taste Receptors

    PubMed Central

    Bachmanov, Alexander A.; Bosak, Natalia P.; Lin, Cailu; Matsumoto, Ichiro; Ohmoto, Makoto; Reed, Danielle R.; Nelson, Theodore M.

    2016-01-01

    Taste receptors function as one of the interfaces between internal and external milieus. Taste receptors for sweet and umami (T1R [taste receptor, type 1]), bitter (T2R [taste receptor, type 2]), and salty (ENaC [epithelial sodium channel]) have been discovered in the recent years, but transduction mechanisms of sour taste and ENaC-independent salt taste are still poorly understood. In addition to these five main taste qualities, the taste system detects such noncanonical “tastes” as water, fat, and complex carbohydrates, but their reception mechanisms require further research. Variations in taste receptor genes between and within vertebrate species contribute to individual and species differences in taste-related behaviors. These variations are shaped by evolutionary forces and reflect species adaptations to their chemical environments and feeding ecology. Principles of drug discovery can be applied to taste receptors as targets in order to develop novel taste compounds to satisfy demand in better artificial sweeteners, enhancers of sugar and sodium taste, and blockers of bitterness of food ingredients and oral medications. PMID:23886383

  6. Genetics of taste receptors.

    PubMed

    Bachmanov, Alexander A; Bosak, Natalia P; Lin, Cailu; Matsumoto, Ichiro; Ohmoto, Makoto; Reed, Danielle R; Nelson, Theodore M

    2014-01-01

    Taste receptors function as one of the interfaces between internal and external milieus. Taste receptors for sweet and umami (T1R [taste receptor, type 1]), bitter (T2R [taste receptor, type 2]), and salty (ENaC [epithelial sodium channel]) have been discovered in the recent years, but transduction mechanisms of sour taste and ENaC-independent salt taste are still poorly understood. In addition to these five main taste qualities, the taste system detects such noncanonical "tastes" as water, fat, and complex carbohydrates, but their reception mechanisms require further research. Variations in taste receptor genes between and within vertebrate species contribute to individual and species differences in taste-related behaviors. These variations are shaped by evolutionary forces and reflect species adaptations to their chemical environments and feeding ecology. Principles of drug discovery can be applied to taste receptors as targets in order to develop novel taste compounds to satisfy demand in better artificial sweeteners, enhancers of sugar and sodium taste, and blockers of bitterness of food ingredients and oral medications. PMID:23886383

  7. Dopamine Receptors and Neurodegeneration

    PubMed Central

    Rangel-Barajas, Claudia; Coronel, Israel; Florán, Benjamín

    2015-01-01

    Dopamine (DA) is one of the major neurotransmitters and participates in a number of functions such as motor coordination, emotions, memory, reward mechanism, neuroendocrine regulation etc. DA exerts its effects through five DA receptors that are subdivided in 2 families: D1-like DA receptors (D1 and D5) and the D2-like (D2, D3 and D4). All DA receptors are widely expressed in the central nervous system (CNS) and play an important role in not only in physiological conditions but also pathological scenarios. Abnormalities in the DAergic system and its receptors in the basal ganglia structures are the basis Parkinson’s disease (PD), however DA also participates in other neurodegenerative disorders such as Huntington disease (HD) and multiple sclerosis (MS). Under pathological conditions reorganization of DAergic system has been observed and most of the times, those changes occur as a mechanism of compensation, but in some cases contributes to worsening the alterations. Here we review the changes that occur on DA transmission and DA receptors (DARs) at both levels expression and signals transduction pathways as a result of neurotoxicity, inflammation and in neurodegenerative processes. The better understanding of the role of DA receptors in neuropathological conditions is crucial for development of novel therapeutic approaches to treat alterations related to neurodegenerative diseases. PMID:26425390

  8. Induction of intermediate mesoderm by retinoic acid receptor signaling from differentiating mouse embryonic stem cells.

    PubMed

    Oeda, Shiho; Hayashi, Yohei; Chan, Techuan; Takasato, Minoru; Aihara, Yuko; Okabayashi, Koji; Ohnuma, Kiyoshi; Asashima, Makoto

    2013-01-01

    Renal lineages including kidney are derived from intermediate mesoderm, which are differentiated from a subset of caudal undifferentiated mesoderm. The inductive mechanisms of mammalian intermediate mesoderm and renal lineages are still poorly understood. Mouse embryonic stem cells (mESCs) can be a good in vitro model to reconstitute the developmental pathway of renal lineages and to analyze the mechanisms of the sequential differentiation. We examined the effects of Activin A and retinoic acid (RA) on the induction of intermediate mesoderm from mESCs under defined, serum-free, adherent, monolayer culture conditions. We measured the expression level of intermediate mesodermal marker genes and examined the developmental potential of the differentiated cells into kidney using an ex vivo transplantation assay. Adding Activin A followed by RA to mESC cultures induced the expression of marker genes and proteins for intermediate mesoderm, odd-skipped related 1 (Osr1) and Wilm’s Tumor 1 (Wt1). These differentiated cells integrated into laminin-positive tubular cells and Pax2-positive renal cells in cultured embryonic kidney explants. We demonstrated that intermediate mesodermal marker expression was also induced by RA receptor (RAR) agonist, but not by retinoid X receptor (RXR) agonists. Furthermore, the expression of these markers was decreased by RAR antagonists. We directed the differentiation of mESCs into intermediate mesoderm using Activin A and RA and revealed the role of RAR signaling in this differentiation. These methods and findings will improve our understanding of renal lineage development and could contribute to the regenerative medicine of kidney.

  9. Modulators of estrogen receptor inhibit proliferation and migration of prostate cancer cells.

    PubMed

    Piccolella, Margherita; Crippa, Valeria; Messi, Elio; Tetel, Marc J; Poletti, Angelo

    2014-01-01

    In the initial stages, human prostate cancer (PC) is an androgen-sensitive disease, which can be pharmacologically controlled by androgen blockade. This therapy often induces selection of androgen-independent PC cells with increased invasiveness. We recently demonstrated, both in cells and mice, that a testosterone metabolite locally synthetized in prostate, the 5α-androstane-3β, 17β-diol (3β-Adiol), inhibits PC cell proliferation, migration and invasion, acting as an anti-proliferative/anti-metastatic agent. 3β-Adiol is unable to bind androgen receptor (AR), but exerts its protection against PC by specifically interacting with estrogen receptor beta (ERβ). Because of its potential retro-conversion to androgenic steroids, 3β-Adiol cannot be used "in vivo", thus, the aims of this study were to investigate the capability of four ligands of ERβ (raloxifen, tamoxifen, genistein and curcumin) to counteract PC progression by mimicking the 3β-Adiol activity. Our results demonstrated that raloxifen, tamoxifen, genistein and curcumin decreased DU145 and PC3 cell proliferation in a dose-dependent manner; in addition, all four compounds significantly decreased the detachment of cells seeded on laminin or fibronectin. Moreover, raloxifen, tamoxifen, genistein and curcumin-treated DU145 and PC3 cells showed a significant decrease in cell migration. Notably, all these effects were reversed by the anti-estrogen, ICI 182,780, suggesting that their actions are mediated by the estrogenic pathway, via the ERβ, the only isoform present in these PCs. In conclusion, these data demonstrate that by selectively activating the ERβ, raloxifen, tamoxifen, genistein and curcumin inhibit human PC cells proliferation and migration favoring cell adesion. These synthetic and natural modulators of ER action may exert a potent protective activity against the progression of PC even in its androgen-independent status. PMID:24184124

  10. Ionotropic Crustacean Olfactory Receptors

    PubMed Central

    Corey, Elizabeth A.; Bobkov, Yuriy; Ukhanov, Kirill; Ache, Barry W.

    2013-01-01

    The nature of the olfactory receptor in crustaceans, a major group of arthropods, has remained elusive. We report that spiny lobsters, Panulirus argus, express ionotropic receptors (IRs), the insect chemosensory variants of ionotropic glutamate receptors. Unlike insects IRs, which are expressed in a specific subset of olfactory cells, two lobster IR subunits are expressed in most, if not all, lobster olfactory receptor neurons (ORNs), as confirmed by antibody labeling and in situ hybridization. Ligand-specific ORN responses visualized by calcium imaging are consistent with a restricted expression pattern found for other potential subunits, suggesting that cell-specific expression of uncommon IR subunits determines the ligand sensitivity of individual cells. IRs are the only type of olfactory receptor that we have detected in spiny lobster olfactory tissue, suggesting that they likely mediate olfactory signaling. Given long-standing evidence for G protein-mediated signaling in activation of lobster ORNs, this finding raises the interesting specter that IRs act in concert with second messenger-mediated signaling. PMID:23573266

  11. Taste Receptors in Innate Immunity

    PubMed Central

    Lee, Robert J.

    2014-01-01

    Taste receptors were first identified on the tongue, where they initiate a signaling pathway that communicates information to the brain about the nutrient content or potential toxicity of ingested foods. However, recent research has shown that taste receptors are also expressed in a myriad of other tissues, from the airway and gastrointestinal epithelia to the pancreas and brain. The functions of many of these extraoral taste receptors remain unknown, but emerging evidence suggests that bitter and sweet taste receptors in the airway are important sentinels of innate immunity. This review discusses taste receptor signaling, focusing on the G-protein coupled–receptors that detect bitter, sweet, and savory tastes, followed by an overview of extraoral taste receptors and in-depth discussion of studies demonstrating the roles of taste receptors in airway innate immunity. Future research on extraoral taste receptors has significant potential for identification of novel immune mechanisms and insights into host-pathogen interactions. PMID:25323130

  12. New insights into receptor regulation.

    PubMed

    Poste, G

    1984-11-01

    This review provides a brief summary of certain recent advances in our understanding of receptor regulation, signal transduction, and the diverse pathways by which receptor-ligand complexes are internalized and delivered to specific organelles, together with recycling of receptors back to the cell surface. Emphasis is also given to the importance of methodological advances in receptor isolation, immunologic analysis of receptor structure and function, the development of new instrumentation for microchemical characterization of very small amounts of receptor material, and the increasing use of genetic engineering techniques to isolate the genes for receptors and their regulatory subunits, to transfer such genes between cells, and to study receptor function by creating structurally modified receptors via subtle changes in gene structure. PMID:6151557

  13. Olfactory receptor signaling.

    PubMed

    Antunes, Gabriela; Simoes de Souza, Fabio Marques

    2016-01-01

    The guanine nucleotide protein (G protein)-coupled receptors (GPCRs) superfamily represents the largest class of membrane protein in the human genome. More than a half of all GPCRs are dedicated to interact with odorants and are termed odorant-receptors (ORs). Linda Buck and Richard Axel, the Nobel Prize laureates in physiology or medicine in 2004, first cloned and characterized the gene family that encode ORs, establishing the foundations to the understanding of the molecular basis for odor recognition. In the last decades, a lot of progress has been done to unravel the functioning of the sense of smell. This chapter gives a general overview of the topic of olfactory receptor signaling and reviews recent advances in this field. PMID:26928542

  14. Progesterone Receptor Signaling Mechanisms.

    PubMed

    Grimm, Sandra L; Hartig, Sean M; Edwards, Dean P

    2016-09-25

    Progesterone receptor (PR) is a master regulator in female reproductive tissues that controls developmental processes and proliferation and differentiation during the reproductive cycle and pregnancy. PR also plays a role in progression of endocrine-dependent breast cancer. As a member of the nuclear receptor family of ligand-dependent transcription factors, the main action of PR is to regulate networks of target gene expression in response to binding its cognate steroid hormone, progesterone. This paper summarizes recent advances in understanding the structure-function properties of the receptor protein and the tissue/cell-type-specific PR signaling pathways that contribute to the biological actions of progesterone in the normal breast and in breast cancer. PMID:27380738

  15. CONTAMINANT INTERACTIONS WITH STEROID RECEPTORS: EVIDENCE FOR RECEPTOR BINDING.

    EPA Science Inventory

    Steroid receptors are important determinants of endocrine disrupter consequences. As the most frequently proposed mechanism of endocrine-disrupting contaminant (EDC) action, steroid receptors are not only targets of natural steroids but are also commonly sites of nonsteroidal com...

  16. Vasopressin receptor antagonists.

    PubMed

    Palmer, Biff F

    2015-01-01

    Arginine vasopressin (AVP) is the principal hormone involved in regulating the tonicity of body fluids. Less appreciated is the role that AVP plays in a variety of other physiologic functions including glucose metabolism, cardiovascular homeostasis, bone metabolism, and cognitive behavior. AVP receptor antagonists are now available and currently approved to treat hyponatremia. There is a great deal of interest in exploring the potential benefits that these drugs may play in blocking AVP-mediated effects in other organ systems. The purpose of this report is to provide an update on the expanding role of AVP receptor antagonists and what disease states these drugs may eventually be used for.

  17. Vasopressin receptor antagonists.

    PubMed

    Palmer, Biff F

    2015-01-01

    Arginine vasopressin (AVP) is the principal hormone involved in regulating the tonicity of body fluids. Less appreciated is the role that AVP plays in a variety of other physiologic functions including glucose metabolism, cardiovascular homeostasis, bone metabolism, and cognitive behavior. AVP receptor antagonists are now available and currently approved to treat hyponatremia. There is a great deal of interest in exploring the potential benefits that these drugs may play in blocking AVP-mediated effects in other organ systems. The purpose of this report is to provide an update on the expanding role of AVP receptor antagonists and what disease states these drugs may eventually be used for. PMID:25604388

  18. Biomimetic Receptors and Sensors

    PubMed Central

    Dickert, Franz L.

    2014-01-01

    In biomimetics, living systems are imitated to develop receptors for ions, molecules and bioparticles. The most pertinent idea is self-organization in analogy to evolution in nature, which created the key-lock principle. Today, modern science has been developing host-guest chemistry, a strategy of supramolecular chemistry for designing interactions of analytes with synthetic receptors. This can be realized, e.g., by self-assembled monolayers (SAMs) or molecular imprinting. The strategies are used for solid phase extraction (SPE), but preferably in developing recognition layers of chemical sensors. PMID:25436653

  19. Exclusion of candidate genes from the chromosome 1q juvenile glaucoma region and mapping of the peripheral cannabis receptor gene (CNR2) to chromosome 1

    SciTech Connect

    Sunden, S.L.F.; Nichols, B.E.; Alward, W.L.M.

    1994-09-01

    Juvenile onset primary open angle glaucoma has been mapped by linkage to 1q21-q31. Several candidate genes were evaluated in the same family used to identify the primary linkage. Atrionatriuretic peptide receptor A (NPR1) and laminin C1 (LAMC1) have been previously mapped to this region and could putatively play a role in the pathogenesis of glaucoma. A third gene, the peripheral cannabis receptor (CNR2) was not initially mapped in humans but was a candidate because of the relief that cannabis affords some patients with primary open angle glaucoma. Microsatellites associated with NPR1 and LAMC1 revealed multiple recombinations in affected members of this pedigree. CNR2 was shown to be on chromosome 1 by PCR amplification of a 150 bp fragment of the 3{prime} untranslated region in monochromosomal somatic cell hybrids (NIGMS panel No. 2). These primers also revealed a two allele single strand conformation polymorphism which showed multiple recombinants with juvenile onset primary open angle glaucoma in large pedigrees, segregating this disorder. The marker was then mapped to 1p34-p36 by linkage, with the most likely location between liver alkaline phosphatase (ALPL) and alpha-L-1 fucosidase (FUCA1).

  20. Computational Biology of Olfactory Receptors

    PubMed Central

    Crasto, Chiquito J.

    2011-01-01

    Olfactory receptors, in addition to being involved in first step of the physiological processes that leads to olfaction, occupy an important place in mammalian genomes. ORs constitute super families in these genomes. Elucidating ol-factory receptor function at a molecular level can be aided by a computationally derived structure and an understanding of its interactions with odor molecules. Experimental functional analyses of olfactory receptors in conjunction with computational studies serve to validate findings and generate hypotheses. We present here a review of the research efforts in: creating computational models of olfactory receptors, identifying binding strategies for these receptors with odorant molecules, performing medium to long range simulation studies of odor ligands in the receptor binding region, and identifying amino acid positions within the receptor that are responsible for ligand-binding and olfactory receptor activation. Written as a primer and a teaching tool, this review will help researchers extend the methodologies described herein to other GPCRs. PMID:21984880

  1. Synaptic Neurotransmitter-Gated Receptors

    PubMed Central

    Smart, Trevor G.; Paoletti, Pierre

    2012-01-01

    Since the discovery of the major excitatory and inhibitory neurotransmitters and their receptors in the brain, many have deliberated over their likely structures and how these may relate to function. This was initially satisfied by the determination of the first amino acid sequences of the Cys-loop receptors that recognized acetylcholine, serotonin, GABA, and glycine, followed later by similar determinations for the glutamate receptors, comprising non-NMDA and NMDA subtypes. The last decade has seen a rapid advance resulting in the first structures of Cys-loop receptors, related bacterial and molluscan homologs, and glutamate receptors, determined down to atomic resolution. This now provides a basis for determining not just the complete structures of these important receptor classes, but also for understanding how various domains and residues interact during agonist binding, receptor activation, and channel opening, including allosteric modulation. This article reviews our current understanding of these mechanisms for the Cys-loop and glutamate receptor families. PMID:22233560

  2. Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor

    PubMed Central

    1988-01-01

    Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'- untranslated region of 2.2 kb. The open reading frame encodes a 1,245- residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asn-linked glycosylation sites, and two potential Ca2+-binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells. PMID:3264556

  3. Human placental calcitonin receptors.

    PubMed Central

    Nicholson, G C; D'Santos, C S; Evans, T; Moseley, J M; Kemp, B E; Michelangeli, V P; Martin, T J

    1988-01-01

    Receptors for the hypocalcaemic hormone, calcitonin (CT), have been identified in a membrane fraction prepared from term human placentae. Binding of 125I-labelled salmon CT (125I-sCT) to the membranes was time- and temperature-dependent, saturable (Bmax. 58 +/- 11 fmol/mg of protein), of high affinity (Kd 80 +/- 21 pM) and poorly reversible. Species-specific CTs and CT analogues competed for 125I-sCT binding with potencies proportional to their known biological potencies. Various unrelated peptide hormones did not compete, indicating that receptor binding was specific for CT. Photoaffinity labelling using a derivatized biologically active sCT analogue, [Arg11,18,3-nitrophenylazide-Lys14]sCT, identified a receptor component of Mr approximately 85,000, comparable with findings in osteoclasts and other target cells. The presence of CT receptors in the human placenta supports other evidence that CT may have a role in the regulation of placental function. PMID:2839149

  4. Nicotinic Receptors in Neurodegeneration

    PubMed Central

    Posadas, Inmaculada; López-Hernández, Beatriz; Ceña, Valentín

    2013-01-01

    Many studies have focused on expanding our knowledge of the structure and diversity of peripheral and central nicotinic receptors. Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of pentameric ligand-gated ion channels, which include GABA (A and C), serotonin, and glycine receptors. Currently, 9 alpha (α2-α10) and 3 beta (β2-β4) subunits have been identified in the central nervous system (CNS), and these subunits assemble to form a variety of functional nAChRs. The pentameric combination of several alpha and beta subunits leads to a great number of nicotinic receptors that vary in their properties, including their sensitivity to nicotine, permeability to calcium and propensity to desensitize. In the CNS, nAChRs play crucial roles in modulating presynaptic, postsynaptic, and extrasynaptic signaling, and have been found to be involved in a complex range of CNS disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), schizophrenia, Tourette´s syndrome, anxiety, depression and epilepsy. Therefore, there is growing interest in the development of drugs that modulate nAChR functions with optimal benefits and minimal adverse effects. The present review describes the main characteristics of nAChRs in the CNS and focuses on the various compounds that have been tested and are currently in phase I and phase II trials for the treatment of neurodegenerative diseases including PD, AD and age-associated memory and mild cognitive impairment. PMID:24179465

  5. Diversification of TAM receptor function

    PubMed Central

    Zagórska, Anna; Través, Paqui G.; Lew, Erin D.; Dransfield, Ian; Lemke, Greg

    2014-01-01

    Apoptotic cell clearance is critical for both tissue homeostasis and the resolution of inflammation. We found that the TAM receptor tyrosine kinases Axl and Mer played distinct roles as phagocytic receptors in these two settings, where they exhibited divergent expression, regulation, and activity. Mer acted as a tolerogenic receptor in resting macrophages and in settings of immune suppression. Conversely, Axl was an inflammatory response receptor whose expression was induced by pro-inflammatory stimuli. Axl and Mer displayed distinct ligand specificities, ligand-receptor complex formation in tissues, and receptor shedding upon activation. These differences notwithstanding, phagocytosis by either protein was strictly dependent on receptor activation that was triggered by bridging TAM receptor–ligand complexes to the ‘eat-me’ signal phosphatidylserine on apoptotic cell surfaces. PMID:25194421

  6. Taste Receptor Genes

    PubMed Central

    Bachmanov, Alexander A.; Beauchamp, Gary K.

    2009-01-01

    In the past several years, tremendous progress has been achieved with the discovery and characterization of vertebrate taste receptors from the T1R and T2R families, which are involved in recognition of bitter, sweet, and umami taste stimuli. Individual differences in taste, at least in some cases, can be attributed to allelic variants of the T1R and T2R genes. Progress with understanding how T1R and T2R receptors interact with taste stimuli and with identifying their patterns of expression in taste cells sheds light on coding of taste information by the nervous system. Candidate mechanisms for detection of salts, acids, fat, complex carbohydrates, and water have also been proposed, but further studies are needed to prove their identity. PMID:17444812

  7. TSH RECEPTOR AUTOANTIBODIES

    PubMed Central

    Michalek, Krzysztof; Morshed, Syed A.; Latif, Rauf; Davies, Terry F.

    2009-01-01

    Thyrotropin receptor autoantibodies (TSHR-Abs) of the stimulating variety are the hallmark of Graves’ disease. The presence of immune defects leading to synthesis of TSHR-Abs causes hyperthyroidism and is associated with other extrathyroidal manifestations. Further characterization of these antibodies has now been made possible by the generation of monoclonal antibodies with this unique stimulating capacity as well as similar TSHR-Abs not associated with hyperthyroidism. Their present classification divides TSHR-Abs into stimulating, blocking (competing with TSH binding) and neutral (no signaling). Recent studies using monoclonal TSHR-Abs has revealed that stimulating and blocking antibodies bind to the receptor using mostly conformational epitopes, whilst neutral antibodies utilize exclusively linear peptides. Subtle differences in epitopes for stimulating and blocking antibodies account for the diversity of their biological actions. Recently non-classical signaling elicited by neutral antibodies has also been described, raising the need for a new classification of TSHR-Abs. PMID:19332151

  8. The interleukin-4 receptor: signal transduction by a hematopoietin receptor.

    PubMed

    Keegan, A D; Pierce, J H

    1994-02-01

    Over the last several years, the receptors for numerous cytokines have been molecularly characterized. Analysis of their amino acid sequences shows that some of these receptors bear certain motifs in their extracellular domains that define a family of receptors called the Hematopoietin receptor superfamily. Significant advances in characterizing the structure, function, and mechanisms of signal transduction have been made for several members of this family. The purpose of this review is to discuss the recent advances made for one of the family members, the interleukin (IL) 4 receptor. Other receptor systems have recently been reviewed elsewhere. The IL-4 receptor consists of, at the minimum, the cloned 140 kDa IL-4-binding chain with the potential for associating with other chains. The IL-4 receptor transduces its signal by activating a tyrosine kinase that phosphorylates cellular substrates, including the receptor itself, and the 170 kDa substrate called 4PS. Phosphorylated 4PS interacts with the SH2 domain of the enzyme PI-3'-kinase and increases its enzymatic activity. These early events in the IL-4 receptor initiated signaling pathway may trigger a series of signals that will ultimately lead to an IL-4 specific biologic outcome.

  9. Quantitative receptor autoradiography

    SciTech Connect

    Boast, C.A.; Snowhill, E.W.; Altar, C.A.

    1986-01-01

    Quantitative receptor autoradiography addresses the topic of technical and scientific advances in the sphere of quantitative autoradiography. The volume opens with a overview of the field from a historical and critical perspective. Following is a detailed discussion of in vitro data obtained from a variety of neurotransmitter systems. The next section explores applications of autoradiography, and the final two chapters consider experimental models. Methodological considerations are emphasized, including the use of computers for image analysis.

  10. Receptors for enterovirus 71

    PubMed Central

    Yamayoshi, Seiya; Fujii, Ken; Koike, Satoshi

    2014-01-01

    Enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease (HFMD). Occasionally, EV71 infection is associated with severe neurological diseases, such as acute encephalitis, acute flaccid paralysis and cardiopulmonary failure. Several molecules act as cell surface receptors that stimulate EV71 infection, including scavenger receptor B2 (SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), sialylated glycan, heparan sulfate and annexin II (Anx2). SCARB2 plays critical roles in attachment, viral entry and uncoating, and it can facilitate efficient EV71 infection. The three-dimensional structures of the mature EV71 virion, procapsid and empty capsid, as well as the exofacial domain of SCARB2, have been elucidated. This structural information has greatly increased our understanding of the early steps of EV71 infection. Furthermore, SCARB2 plays essential roles in the development of EV71 neurological disease in vivo. Adult mice are not susceptible to infection by EV71, but transgenic mice that express human SCARB2 become susceptible to EV71 infection and develop similar neurological diseases to those found in humans. This mouse model facilitates the in vivo investigation of many issues related to EV71. PSGL-1, sialylated glycan, heparan sulfate and Anx2 are attachment receptors, which enhance viral infection by retaining the virus on the cell surface. These molecules also contribute to viral infection in vitro either by interacting with SCARB2 or independently of SCARB2. However, the cooperative effects of these receptors, and their contribution to EV71 pathogenicity in vivo, remain to be elucidated. PMID:26038749

  11. Galectin-1 stimulates motility of human umbilical cord blood-derived mesenchymal stem cells by downregulation of smad2/3-dependent collagen 3/5 and upregulation of NF-κB-dependent fibronectin/laminin 5 expression.

    PubMed

    Yun, S P; Lee, S-J; Jung, Y H; Han, H J

    2014-01-01

    Galectin-1 (Gal-1) belongs to a family of endogenous lectins with conserved carbohydrate recognition domains binding β-galactosidase sugars and plays a vital role in regulating stem cell functions including determination of cell fate. However, our understanding of the functional roles of Gal-1 in human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) is still fragmentary and incomplete. Gal-1 significantly increased motility after a 24-h incubation, and this effect was inhibited by β-lactose. We analyzed 17 extracellular matrix (ECM) genes in UCB-MSCs. Gal-1 decreased the expression of collagen genes COL3A1 (COL-3) and COL5A1 (COL-5) but increased the expression of fibronectin (FN) and laminin 5 (LM-5), that were reversed by β-lactose. Gal-1 increased protein kinase C (PKC), c-Src, and caveolin-1 (Cav-1) phosphorylation that was attenuated by β-lactose and the Src inhibitor PP2. In addition, pretreatment with the lipid raft disruptor Mβ-CD and the PKC inhibitors inhibited Gal-1-induced UCB-MSC motility. In addition, Gal-1 reduced smad2/3 phosphorylation and induced nuclear factor (NF)-κB phosphorylation. Pretreatment with Mβ-CD attenuated Gal-1-reduced smad2/3 phosphorylation, COL-3, and COL-5 expression but did not affect NF-κB phosphorylation, FN, or LM-5 expression. In contrast, PKC inhibitors only attenuated NF-κB phosphorylation, FN, and LM-5 expression. Reconstructing Gal-1-induced genetic changes by replacing it with siRNA specific for COL-3 or COL-5, or treatment of the cells with FN and LM-5 proteins, increased motility and its related proteins such as focal adhesion kinase, Akt, Erk, integrins, and matrix metalloproteinase-2. A combined treatment with COL-3/COL-5 siRNA or FN/LM-5 compared with that of single treatments was synergistic. However, a single Gal-1 treatment maximally stimulated motility and related protein phosphorylation/expression. These results demonstrate that Gal-1 stimulated human UCB-MSC motility by decreasing COL

  12. [Lipoprotein receptors. Old acquaintances and newcomers].

    PubMed

    Ducobu, J

    1997-02-01

    Lipoprotein receptors are plasma membrane proteins of high affinity which interact with circulating lipoprotein particles. The well characterized LDL receptor continues to be analysed and some new findings on its intracellular mechanisms of action have emerged. New lipoprotein receptors have recently been described: the chylomicron remnant receptor or LDL-related protein (LRP), the lipolysis stimulated receptor (LSR), the very low density lipoprotein receptor (VLDLR), the HDL receptor (HDLR) and the scavenger receptor (SR). The molecular details of the receptors will facilitate the development of new therapeutic means to improve receptor-mediated clearance of lipoproteins.

  13. Role of the prostaglandin E2 receptor agonists in TGF-β1-induced mesangial cell damage

    PubMed Central

    Xi, Pei-pei; Xu, Yu-yin; Chen, Xiao-lan; Fan, Ya-ping; Wu, Jian-hua

    2016-01-01

    PGE2 exerts its biological effect through binding to various EP receptors that result inactivation of various signal transduction pathways. It also plays an important role in mice glomerular mesangial cells (MCs) damage induced by transforming growth factor-β1 (TGF-β1); however, the molecular mechanisms remain unknown. In the present study, we tested the efficacy of four selective agonists of PGE2 receptor, EP1A (17-phenyl trinor prostaglandin E2 ethyl amid), EP2A (butaprost), EP3A (sulprostone) and EP4A (cay10580), on mice MCs. Compared with the cAMP produced by TGF-β1, additional pretreatment of EP3A decreased the cAMP level. MCs treated with EP1A and EP3A augmented PGE2, cyclooxygenase-2 (COX-2), membrane-bound PGE synthase 1 (mPGES1), laminin (LN), connective tissue growth factor (CTGF) and cyclin D1 expression stimulated by TGFβ1. EP1A and EP3A increased the number of cells in S+G2/M phase and reduced cells in G0/G1 phase. EP1 and EP3 agonists also strengthened TGFβ1-induced mitogen-activated protein kinase (p38MAPK) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Whereas MCs treated with EP2A and EP4A weakened PGE2, COX-2, mPGES1, LN, CTGF and cyclin D1 expression stimulated by TGFβ1. EP2A and EP4A decreased the number of cells in S+G2/M phase and increased cells in G0/G1 phase. EP2 and EP4 agonists weakened TGFβ1-induced p38MAPK and ERK1/2 phosphorylation. These findings suggest that PGE2 has an important role in the progression of kidney disease via the EP1/EP3 receptor, whereas EP2 and EP4 receptors are equally important in preserving the progression of chronic kidney failure. Thus, agonists of EP2 and EP4 receptors may provide a basis for treating kidney damage induced by TGF-β1. PMID:27512093

  14. Melatonin Receptor Genes in Vertebrates

    PubMed Central

    Li, Di Yan; Smith, David Glenn; Hardeland, Rüdiger; Yang, Ming Yao; Xu, Huai Liang; Zhang, Long; Yin, Hua Dong; Zhu, Qing

    2013-01-01

    Melatonin receptors are members of the G protein-coupled receptor (GPCR) family. Three genes for melatonin receptors have been cloned. The MT1 (or Mel1a or MTNR1A) and MT2 (or Mel1b or MTNR1B) receptor subtypes are present in humans and other mammals, while an additional melatonin receptor subtype, Mel1c (or MTNR1C), has been identified in fish, amphibians and birds. Another melatonin related orphan receptor, GPR50, which does not bind melatonin, is found exclusively in mammals. The hormone melatonin is secreted primarily by the pineal gland, with highest levels occurring during the dark period of a circadian cycle. This hormone acts systemically in numerous organs. In the brain, it is involved in the regulation of various neural and endocrine processes, and it readjusts the circadian pacemaker, the suprachiasmatic nucleus. This article reviews recent studies of gene organization, expression, evolution and mutations of melatonin receptor genes of vertebrates. Gene polymorphisms reveal that numerous mutations are associated with diseases and disorders. The phylogenetic analysis of receptor genes indicates that GPR50 is an outgroup to all other melatonin receptor sequences. GPR50 may have separated from a melatonin receptor ancestor before the split between MTNR1C and the MTNR1A/B ancestor. PMID:23712359

  15. Receptor binding properties of amperozide.

    PubMed

    Svartengren, J; Simonsson, P

    1990-01-01

    The receptor pharmacology of amperozide was investigated with in vitro radioligand binding technique. Amperozide possessed a high affinity to the 5-HT2 receptors (Ki = 16.5 +/- 2.1 nM) and a moderate affinity to alpha 1-adrenergic receptors of rat cerebral cortical membranes (Ki = 172 +/- 14 nM). The affinity of amperozide for striatal and limbic dopamine D2 receptors was low and not significantly different (Ki +/- S.E.M. = 540 +/- 59 nM vs 403 +/- 42 nM; p less than 0.11, n = 4). The affinity for striatal and limbic 5-HT2 receptors was measured as well and found to be very close to the affinity to the cerebral cortical 5-HT2 receptor. The drug affinity for D2 and 5-HT2 receptors seems thus not to be influenced by the location of the receptor moiety. The affinity for several other rat brain receptors such as 5-HT1A, alpha 2-adrenergic, dopamine D1, muscarinic M1 and M2, opiate sigma and beta 2-adrenergic was low. The pseudo-Hill coefficient of the amperozide competition binding curve was consistently higher than one indicating antagonistic and complex interactions with the 5-HT2 receptor or with alpha 1-adrenergic and dopamine D2 receptors. The antagonistic properties of amperozide were investigated by its ability to antagonize the serotonin-induced formation of inositol-1-phosphate in human blood platelets. Amperozide inhibited this 5-HT2 receptor-mediated intracellular response with similar potency as ketanserin. These results suggest that amperozide is a selective 5-HT2 receptor antagonist.

  16. Bone marrow-derived mesenchymal stem cells enhance angiogenesis via their α6β1 integrin receptor

    SciTech Connect

    Carrion, Bita; Kong, Yen P.; Kaigler, Darnell; Putnam, Andrew J.

    2013-11-15

    Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6β1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes their failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis. Highlights: • BMSCs stimulate angiogenesis, but the mechanisms remain unclear. • We silenced the expression of the α6 integrin subunit in BMSCs. • Silencing this receptor subunit significantly inhibited angiogenic sprouting. • Knocking down α6 integrin affected laminin and αSMA expression. • Silencing α6 integrin expression also reduced BMSC proliferation.

  17. Angiotensin II receptors in testes

    SciTech Connect

    Millan, M.A.; Aguilera, G.

    1988-05-01

    Receptors for angiotensin II (AII) were identified and characterized in testes of rats and several primate species. Autoradiographic analysis of the binding of 125I-labeled (Sar1,Ile8)AII to rat, rhesus monkey, cebus monkey, and human testicular slide-mounted frozen sections indicated specific binding to Leydig cells in the interstitium. In rat collagenase-dispersed interstitial cells fractionated by Percoll gradient, AII receptor content was parallel to that of hCG receptors, confirming that the AII receptors are in the Leydig cells. In rat dispersed Leydig cells, binding was specific for AII and its analogs and of high affinity (Kd, 4.8 nM), with a receptor concentration of 15 fmol/10(6) cells. Studies of AII receptors in rat testes during development reveals the presence of high receptor density in newborn rats which decreases toward the adult age (4934 +/- 309, 1460 +/- 228, 772 +/- 169, and 82 +/- 12 fmol/mg protein at 5, 15, 20, and 30 days of age, respectively) with no change in affinity. At all ages receptors were located in the interstitium, and the decrease in binding was parallel to the decrease in the interstitial to tubular ratio observed with age. AII receptor properties in membrane-rich fractions from prepuberal testes were similar in the rat and rhesus monkey. Binding was time and temperature dependent, reaching a plateau at 60 min at 37 C, and was increased by divalent cations, EGTA, and dithiothreitol up to 0.5 mM. In membranes from prepuberal monkey testes, AII receptors were specific for AII analogs and of high affinity (Kd, 4.2 nM) with a receptor concentration of 7599 +/- 1342 fmol/mg protein. The presence of AII receptors in Leydig cells in rat and primate testes in conjunction with reports of the presence of other components of the renin-angiotensin system in the testes suggests that the peptide has a physiological role in testicular function.

  18. Angiotensin II receptor signalling.

    PubMed

    Daniels, Derek; Yee, Daniel K; Fluharty, Steven J

    2007-05-01

    Angiotensin II plays a key role in the regulation of body fluid homeostasis. To correct body fluid deficits that occur during hypovolaemia, an animal needs to ingest both water and electrolytes. Thus, it is not surprising that angiotensin II, which is synthesized in response to hypovolaemia, acts centrally to increase both water and NaCl intake. Here, we review findings relating to the properties of angiotensin II receptors that give rise to changes in behaviour. Data are described to suggest that divergent signal transduction pathways are responsible for separable behavioural responses to angiotensin II, and a hypothesis is proposed to explain how this divergence may map onto neural circuits in the brain.

  19. The human olfactory receptor repertoire

    PubMed Central

    Zozulya, Sergey; Echeverri, Fernando; Nguyen, Trieu

    2001-01-01

    Background The mammalian olfactory apparatus is able to recognize and distinguish thousands of structurally diverse volatile chemicals. This chemosensory function is mediated by a very large family of seven-transmembrane olfactory (odorant) receptors encoded by approximately 1,000 genes, the majority of which are believed to be pseudogenes in humans. Results The strategy of our sequence database mining for full-length, functional candidate odorant receptor genes was based on the high overall sequence similarity and presence of a number of conserved sequence motifs in all known mammalian odorant receptors as well as the absence of introns in their coding sequences. We report here the identification and physical cloning of 347 putative human full-length odorant receptor genes. Comparative sequence analysis of the predicted gene products allowed us to identify and define a number of consensus sequence motifs and structural features of this vast family of receptors. A new nomenclature for human odorant receptors based on their chromosomal localization and phylogenetic analysis is proposed. We believe that these sequences represent the essentially complete repertoire of functional human odorant receptors. Conclusions The identification and cloning of all functional human odorant receptor genes is an important initial step in understanding receptor-ligand specificity and combinatorial encoding of odorant stimuli in human olfaction. PMID:11423007

  20. The spatial and temporal expression of VEGF and its receptors 1 and 2 in post-traumatic bone bridge formation of the growth plate.

    PubMed

    Fischerauer, Eva; Heidari, Nima; Neumayer, Bernhard; Deutsch, Alexander; Weinberg, Annelie M

    2011-12-01

    Injuries to growth plates may initiate the formation of reversible or irreversible bone-bridges, may leading to bone length discrepancy or axis deviation. As vascular invasion is essential for the formation of bone tissue, the aim of our study was to investigate the kinetic expression of Vascular Endothelial Growth Factor (VEGF) and its receptors R1 and R2 and the ingrowth of vessels in the formation of bone bridges in a rat physeal injury model. Quantitative Real-Time Polymerase Chain Reaction was performed for VEGF and its receptors. Samples from the proximal physis of the tibial bone were immunohistochemically evaluated for the expression of VEGF and its R1 and R2 receptors and Laminin. Morphologically, physeal bone bridge formation was validated by means of Magnetic Resonance Imaging. Kinetic expression of VEGF and VEGF-R1 mRNA documented a tendency towards an increase in expression on day 7. Histological analyses showed a hematoma containing bone debris on day 1 which was replaced with bony trabeculae by day 14, forming a bone bridge by day 28 which was preceded and accompanied by angiogenesis and consistent with MRI data. VEGF and VEGF-R2 was expressed on the debris within the hematoma and bone trabeculae from days 1 to 28. VEGF-R1 expression was only noted until day 14. The findings of our study suggest that physeal bone bridge formation is in part triggered by VEGF expression and associated with angiogenesis, which was shown to precede bone bridge formation and may be further stimulated through VEGF-positive bone debris.

  1. Chemokine receptors in airway disease: which receptors to target?

    PubMed

    Owen, C

    2001-01-01

    Many disease states within the airway result in the co-ordinated infiltration of key inflammatory cells. The cellular influx is choreographed through the temporal and spatially-regulated expression of chemokines, which potentiate the migration of cells along gradients of chemotactic ligands. Chemokines act as ligands for the chemokine receptors; a distinct class of G-protein-coupled receptor. Over 40 chemokine ligands and 18 chemokine receptors have been identified on human cells. Chemokine receptors are divided into several classes; the two most prominent of which are the CC- and CXC-chemokine receptors, classified through the spatial arrangement of two conserved cysteine residues. The role of chemokine receptors such as CCR2, CCR3, CCR4, CCR8 and the CXC chemokine receptors; CXCR1 and CXCR2 on cell types of relevance to respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and chronic bronchitis will be explored in this review. Chemokines have proven to be amenable drug targets for the development of low molecular weight antagonists by the pharmaceutical industry. So far, no chemokine receptor antagonist has entered the clinic in trials for respiratory disease, but over the next few years it is expected that many will do so, at which time the potential of these exciting new targets will be fully realised.

  2. GABAB receptors modulate NMDA receptor calcium signals in dendritic spines.

    PubMed

    Chalifoux, Jason R; Carter, Adam G

    2010-04-15

    Metabotropic GABA(B) receptors play a fundamental role in modulating the excitability of neurons and circuits throughout the brain. These receptors influence synaptic transmission by inhibiting presynaptic release or activating postsynaptic potassium channels. However, their ability to directly influence different types of postsynaptic glutamate receptors remains unresolved. Here we examine GABA(B) receptor modulation in layer 2/3 pyramidal neurons from the mouse prefrontal cortex. We use two-photon laser-scanning microscopy to study synaptic modulation at individual dendritic spines. Using two-photon optical quantal analysis, we first demonstrate robust presynaptic modulation of multivesicular release at single synapses. Using two-photon glutamate uncaging, we then reveal that GABA(B) receptors strongly inhibit NMDA receptor calcium signals. This postsynaptic modulation occurs via the PKA pathway and does not affect synaptic currents mediated by AMPA or NMDA receptors. This form of GABA(B) receptor modulation has widespread implications for the control of calcium-dependent neuronal function.

  3. Axonal GABAA receptors.

    PubMed

    Trigo, Federico F; Marty, Alain; Stell, Brandon M

    2008-09-01

    Type A GABA receptors (GABA(A)Rs) are well established as the main inhibitory receptors in the mature mammalian forebrain. In recent years, evidence has accumulated showing that GABA(A)Rs are prevalent not only in the somatodendritic compartment of CNS neurons, but also in their axonal compartment. Evidence for axonal GABA(A)Rs includes new immunohistochemical and immunogold data: direct recording from single axonal terminals; and effects of local applications of GABA(A)R modulators on action potential generation, on axonal calcium signalling, and on neurotransmitter release. Strikingly, whereas presynaptic GABA(A)Rs have long been considered inhibitory, the new studies in the mammalian brain mostly indicate an excitatory action. Depending on the neuron that is under study, axonal GABA(A)Rs can be activated by ambient GABA, by GABA spillover, or by an autocrine action, to increase either action potential firing and/or transmitter release. In certain neurons, the excitatory effects of axonal GABA(A)Rs persist into adulthood. Altogether, axonal GABA(A)Rs appear as potent neuronal modulators of the mammalian CNS.

  4. The Multifaceted Mineralocorticoid Receptor

    PubMed Central

    Gomez-Sanchez, Elise; Gomez-Sanchez, Celso E.

    2015-01-01

    The primary adrenal cortical steroid hormones, aldosterone, and the glucocorticoids cortisol and corticosterone, act through the structurally similar mineralocorticoid (MR) and glucocorticoid receptors (GRs). Aldosterone is crucial for fluid, electrolyte, and hemodynamic homeostasis and tissue repair; the significantly more abundant glucocorticoids are indispensable for energy homeostasis, appropriate responses to stress, and limiting inflammation. Steroid receptors initiate gene transcription for proteins that effect their actions as well as rapid non-genomic effects through classical cell signaling pathways. GR and MR are expressed in many tissues types, often in the same cells, where they interact at molecular and functional levels, at times in synergy, others in opposition. Thus the appropriate balance of MR and GR activation is crucial for homeostasis. MR has the same binding affinity for aldosterone, cortisol, and corticosterone. Glucocorticoids activate MR in most tissues at basal levels and GR at stress levels. Inactivation of cortisol and corticosterone by 11β-HSD2 allows aldosterone to activate MR within aldosterone target cells and limits activation of the GR. Under most conditions, 11β-HSD1 acts as a reductase and activates cortisol/corticosterone, amplifying circulating levels. 11β-HSD1 and MR antagonists mitigate inappropriate activation of MR under conditions of oxidative stress that contributes to the pathophysiology of the cardiometabolic syndrome; however, MR antagonists decrease normal MR/GR functional interactions, a particular concern for neurons mediating cognition, memory, and affect. PMID:24944027

  5. Kinins and peptide receptors.

    PubMed

    Regoli, Domenico; Gobeil, Fernand

    2016-04-01

    This paper is divided into two sections: the first contains the essential elements of the opening lecture presented by Pr. Regoli to the 2015 International Kinin Symposium in S. Paulo, Brazil on June 28th and the second is the celebration of Dr. Regoli's 60 years of research on vasoactive peptides. The cardiovascular homeostasis derives from a balance of two systems, the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). The biologically active effector entity of RAS is angiotensin receptor-1 (AT-1R), and that of KKS is bradykinin B2 receptor (B2R). The first mediates vasoconstriction, the second is the most potent and efficient vasodilator. Thanks to its complex and multi-functional mechanism of action, involving nitric oxide (NO), prostacyclin and endothelial hyperpolarizing factor (EDHF). B2R is instrumental for the supply of blood, oxygen and nutrition to tissues. KKS is present on the vascular endothelium and functions as an autacoid playing major roles in cardiovascular diseases (CVDs) and diabetes. KKS exerts a paramount role in the prevention of thrombosis and atherosclerosis. Such knowledge emphasizes the already prominent value of the ACE-inhibitors (ACEIs) for the treatment of CVDs and diabetes. Indeed, the ACEIs, thanks to their double action (block of the RAS and potentiation of the KKS) are the ideal agents for a rational treatment of these diseases. PMID:26408609

  6. Leptin and its receptors.

    PubMed

    Wada, Nobuhiro; Hirako, Satoshi; Takenoya, Fumiko; Kageyama, Haruaki; Okabe, Mai; Shioda, Seiji

    2014-11-01

    Leptin is mainly produced in the white adipose tissue before being secreted into the blood and transported across the blood-brain barrier. Leptin binds to a specific receptor (LepR) that has numerous subtypes (LepRa, LepRb, LepRc, LepRd, LepRe, and LepRf). LepRb, in particular, is expressed in several brain nuclei, including the arcuate nucleus, the paraventricular nucleus, and the dorsomedial, lateral and ventromedial regions of the hypothalamus. LepRb is also co-expressed with several neuropeptides, including proopiomelanocortin, neuropeptide Y, galanin, galanin-like peptide, gonadotropin-releasing hormone, tyrosine hydroxylase and neuropeptide W. Functionally, LepRb induces activation of the JAK2/ERK, /STAT3, /STAT5 and IRS/PI3 kinase signaling cascades, which are important for the regulation of energy homeostasis and appetite in mammals. In this review, we discuss the structure, genetics and distribution of the leptin receptors, and their role in cell signaling mechanisms. PMID:25218975

  7. Angiotensin II receptor heterogeneity

    SciTech Connect

    Herblin, W.F.; Chiu, A.T.; McCall, D.E.; Ardecky, R.J.; Carini, D.J.; Duncia, J.V.; Pease, L.J.; Wong, P.C.; Wexler, R.R.; Johnson, A.L. )

    1991-04-01

    The possibility of receptor heterogeneity in the angiotensin II (AII) system has been suggested previously, based on differences in Kd values or sensitivity to thiol reagents. One of the authors earliest indications was the frequent observation of incomplete inhibition of the binding of AII to adrenal cortical membranes. Autoradiographic studies demonstrated that all of the labeling of the rat adrenal was blocked by unlabeled AII or saralasin, but not by DuP 753. The predominant receptor in the rat adrenal cortex (80%) is sensitive to dithiothreitol (DTT) and DuP 753, and is designated AII-1. The residual sites in the adrenal cortex and almost all of the sites in the rat adrenal medulla are insensitive to both DTT and DuP 753, but were blocked by EXP655. These sites have been confirmed by ligand binding studies and are designated AII-2. The rabbit adrenal cortex is unique in yielding a nonuniform distribution of AII-2 sites around the outer layer of glomerulosa cells. In the rabbit kidney, the sites on the glomeruli are AII-1, but the sites on the kidney capsule are AII-2. Angiotensin III appears to have a higher affinity for AII-2 sites since it inhibits the binding to the rabbit kidney capsule but not the glomeruli. Elucidation of the distribution and function of these diverse sites should permit the development of more selective and specific therapeutic strategies.

  8. [Adrenergic receptors of blood platelets].

    PubMed

    Lanza, F; Cazenave, J P

    1987-01-01

    Blood platelets possess adrenergic receptors and are stimulated by adrenaline in the circulation. This review summarizes the state of knowledge of the pharmacology of adrenergic receptors and the biochemical mechanisms of platelet activation by adrenaline in various physiological and pathological conditions. PMID:2837727

  9. Sensory receptors in monotremes.

    PubMed

    Proske, U; Gregory, J E; Iggo, A

    1998-07-29

    This is a summary of the current knowledge of sensory receptors in skin of the bill of the platypus, Ornithorhynchus anatinus, and the snout of the echidna, Tachyglossus aculeatus. Brief mention is also made of the third living member of the monotremes, the long-nosed echidna, Zaglossus bruijnii. The monotremes are the only group of mammals known to have evolved electroreception. The structures in the skin responsible for the electric sense have been identified as sensory mucous glands with an expanded epidermal portion that is innervated by large-diameter nerve fibres. Afferent recordings have shown that in both platypuses and echidnas the receptors excited by cathodal (negative) pulses and inhibited by anodal (positive) pulses. Estimates give a total of 40,000 mucous sensory glands in the upper and lower bill of the platypus, whereas there are only about 100 in the tip of the echidna snout. Recording of electroreceptor-evoked activity from the brain of the platypus have shown that the largest area dedicated to somatosensory input from the bill, S1, shows alternating rows of mechanosensory and bimodal neurons. The bimodal neurons respond to both electrosensory and mechanical inputs. In skin of the platypus bill and echidna snout, apart from the electroreceptors, there are structures called push rods, which consist of a column of compacted cells that is able to move relatively independently of adjacent regions of skin. At the base of the column are Merkel cell complexes, known to be type I slowly adapting mechanoreceptors, and lamellated corpuscles, probably vibration receptors. It has been speculated that the platypus uses its electric sense to detect the electromyographic activity from moving prey in the water and for obstacle avoidance. Mechanoreceptors signal contact with the prey. For the echidna, a role for the electrosensory system has not yet been established during normal foraging behaviour, although it has been shown that it is able to detect the presence

  10. Allosteric Modulation of Chemoattractant Receptors

    PubMed Central

    Allegretti, Marcello; Cesta, Maria Candida; Locati, Massimo

    2016-01-01

    Chemoattractants control selective leukocyte homing via interactions with a dedicated family of related G protein-coupled receptor (GPCR). Emerging evidence indicates that the signaling activity of these receptors, as for other GPCR, is influenced by allosteric modulators, which interact with the receptor in a binding site distinct from the binding site of the agonist and modulate the receptor signaling activity in response to the orthosteric ligand. Allosteric modulators have a number of potential advantages over orthosteric agonists/antagonists as therapeutic agents and offer unprecedented opportunities to identify extremely selective drug leads. Here, we resume evidence of allosterism in the context of chemoattractant receptors, discussing in particular its functional impact on functional selectivity and probe/concentration dependence of orthosteric ligands activities. PMID:27199992

  11. Nicotinic receptors and schizophrenia.

    PubMed

    Ripoll, Nadège; Bronnec, Marie; Bourin, Michel

    2004-07-01

    The incidence of smoking is very high in non-schizophrenic subjects presenting various psychiatric disorders (35 to 54%). However, the incidence of smoking is extremely high in schizophrenic patients: 80% to 90%, versus 25% to 30% of the general population. Various studies have demonstrated that the use of tobacco transiently restores the schizophrenic patient's cognitive and sensory deficits. Smoking cessation also appears to exacerbate the symptoms of the disease. Post-mortem binding studies have revealed a disturbance of nicotinic receptor expression, affecting the alpha(7) and alpha(4)beta(2) subunits, in various cerebral areas. Genetic linkage studies have also shown that the alpha(7) subunit is involved in schizophrenia. This review assesses the involvement of the nicotinic system in schizophrenia and suggests ways in which this system may participate in the pathophysiology of this disease.

  12. Androgen receptor genomic regulation

    PubMed Central

    Jin, Hong-Jian; Kim, Jung

    2013-01-01

    The transcriptional activity of the androgen receptor (AR) is not only critical for the normal development and function of the prostate but also pivotal to the onset and progression of prostate cancer (PCa). The studies of AR transcriptional regulation were previously limited to a handful of AR-target genes. Owing to the development of various high-throughput genomic technologies, significant advances have been made in recent years. Here we discuss the discoveries of genome-wide androgen-regulated genes in PCa cell lines, animal models and tissues using expression microarray and sequencing, the mapping of genomic landscapes of AR using Combining Chromatin Immunoprecipitation (ChIP)-on-chip and ChIP-seq assays, the interplay of transcriptional cofactors in defining AR binding profiles, and the genomic regulation and AR reprogramming in advanced PCa. PMID:25237629

  13. Exon organization of the mouse entactin gene corresponds to the structural domains of the polypeptide and has regional homology to the low-density lipoprotein receptor gene

    SciTech Connect

    Durkin, M.E.; Chung, A.E.; Wewer, U.M.

    1995-03-20

    Entactin is a widespread basement membrane protein of 150 kDa that binds to type IV collagen and laminin. The complete exon-intron structure of the mouse entactin gene has been determined from {lambda} genomic DNA clones. The gene spans at least 65 kb and contains 20 exons. The exon organization of the mouse entactin gene closely corresponds to the organization of the polypeptide into distinct structural and functional domains. The two amino-terminal globular domains are encoded by three exons each. Single exons encode the two protease-sensitive, O-glycosylated linking regions. The six EGF-like repeats and the single thyroglobulin-type repeat are each encoded by separate exons. The carboxyl-terminal half of entactin displays sequence homology to the growth factor-like region of the low-density lipoprotein receptor, and in both genes this region is encoded by eight exons. The positions of four introns are also conserved in the homologous region of the two genes. These observations suggest that the entactin gene has evolved via exon shuffling. Finally, several sequence polymorphisms useful for gene linkage analysis were found in the 3{prime} noncoding region of the last exon. 52 refs., 8 figs.

  14. Malignant Peripheral Nerve Sheath Tumor Invasion Requires Aberrantly Expressed Epidermal Growth Factor (EGF) Receptors and is Variably Enhanced by Multiple EGF Family Ligands

    PubMed Central

    Byer, Stephanie J.; Brossier, Nicole M.; Peavler, Lafe T.; Eckert, Jenell M.; Watkins, Stacey; Roth, Kevin A.; Carroll, Steven L.

    2013-01-01

    Aberrant epidermal growth factor receptor (EGFR) expression promotes the pathogenesis of malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1, but the mechanisms by which EGFR expression promotes MPNST pathogenesis are poorly understood. We hypothesized that inappropriately expressed EGFRs promote MPNST invasion and found that these kinases are concentrated in MPNST invadopodia in vitro. EGFR knockdown inhibited the migration of unstimulated MPNST cells in vitro and exogenous EGF further enhanced MPNST migration in a substrate-specific manner, promoting migration on laminin and, to a lesser extent, collagen. Thus, in this setting, EGF acts as a chemotactic factor. We also found that the 7 known EGFR ligands (EGF, betacellulin, epiregulin, heparin-binding EGF, transforming growth factor α [TGFα], amphiregulin, and epigen) variably enhanced MPNST migration in a concentration-dependent manner, with TGFα being particularly potent. With the exception of epigen, these factors similarly promoted the migration of non-neoplastic Schwann cells. Although transcripts encoding all 7 EGFR ligands were detected in human MPNST cells and tumor tissues, only TGFα was consistently overexpressed and was found to colocalize with EGFR in situ. These data indicate that constitutive EGFR activation, potentially driven by autocrine or paracrine TGFα signaling, promotes the aggressive invasive behavior characteristic of MPNSTs. PMID:23399900

  15. The somatostatin receptor family.

    PubMed

    Patel, Y C; Greenwood, M T; Panetta, R; Demchyshyn, L; Niznik, H; Srikant, C B

    1995-01-01

    The diverse biological effects of somatostatin (SST) are mediated through a family of G protein coupled receptors of which 5 members have been recently identified by molecular cloning. This review focuses on the molecular biology, pharmacology, expression, and function of these receptors with particular emphasis on the human (h) homologs. hSSTRs are encoded by a family of 5 genes which map to separate chromosomes and which, with one exception, are intronless. SSTR2 gives rise to spliced variants, SSTR2A and 2B. hSSTR1-4 display weak selectivity for SST-14 binding whereas hSSTR5 is SST-28 selective. Based on structural similarity and reactivity for octapeptide and hexapeptide SST analogs, hSSTR2,3, and 5 belong to a similar SSTR subclass. hSSTR1 and 4 react poorly with these analogs and belong to a separate subclass. All 5 hSSTRs are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin sensitive GTP binding proteins. Some of the subtypes are also coupled to tyrosine phosphatase (SSTR1,2), Ca2+ channels (SSTR2), Na+/H+ exchanger (SSTR1), PLA-2 (SSTR4), and MAP kinase (SSTR4). mRNA for SSTR1-5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective, and tissue- and species-specific. Pituitary and islet tumors express several SSTR genes suggesting that multiple SSTR subtypes are coexpressed in the same cell. Structure-function studies indicate that the core residues in SST-14 ligand Phe6-Phe11 dock within a ligand binding pocket located in TMDs 3-7 which is lined by hydrophobic and charged amino acid residues.

  16. Estrogen-related receptor β (ERRβ) – renaissance receptor or receptor renaissance?

    PubMed Central

    Divekar, Shailaja D.; Tiek, Deanna M.; Fernandez, Aileen; Riggins, Rebecca B.

    2016-01-01

    Estrogen-related receptors (ERRs) are founding members of the orphan nuclear receptor (ONR) subgroup of the nuclear receptor superfamily. Twenty-seven years of study have yet to identify cognate ligands for the ERRs, though they have firmly placed ERRα and ERRγ at the intersection of cellular metabolism and oncogenesis. The pace of discovery for novel functions of ERRβ, however, has until recently been somewhat slower than that of its family members. ERRβ has also been largely ignored in summaries and perspectives of the ONR literature. Here, we provide an overview of established and emerging knowledge of ERRβ in mouse, man, and other species, highlighting unique aspects of ERRβ biology that set it apart from the other two estrogen-related receptors, with a focus on the impact of alternative splicing on the structure and function of this receptor. PMID:27507929

  17. Estrogen-related receptor β (ERRβ) - renaissance receptor or receptor renaissance?

    PubMed

    Divekar, Shailaja D; Tiek, Deanna M; Fernandez, Aileen; Riggins, Rebecca B

    2016-01-01

    Estrogen-related receptors (ERRs) are founding members of the orphan nuclear receptor (ONR) subgroup of the nuclear receptor superfamily. Twenty-seven years of study have yet to identify cognate ligands for the ERRs, though they have firmly placed ERRα and ERRγ at the intersection of cellular metabolism and oncogenesis. The pace of discovery for novel functions of ERRβ, however, has until recently been somewhat slower than that of its family members. ERRβ has also been largely ignored in summaries and perspectives of the ONR literature. Here, we provide an overview of established and emerging knowledge of ERRβ in mouse, man, and other species, highlighting unique aspects of ERRβ biology that set it apart from the other two estrogen-related receptors, with a focus on the impact of alternative splicing on the structure and function of this receptor.

  18. Estrogen-related receptor β (ERRβ) - renaissance receptor or receptor renaissance?

    PubMed

    Divekar, Shailaja D; Tiek, Deanna M; Fernandez, Aileen; Riggins, Rebecca B

    2016-01-01

    Estrogen-related receptors (ERRs) are founding members of the orphan nuclear receptor (ONR) subgroup of the nuclear receptor superfamily. Twenty-seven years of study have yet to identify cognate ligands for the ERRs, though they have firmly placed ERRα and ERRγ at the intersection of cellular metabolism and oncogenesis. The pace of discovery for novel functions of ERRβ, however, has until recently been somewhat slower than that of its family members. ERRβ has also been largely ignored in summaries and perspectives of the ONR literature. Here, we provide an overview of established and emerging knowledge of ERRβ in mouse, man, and other species, highlighting unique aspects of ERRβ biology that set it apart from the other two estrogen-related receptors, with a focus on the impact of alternative splicing on the structure and function of this receptor. PMID:27507929

  19. Immunohistochemical localization of metallothionein in human breast cancer in comparison with cathepsin D, stromelysin-1, CD44, extracellular matrix components, P53, Rb, C-erbB-2, EGFR, steroid receptor content and proliferation.

    PubMed

    Ioachim, E; Kamina, S; Demou, A; Kontostolis, M; Lolis, D; Agnantis, N J

    1999-01-01

    Metallothionein (MT) is a low molecular weight, cysteine-rich, zinc-binding protein that may have a function in cellular repair processes, growth and differentiation. Using a monoclonal antibody (E9) to metallothionein, we investigated the immunohistochemical expression of MT in routinely fixed and paraffin-embedded tissue from 98 cases of female breast carcinomas. The MT expression was studied in comparison with the expression of the basement membrane (BM) antigens (type IV collagen, laminin), fibronectin, cathepsin D, adhesion molecule CD44, p53 protein, the pRb, c-erbB-2 oncoprotein, EGFR, stromelysin-1, proliferation indices (Ki-67, PCNA), steroid receptor content as well as with other conventional clinicopathological parameters of breast cancer. Strong MT expression was observed in the majority of tumour cells in 18.4% of tumours, focal MT positivity in 13.3% and almost complete lack of MT expression in 68.4% of cases (mean value 33.36 +/- 26.36). The MT expression in carcinoma cells was strongly associated with the DCIS component of the tumour (p < 0.0001). High values of MT were correlated with low steroid receptor status (p = 0.08 for ER receptor and p = 0.019 for PgR receptor content). MT positive cases were correlated with stromelysin-1 expression (p = 0.059) and cathepsin D (p = 0.058). These findings suggest that MT expression is characteristic of the early phase