46 results on '"Durkin, M E"'
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2. Deleted in liver cancer 3 (DLC-3), a novel Rho GTPase-activating protein, is downregulated in cancer and inhibits tumor cell growth
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Durkin, M E, Ullmannova, V, Guan, M, and Popescu, N C
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- 2007
- Full Text
- View/download PDF
3. Mice with a targeted deletion of the tetranectin gene exhibit a spinal deformity.
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Iba, K, Durkin, M E, Johnsen, L, Hunziker, E, Damgaard-Pedersen, K, Zhang, H, Engvall, E, Albrechtsen, R, Wewer, U M, Iba, K, Durkin, M E, Johnsen, L, Hunziker, E, Damgaard-Pedersen, K, Zhang, H, Engvall, E, Albrechtsen, R, and Wewer, U M
- Abstract
Udgivelsesdato: 2001-Nov, Tetranectin is a plasminogen-binding, homotrimeric protein belonging to the C-type lectin family of proteins. Tetranectin has been suggested to play a role in tissue remodeling, due to its ability to stimulate plasminogen activation and its expression in developing tissues such as developing bone and muscle. To test the functional role of tetranectin directly, we have generated mice with a targeted disruption of the gene. We report that the tetranectin-deficient mice exhibit kyphosis, a type of spinal deformity characterized by an increased curvature of the thoracic spine. The kyphotic angles were measured on radiographs. In 6-month-old normal mice (n = 27), the thoracic angle was 73 degrees +/- 2 degrees, while in tetranectin-deficient 6-month-old mice (n = 35), it was 93 degrees +/- 2 degrees (P < 0.0001). In approximately one-third of the mutant mice, X-ray analysis revealed structural changes in the morphology of the vertebrae. Histological analysis of the spines of these mice revealed an apparently asymmetric development of the growth plate and of the intervertebral disks of the vertebrae. In the most advanced cases, the growth plates appeared disorganized and irregular, with the disk material protruding through the growth plate. Tetranectin-null mice had a normal peak bone mass density and were not more susceptible to ovariectomy-induced osteoporosis than were their littermates as determined by dual-emission X-ray absorptiometry scanning. These results demonstrate that tetranectin plays a role in tissue growth and remodeling. The tetranectin-deficient mouse is the first mouse model that resembles common human kyphotic disorders, which affect up to 8% of the population.
- Published
- 2001
4. Regulation of laminin beta2 chain gene expression in human cancer cell lines.
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Durkin, M E, Nielsen, F C, Loechel, F, Albrechtsen, R, Wewer, U M, Durkin, M E, Nielsen, F C, Loechel, F, Albrechtsen, R, and Wewer, U M
- Abstract
Udgivelsesdato: 2001-Jul, The laminin beta2 chain is a basement membrane component expressed in a tissue- and developmental stage-specific manner. In this report we have examined the transcriptional and post-transcriptional regulation of the human laminin beta2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma and clone A colon carcinoma cells express the laminin beta2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the beta2 chain. Segments of the beta2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested by transient transfection. Sequences downstream of the transcription start site between nucleotides +91 and +120 were found to be essential for luciferase activity in the two cell lines. Additional positive regulatory regions were present further upstream, between nucleotides -164 to -667 and between nucleotides -667 to -1724. Genomic DNA at the 3' end of the gene also appeared to have enhancer activity, as a 1.1-kb fragment located downstream of the last exon stimulated the luciferase activity of the nucleotides -667/+297 promoter segment approximately threefold. Alternative splicing of the first intron of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain mRNA, transient transfection of chimeric beta2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates.
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- 2001
5. Risk factors for injuries due to the 1990 earthquake in Luzon, Philippines
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Roces, M. C., White, M. E., Dayrit, M. M., and Durkin, M. E.
- Subjects
Disasters ,Emergency Medical Services ,Socioeconomic Factors ,Risk Factors ,Case-Control Studies ,Philippines ,Confidence Intervals ,Odds Ratio ,Humans ,Wounds and Injuries ,Disaster Planning ,Research Article ,Demography - Abstract
On 16 July 1990, an earthquake measuring 7.7 on the Richter scale struck the island of Luzon, Philippines. A case-control study was carried out to identify the risk factors for earthquake-related injuries and at the same time observations were made on the rescue efforts. Being hit by falling objects was the leading cause of injury (34%). Those injured during the tremor were more likely to have been inside buildings constructed of concrete or mixed materials (odds ratio, 2.6; 95% confidence interval (CI), 1.7-4.1) and to have been on the middle floors of multistorey buildings (odds ratio, 3.4; 95% CI, 2.2-5.5). Leaving a building during the earthquake was a protective behaviour (odds ratio, 0.3; 95% CI, 0.2-0.8). Of the 235 survivors who were trapped and rescued alive from the rubble, 99% were rescued within 48 hours of the impact of the tremor. These findings should prove useful in developing seismic safety codes. People should be taught proper evasive actions to take during earthquakes, and training in basic first aid and methods of rescue should be an integral part of community preparedness programmes.
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- 1992
6. Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS).
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Durkin, M E, Jäger, A C, Khurana, T S, Nielsen, F C, Albrechtsen, R, Wewer, U M, Durkin, M E, Jäger, A C, Khurana, T S, Nielsen, F C, Albrechtsen, R, and Wewer, U M
- Abstract
Udgivelsesdato: 1999-null, The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).
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- 1999
7. Frequent downregulation and loss of WWOX gene expression in human hepatocellular carcinoma
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Park, S-W, primary, Ludes-Meyers, J, additional, Zimonjic, D B, additional, Durkin, M E, additional, Popescu, N C, additional, and Aldaz, C M, additional
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- 2004
- Full Text
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8. Evaluation of Lama5 as a candidate for the mouse ragged (Ra) mutation.
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Durkin, M E, Albrechtsen, R, Chambers, D M, Abbott, C M, Wewer, U M, Durkin, M E, Albrechtsen, R, Chambers, D M, Abbott, C M, and Wewer, U M
- Abstract
Udgivelsesdato: 1998-Sep-8, The laminin alpha5 chain is a component of the basement membranes of many developing and adult tissues. The mouse laminin alpha5 chain gene (Lama5) has been mapped close to the locus of the semidominant ragged (Ra) mutation on distal chromosome 2. The cause of the Ra mutation, which is usually lethal in the homozygous state, has not been determined. We have investigated whether a defect in Lama5 is responsible for the ragged mutation, using the RaJ strain. No differences in the level of the laminin alpha5 chain transcript were found in placental RNA from homozygous RaJ mutant embryos compared to normal littermates. Antiserum raised against a recombinant laminin alpha5 chain polypeptide stained the basement membranes of both normal and homozygous mutant embryos to a similar extent. More precise mapping of Lama5 on an interspecific Ra backcross indicated that Lama5 is proximal to the Ra locus. These results exclude Lama5 as a candidate gene for the Ra mutation.
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- 1998
9. Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro.
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Wewer, U M, Iba, K, Durkin, M E, Nielsen, F C, Loechel, F, Gilpin, B J, Kuang, W, Engvall, E, Albrechtsen, R, Wewer, U M, Iba, K, Durkin, M E, Nielsen, F C, Loechel, F, Gilpin, B J, Kuang, W, Engvall, E, and Albrechtsen, R
- Abstract
Udgivelsesdato: 1998-Aug-15, Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.
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- 1998
10. Assignment of the gene for human tetranectin (TNA) to chromosome 3p22-->p21.3 by somatic cell hybrid mapping.
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Durkin, M E, Naylor, S L, Albrechtsen, R, Wewer, U M, Durkin, M E, Naylor, S L, Albrechtsen, R, and Wewer, U M
- Abstract
Udgivelsesdato: 1997-null, Tetranectin is a plasminogen-binding protein that is induced during the mineralization phase of osteogenesis. By screening a human chromosome 3 somatic cell hybrid mapping panel, we have localized the human tetranectin gene (TNA) to 3p22-->p21.3, which is distinct from the loci of two human connective tissue disorders that map to the short arm of chromosome 3, MFS2 and LRS1.
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- 1997
11. Extrasynaptic location of laminin beta 2 chain in developing and adult human skeletal muscle.
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Wewer, U M, Thornell, L E, Loechel, F, Zhang, X, Durkin, M E, Amano, S, Burgeson, R E, Engvall, E, Albrechtsen, R, Virtanen, I, Wewer, U M, Thornell, L E, Loechel, F, Zhang, X, Durkin, M E, Amano, S, Burgeson, R E, Engvall, E, Albrechtsen, R, and Virtanen, I
- Abstract
Udgivelsesdato: 1997-Aug, We have investigated the distribution of the laminin beta 2 chain (previously s-laminin) in human fetal and adult skeletal muscle and compared it to the distribution of laminin beta 1. Immunoblotting and transfection assays were used to characterize a panel of monoclonal and polyclonal antibodies to the laminin beta 2 chain. We found that laminin beta 1 chain was detected at all times during development from 10 weeks of gestation. Laminin beta 2 chain was first detected in 15 to 22-week-old fetal skeletal muscle as distinct focal immunoreactivity in the sarcolemmal basement membrane area of some myofibers. In the adult skeletal muscle, laminin beta 2 chain immunoreactivity was found along the entire perimeter of each of the individual myofibers in a large series of different muscles studied. Laminin beta 2 chain was similarly found in the skeletal muscle basement membranes in patients with Duchenne and Becker muscular dystrophy. Immunoaffinity chromatography of muscle extracts with a monoclonal antibody to the laminin alpha 2 chain followed by immunoblotting with various antibodies to the beta 2 chain demonstrated the presence of the laminin-4 (alpha 2-beta 2-gamma 1) isoform. Together the present results demonstrate a prominent extrasynaptic localization of laminin beta 2 in the human muscle, suggesting that it may have an important function in the sarcolemmal basement membrane.
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- 1997
12. Tissue-specific expression of the human laminin alpha5-chain, and mapping of the gene to human chromosome 20q13.2-13.3 and to distal mouse chromosome 2 near the locus for the ragged (Ra) mutation.
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Durkin, M E, Loechel, F, Mattei, M G, Gilpin, B J, Albrechtsen, R, Wewer, U M, Durkin, M E, Loechel, F, Mattei, M G, Gilpin, B J, Albrechtsen, R, and Wewer, U M
- Abstract
Udgivelsesdato: 1997-Jul-14, To investigate the function of the laminin alpha5-chain, previously identified in mice, cDNA clones encoding the 953-amino-acid carboxy terminal G-domain of the human laminin alpha5-chain were characterized. Northern blot analysis showed that the laminin alpha5-chain is expressed in human placenta, heart, lung, skeletal muscle, kidney, and pancreas. The human laminin alpha5-chain gene (LAMA5) was assigned to chromosome 20q13.2-q13.3 by in situ hybridization, and the mouse gene (Lama5) was mapped by linkage analysis to a syntonic region of distal chromosome 2, close to the locus for the ragged (Ra) mutation.
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- 1997
13. Structural organization of the human and mouse laminin beta2 chain genes, and alternative splicing at the 5' end of the human transcript.
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Durkin, M E, Gautam, M, Loechel, F, Sanes, J R, Merlie, J P, Albrechtsen, R, Wewer, U M, Durkin, M E, Gautam, M, Loechel, F, Sanes, J R, Merlie, J P, Albrechtsen, R, and Wewer, U M
- Abstract
Udgivelsesdato: 1996-Jun-7, We have determined the structural organization of the human and mouse genes that encode the laminin beta2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon-intron organization and are the smallest laminin chain genes characterized to date, due to the unusually small size of their introns. The laminin beta2 chain genes of both species consist of 33 exons that span
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- 1996
14. Laminin beta 2 chain and adhalin deficiency in the skeletal muscle of Walker-Warburg syndrome (cerebro-ocular dysplasia-muscular dystrophy).
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Wewer, U M, Durkin, M E, Zhang, X, Laursen, H, Nielsen, N H, Towfighi, J, Engvall, E, Albrechtsen, R, Wewer, U M, Durkin, M E, Zhang, X, Laursen, H, Nielsen, N H, Towfighi, J, Engvall, E, and Albrechtsen, R
- Abstract
Udgivelsesdato: 1995-Nov, Muscular dystrophy may be caused by disturbances in a number of muscle proteins that appear to be part of a chain of interacting molecules that includes cytoskeletal, cell membrane, and basement membrane components. We found that the skeletal muscle cells in two cases of Walker-Warburg syndrome were severely deficient in the laminin beta 2 chain and in adhalin. The findings indicate that these two proteins are key molecules in the interactive protein complex conferring muscle stability and cell survival.
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- 1995
15. 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
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Durkin, M E, Wewer, U M, Chung, A E, Durkin, M E, Wewer, U M, and Chung, A E
- Abstract
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' noncoding region of the last exon.
- Published
- 1995
16. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas.
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Wewer, U M, Gerecke, D R, Durkin, M E, Kurtz, K S, Mattei, M G, Champliaud, M F, Burgeson, R E, Albrechtsen, R, Wewer, U M, Gerecke, D R, Durkin, M E, Kurtz, K S, Mattei, M G, Champliaud, M F, Burgeson, R E, and Albrechtsen, R
- Abstract
Udgivelsesdato: 1994-Nov-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 the 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.
- Published
- 1994
17. A potential role for tetranectin in mineralization during osteogenesis.
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Wewer, U M, Ibaraki, K, Schjørring, P, Durkin, M E, Young, M F, Albrechtsen, R, Wewer, U M, Ibaraki, K, Schjørring, P, Durkin, M E, Young, M F, and Albrechtsen, R
- Abstract
Udgivelsesdato: 1994-Dec, Tetranectin is a protein shared by the blood and the extracellular matrix. Tetranectin is composed of four identical, noncovalently bound polypeptides each with a molecular mass of approximately 21 kD. There is some evidence that tetranectin may be involved in fibrinolysis and proteolysis during tissue remodeling, but its precise biological function is not known. Tetranectin is enriched in the cartilage of the shark, but the gene expression pattern in the mammalian skeletal system has not been determined. In the present study we have examined the expression pattern and putative function of tetranectin during osteogenesis. In the newborn mouse, strong tetranectin immunoreactivity was found in the newly formed woven bone around the cartilage anlage in the future bone marrow and along the periosteum forming the cortex. No tetranectin immunoreactivity was found in the proliferating and hypertrophic cartilage or in the surrounding skeletal muscle. Using an in vitro mineralizing system, we examined osteoblastic cells at different times during their growth and differentiation. Tetranectin mRNA appeared in the cultured osteoblastic cells in parallel with mineralization, in a pattern similar to that of bone sialoprotein, which is regarded as one of the late bone differentiation markers. To explore the putative biological role of tetranectin in osteogenesis we established stably transfected cell lines (PC12-tet) overexpressing recombinant tetranectin as evidenced by Northern and Western blot analysis and immunoprecipitation. Both control PC12 cells and PC12-tet cells injected into nude mice produced tumors containing bone material, as evidenced by von Kossa staining for calcium and immunostaining with bone sialoprotein and alkaline phosphatase antiserum. Nude mice tumors established from PC12-tet cells contained approximately fivefold more bone material than those produced by the untransfected PC12 cell line or by the PC12 cells transfected with the expression vector with no
- Published
- 1994
18. Laminin 2 chain and adhalin deficiency in the skeletal muscle of Walker-Warburg syndrome (cerebroocular dysplasia-muscular dystrophy)
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Wewer, U. M., primary, Durkin, M. E., additional, Zhang, X., additional, Laursen, H., additional, Nielsen, N. H., additional, Towfighi, J., additional, Engvall, E., additional, and Albrechtsen, R., additional
- Published
- 1995
- Full Text
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19. A potential role for tetranectin in mineralization during osteogenesis.
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Wewer, U M, primary, Ibaraki, K, additional, Schjørring, P, additional, Durkin, M E, additional, Young, M F, additional, and Albrechtsen, R, additional
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- 1994
- Full Text
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20. Characterization of the basement membrane glycoprotein entactin synthesized in a baculovirus expression system.
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Tsao, T, primary, Hsieh, J C, additional, Durkin, M E, additional, Wu, C Y, additional, Chakravarti, S, additional, Dong, L J, additional, Lewis, M, additional, and Chung, A E, additional
- Published
- 1990
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21. Characterization of the human laminin β2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS).
- Author
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Durkin, M. E., Jäger, A. C., Khurana, T. S., Nielsen, F. C., Albrechtsen, R., and Wewer, U. M.
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LINKAGE (Genetics) , *MICROSATELLITE repeats , *DNA , *TRANSGLUTAMINASES , *GENETIC markers , *TELOMERES - Abstract
The laminin β2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin β2 chain gene (LAMB2) on chromosome 3p21.3→p21.2, a region paralogous with the chromosome 7q22→q31 region that contains the laminin β1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3′ end of LAMB2 lies 16 kb from the 5′ end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3′ end of LAMB2L was similar to exons 8–12 of the laminin β2 chain gene. The LAMB2L–LAMB2–QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1). [ABSTRACT FROM AUTHOR]
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- 1999
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22. Carboxyl-terminal sequence of entactin deduced from a cDNA clone.
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Durkin, M E, Carlin, B E, Vergnes, J, Bartos, B, Merlie, J, and Chung, A E
- Abstract
Entactin is a widely distributed basement membrane sulfated glycoprotein of approximately equal to 150 kDa. The entactin gene is expressed early in mouse embryogenesis. Two cDNA clones complementary to rat entactin mRNA were isolated by antibody screening of an oligo(dT)-primed cDNA library constructed in the lambda gt11 expression vector. One of the clones, lambda 1E, was subcloned into plasmid pBR322 and further characterized. The clone contained sequences complementary to an mRNA species 6 kilobases in length. This mRNA was translated in rabbit reticulocyte lysates to yield a polypeptide of 143 kDa that was precipitated with anti-entactin antiserum. The cDNA insert, 1328 base pairs long, was sequenced and found to contain an open reading frame of 729 base pairs that coded for 243 amino acids at the carboxyl terminus of entactin. Analysis of the peptide revealed no extended alpha-helical or beta-sheet secondary structures. Radiolabeled probes prepared by nicktranslation of p lambda 1E were used to monitor the steady-state levels of entactin mRNA in F9 embryonal carcinoma cells that were induced to differentiate by exposure to retinoic acid and dibutyryl cyclic AMP. The increase in steady-state levels of entactin mRNA lagged behind the increase in mRNA for the B2 chain of laminin, suggesting that laminin and entactin are independently rather than coordinately regulated.
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- 1987
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23. Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor.
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Durkin, M E, Chakravarti, S, Bartos, B B, Liu, S H, Friedman, R L, and Chung, A E
- Abstract
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.
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- 1988
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24. Synthesis of laminin and entactin by F9 cells induced with retinoic acid and dibutyryl cyclic AMP.
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Carlin, B E, Durkin, M E, Bender, B, Jaffe, R, and Chung, A E
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Mouse embryonal carcinoma F9 cells were exposed to retinoic acid and dibutyryl cyclic AMP. The treated cells synthesized and secreted into the culture medium the basal lamina components, laminin (GP-1 and GP-2) and entactin. The time course of secretion of the basal lamina components was examined by electron microscopic and immunochemical procedures. The induction of the cells resulted in major morphological changes and the deposition of both laminin and entactin at the cell surface and cell junctions. Intracellular deposits of laminin could be localized to the endoplasmic reticulum and membrane-bound intracytoplasmic vacuoles. Concomitant with the appearance of laminin and entactin, there was a loss of fibronectin synthesis and a marked decrease in a 190,000-Da sulfated glycoprotein that appeared to be related to entactin. In the induced cells, laminin and entactin were associated in a complex that could be dissociated with low concentrations of sodium dodecyl sulfate. The induction of laminin and entactin seem to be independent. The enhanced synthesis of laminin appeared to be under transcriptional regulation since it was found that induced F9 cells contained translatable mRNA for GP-2 when tested in a rabbit reticulocyte lysate system. The uninduced cells did not contain detectable quantities of translatable GP-2 mRNA.
- Published
- 1983
- Full Text
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26. Laminin β2 chain and adhalin deficiency in the skeletal muscle of WalkerWarburg syndrome cerebroocular dysplasiamuscular dystrophy
- Author
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Wewer, U. M., Durkin, M. E., Zhang, X., Laursen, H., Nielsen, N. H., Towfighi, J., Engvall, E., and Albrechtsen, R.
- Abstract
Muscular dystrophy may be caused by disturbances in a number of muscle proteins that appear to be part of a chain of interacting molecules that includes cytoskeletal, cell membrane, and basement membrane components. We found that the skeletal muscle cells in two cases of Walker-Warburg syndrome were severely deficient in the laminin β2 chain and in adhalin. The findings indicate that these two proteins are key molecules in the interactive protein complex conferring muscle stability and cell survival.
- Published
- 1995
27. Characterization of the 5' end of the mouse Ent gene encoding the basement membrane protein, entactin
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Durkin, M. E., Liu, S.-H., Reing, J., and Chung, A. E.
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- 1993
- Full Text
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28. Mice with a targeted deletion of the tetranectin gene exhibit a spinal deformity.
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Iba K, Durkin ME, Johnsen L, Hunziker E, Damgaard-Pedersen K, Zhang H, Engvall E, Albrechtsen R, and Wewer UM
- Subjects
- Animals, Blood Proteins genetics, Bone Density, Disease Susceptibility, Female, Gene Deletion, Gene Targeting methods, Kyphosis genetics, Kyphosis pathology, Lectins genetics, Mice, Mice, Inbred C57BL, Mice, Knockout, Osteoporosis etiology, Ovariectomy, Thoracic Vertebrae abnormalities, Thoracic Vertebrae pathology, Blood Proteins physiology, Kyphosis etiology, Lectins physiology, Lectins, C-Type
- Abstract
Tetranectin is a plasminogen-binding, homotrimeric protein belonging to the C-type lectin family of proteins. Tetranectin has been suggested to play a role in tissue remodeling, due to its ability to stimulate plasminogen activation and its expression in developing tissues such as developing bone and muscle. To test the functional role of tetranectin directly, we have generated mice with a targeted disruption of the gene. We report that the tetranectin-deficient mice exhibit kyphosis, a type of spinal deformity characterized by an increased curvature of the thoracic spine. The kyphotic angles were measured on radiographs. In 6-month-old normal mice (n = 27), the thoracic angle was 73 degrees +/- 2 degrees, while in tetranectin-deficient 6-month-old mice (n = 35), it was 93 degrees +/- 2 degrees (P < 0.0001). In approximately one-third of the mutant mice, X-ray analysis revealed structural changes in the morphology of the vertebrae. Histological analysis of the spines of these mice revealed an apparently asymmetric development of the growth plate and of the intervertebral disks of the vertebrae. In the most advanced cases, the growth plates appeared disorganized and irregular, with the disk material protruding through the growth plate. Tetranectin-null mice had a normal peak bone mass density and were not more susceptible to ovariectomy-induced osteoporosis than were their littermates as determined by dual-emission X-ray absorptiometry scanning. These results demonstrate that tetranectin plays a role in tissue growth and remodeling. The tetranectin-deficient mouse is the first mouse model that resembles common human kyphotic disorders, which affect up to 8% of the population.
- Published
- 2001
- Full Text
- View/download PDF
29. Regulation of laminin beta2 chain gene expression in human cancer cell lines.
- Author
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Durkin ME, Nielsen FC, Loechel F, Albrechtsen R, and Wewer UM
- Subjects
- Alternative Splicing, Amino Acid Sequence, Base Sequence, Choriocarcinoma, Colonic Neoplasms, Enhancer Elements, Genetic, Fluorescent Antibody Technique, Indirect, Humans, Laminin analysis, Molecular Sequence Data, Promoter Regions, Genetic, Protein Biosynthesis, RNA Splicing, RNA, Messenger genetics, Recombinant Proteins analysis, Recombinant Proteins biosynthesis, Regulatory Sequences, Nucleic Acid, Rhabdomyosarcoma, Transfection, Tumor Cells, Cultured, Gene Expression Regulation, Neoplastic, Laminin genetics, Transcription, Genetic
- Abstract
The laminin beta2 chain is a basement membrane component expressed in a tissue- and developmental stage-specific manner. In this report we have examined the transcriptional and post-transcriptional regulation of the human laminin beta2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma and clone A colon carcinoma cells express the laminin beta2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the beta2 chain. Segments of the beta2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested by transient transfection. Sequences downstream of the transcription start site between nucleotides +91 and +120 were found to be essential for luciferase activity in the two cell lines. Additional positive regulatory regions were present further upstream, between nucleotides -164 to -667 and between nucleotides -667 to -1724. Genomic DNA at the 3' end of the gene also appeared to have enhancer activity, as a 1.1-kb fragment located downstream of the last exon stimulated the luciferase activity of the nucleotides -667/+297 promoter segment approximately threefold. Alternative splicing of the first intron of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain mRNA, transient transfection of chimeric beta2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates.
- Published
- 2001
- Full Text
- View/download PDF
30. Integration of a c-myc transgene results in disruption of the mouse Gtf2ird1 gene, the homologue of the human GTF2IRD1 gene hemizygously deleted in Williams-Beuren syndrome.
- Author
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Durkin ME, Keck-Waggoner CL, Popescu NC, and Thorgeirsson SS
- Subjects
- Animals, Base Sequence, Chromosome Mapping methods, Chromosomes, Human, Pair 7, DNA, Exons, Gene Deletion, Gene Expression, Genetic Linkage, Helix-Loop-Helix Motifs, Humans, Mice, Mice, Transgenic, Models, Animal, Molecular Sequence Data, RNA, Messenger genetics, Transgenes, Translocation, Genetic, Genes, myc, Transcription Factors genetics, Williams Syndrome genetics
- Abstract
Transgenic mice expressing c-myc under the control of the albumin promoter and enhancer develop liver tumors and have served as a useful model for studying the progression of hepatocarcinogenesis. The chromosomes of one line of c-myc transgenic mice carry the reciprocal translocation t(5;6)(G1;F2) adjacent to the transgene insertion site on the 5G1-ter segment translocated to chromosome 6. To characterize the genomic alterations in the c-myc transgenic animals, we have cloned the mouse DNA flanking the transgene array. By linkage mapping, the transgene integration site was localized to the region of distal chromosome 5 syntenic to the region on human chromosome 7q11.23 that is hemizgygously deleted in Williams-Beuren syndrome, a multisystemic developmental disorder. Comparison of the genomic DNA structure in wildtype and transgenic mice revealed that the transgene integration had induced an approximately 40-kb deletion, starting downstream of the Cyln2 gene and including the first exon of the Gtf2ird1 gene. Gtf2ird1 encodes a polypeptide related to general transcription factor TFII-I, and it is the mouse orthologue of GTF2IRD1 (WBSCR11), one of the genes commonly deleted in Williams-Beuren syndrome patients. Loss of the 5' end of the Gtf2ird1 gene resulted in greatly reduced expression of Gtf2ird1 mRNA in mice homozygous for the transgene.
- Published
- 2001
- Full Text
- View/download PDF
31. Evaluation of Lama5 as a candidate for the mouse ragged (Ra) mutation.
- Author
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Durkin ME, Albrechtsen R, Chambers DM, Abbott CM, and Wewer UM
- Subjects
- Animals, Base Sequence, Chromosome Mapping, DNA, Female, Heterozygote, Homozygote, Male, Mice, Mice, Inbred C3H, Mice, Mutant Strains, Molecular Sequence Data, Laminin genetics, Mutation
- Abstract
The laminin alpha5 chain is a component of the basement membranes of many developing and adult tissues. The mouse laminin alpha5 chain gene (Lama5) has been mapped close to the locus of the semidominant ragged (Ra) mutation on distal chromosome 2. The cause of the Ra mutation, which is usually lethal in the homozygous state, has not been determined. We have investigated whether a defect in Lama5 is responsible for the ragged mutation, using the RaJ strain. No differences in the level of the laminin alpha5 chain transcript were found in placental RNA from homozygous RaJ mutant embryos compared to normal littermates. Antiserum raised against a recombinant laminin alpha5 chain polypeptide stained the basement membranes of both normal and homozygous mutant embryos to a similar extent. More precise mapping of Lama5 on an interspecific Ra backcross indicated that Lama5 is proximal to the Ra locus. These results exclude Lama5 as a candidate gene for the Ra mutation., (Copyright 1998 Academic Press.)
- Published
- 1998
- Full Text
- View/download PDF
32. Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro.
- Author
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Wewer UM, Iba K, Durkin ME, Nielsen FC, Loechel F, Gilpin BJ, Kuang W, Engvall E, and Albrechtsen R
- Subjects
- Animals, Bupivacaine metabolism, Cell Line, Embryonic and Fetal Development, Gene Expression Regulation, Developmental genetics, Immunohistochemistry, Mice, Mice, Inbred Strains, Mice, Inbred mdx, Polyribosomes metabolism, Protein Biosynthesis genetics, RNA, Messenger metabolism, Regeneration, Biomarkers, Tumor metabolism, Blood Proteins metabolism, Cell Differentiation physiology, Lectins, C-Type, Muscle Development, Muscle, Skeletal growth & development
- Abstract
Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis., (Copyright 1998 Academic Press.)
- Published
- 1998
- Full Text
- View/download PDF
33. Extrasynaptic location of laminin beta 2 chain in developing and adult human skeletal muscle.
- Author
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Wewer UM, Thornell LE, Loechel F, Zhang X, Durkin ME, Amano S, Burgeson RE, Engvall E, Albrechtsen R, and Virtanen I
- Subjects
- Adult, Antibodies, Monoclonal, Female, Humans, Immunohistochemistry, Laminin immunology, Muscle, Skeletal cytology, Muscle, Skeletal embryology, Muscle, Skeletal innervation, Muscular Dystrophies metabolism, Muscular Dystrophies pathology, Neuromuscular Junction metabolism, Pregnancy, Transfection, Laminin analysis, Muscle, Skeletal metabolism, Sarcolemma metabolism
- Abstract
We have investigated the distribution of the laminin beta 2 chain (previously s-laminin) in human fetal and adult skeletal muscle and compared it to the distribution of laminin beta 1. Immunoblotting and transfection assays were used to characterize a panel of monoclonal and polyclonal antibodies to the laminin beta 2 chain. We found that laminin beta 1 chain was detected at all times during development from 10 weeks of gestation. Laminin beta 2 chain was first detected in 15 to 22-week-old fetal skeletal muscle as distinct focal immunoreactivity in the sarcolemmal basement membrane area of some myofibers. In the adult skeletal muscle, laminin beta 2 chain immunoreactivity was found along the entire perimeter of each of the individual myofibers in a large series of different muscles studied. Laminin beta 2 chain was similarly found in the skeletal muscle basement membranes in patients with Duchenne and Becker muscular dystrophy. Immunoaffinity chromatography of muscle extracts with a monoclonal antibody to the laminin alpha 2 chain followed by immunoblotting with various antibodies to the beta 2 chain demonstrated the presence of the laminin-4 (alpha 2-beta 2-gamma 1) isoform. Together the present results demonstrate a prominent extrasynaptic localization of laminin beta 2 in the human muscle, suggesting that it may have an important function in the sarcolemmal basement membrane.
- Published
- 1997
34. Tissue-specific expression of the human laminin alpha5-chain, and mapping of the gene to human chromosome 20q13.2-13.3 and to distal mouse chromosome 2 near the locus for the ragged (Ra) mutation.
- Author
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Durkin ME, Loechel F, Mattei MG, Gilpin BJ, Albrechtsen R, and Wewer UM
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cloning, Molecular, Gene Expression Regulation, Genetic Linkage, Humans, In Situ Hybridization, Laminin chemistry, Mice, Molecular Sequence Data, Mutation, RNA, Messenger analysis, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Chromosome Mapping, Chromosomes, Human, Pair 20 genetics, Laminin genetics
- Abstract
To investigate the function of the laminin alpha5-chain, previously identified in mice, cDNA clones encoding the 953-amino-acid carboxy terminal G-domain of the human laminin alpha5-chain were characterized. Northern blot analysis showed that the laminin alpha5-chain is expressed in human placenta, heart, lung, skeletal muscle, kidney, and pancreas. The human laminin alpha5-chain gene (LAMA5) was assigned to chromosome 20q13.2-q13.3 by in situ hybridization, and the mouse gene (Lama5) was mapped by linkage analysis to a syntonic region of distal chromosome 2, close to the locus for the ragged (Ra) mutation.
- Published
- 1997
- Full Text
- View/download PDF
35. Assignment of the gene for human tetranectin (TNA) to chromosome 3p22-->p21.3 by somatic cell hybrid mapping.
- Author
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Durkin ME, Naylor SL, Albrechtsen R, and Wewer UM
- Subjects
- Base Sequence, Chromosome Mapping, DNA Primers, Humans, Hybrid Cells, Molecular Sequence Data, Biomarkers, Tumor genetics, Blood Proteins genetics, Chromosomes, Human, Pair 3, Lectins, C-Type
- Abstract
Tetranectin is a plasminogen-binding protein that is induced during the mineralization phase of osteogenesis. By screening a human chromosome 3 somatic cell hybrid mapping panel, we have localized the human tetranectin gene (TNA) to 3p22-->p21.3, which is distinct from the loci of two human connective tissue disorders that map to the short arm of chromosome 3, MFS2 and LRS1.
- Published
- 1997
- Full Text
- View/download PDF
36. Structural organization of the human and mouse laminin beta2 chain genes, and alternative splicing at the 5' end of the human transcript.
- Author
-
Durkin ME, Gautam M, Loechel F, Sanes JR, Merlie JP, Albrechtsen R, and Wewer UM
- Subjects
- Alternative Splicing, Amino Acid Sequence, Animals, Base Sequence, Basement Membrane chemistry, Chromosome Mapping, Conserved Sequence, DNA Primers genetics, Exons, Gene Expression Regulation, Humans, Introns, Laminin chemistry, Mice, Molecular Sequence Data, Promoter Regions, Genetic, Sequence Homology, Amino Acid, Species Specificity, Transcription, Genetic, Laminin genetics
- Abstract
We have determined the structural organization of the human and mouse genes that encode the laminin beta2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon-intron organization and are the smallest laminin chain genes characterized to date, due to the unusually small size of their introns. The laminin beta2 chain genes of both species consist of 33 exons that span =12 kilobase pairs of genomic DNA. The exon-intron pattern of the laminin beta2 chain gene is also highly similar to that of the human genes encoding the homologous laminin beta1 and beta3 chains. The putative promoter regions of the human and mouse laminin beta2 chain genes have features characteristic of the promoters of genes that have a limited tissue expression. Considerable conservation of the intron sequences of the mouse and human genes was observed. The first intron of the human gene, located 1 base pair upstream of the translation start codon, contains a non-consensus 5' splice site. This intron was shown to be inefficiently spliced in humans, suggesting that post-transcriptional mechanisms may be involved in the regulation of laminin beta2 chain gene expression.
- Published
- 1996
- Full Text
- View/download PDF
37. Laminin beta 2 chain and adhalin deficiency in the skeletal muscle of Walker-Warburg syndrome (cerebro-ocular dysplasia-muscular dystrophy).
- Author
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Wewer UM, Durkin ME, Zhang X, Laursen H, Nielsen NH, Towfighi J, Engvall E, and Albrechtsen R
- Subjects
- Basement Membrane metabolism, Child, Preschool, Humans, Immunohistochemistry, Male, Sarcoglycans, Syndrome, Abnormalities, Multiple metabolism, Cytoskeletal Proteins analysis, Eye Abnormalities metabolism, Laminin analysis, Membrane Glycoproteins analysis, Muscles metabolism, Muscular Dystrophies metabolism
- Abstract
Muscular dystrophy may be caused by disturbances in a number of muscle proteins that appear to be part of a chain of interacting molecules that includes cytoskeletal, cell membrane, and basement membrane components. We found that the skeletal muscle cells in two cases of Walker-Warburg syndrome were severely deficient in the laminin beta 2 chain and in adhalin. The findings indicate that these two proteins are key molecules in the interactive protein complex conferring muscle stability and cell survival.
- Published
- 1995
- Full Text
- View/download PDF
38. 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.
- Author
-
Durkin ME, Wewer UM, and Chung AE
- Subjects
- Amino Acid Sequence, Animals, Consensus Sequence, Genes, Membrane Glycoproteins chemistry, Mice, Inbred AKR, Mice, Inbred C57BL, Mice, Inbred CBA, Molecular Sequence Data, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Exons genetics, Membrane Glycoproteins genetics, Mice genetics, Receptors, LDL genetics
- Abstract
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' noncoding region of the last exon.
- Published
- 1995
- Full Text
- View/download PDF
39. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas.
- Author
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Wewer UM, Gerecke DR, Durkin ME, Kurtz KS, Mattei MG, Champliaud MF, Burgeson RE, and Albrechtsen R
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Chromosome Mapping, Cloning, Molecular, DNA, Complementary, Female, Gene Expression Regulation, Neoplastic, Humans, Laminin chemistry, Molecular Sequence Data, Rats, Tumor Cells, Cultured, Chromosomes, Human, Pair 3, Laminin genetics, Neoplasms genetics
- Abstract
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 the 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.
- Published
- 1994
- Full Text
- View/download PDF
40. Biological functions of entactin.
- Author
-
Chung AE, Dong LJ, Wu C, and Durkin ME
- Subjects
- Animals, Basement Membrane physiology, Cell Line, Collagen metabolism, Extracellular Matrix metabolism, Fibrinogen metabolism, Hemostasis physiology, Humans, Laminin metabolism, Membrane Glycoproteins genetics, Membrane Glycoproteins physiology
- Abstract
Entactin is a sulfated multidomain glycoprotein component of basement membranes. The molecule consists of 1217 amino acids which are organized into two terminal globular domains linked by a rod-like structure largely composed of four EGF- and one thyroglobulin-like cysteine-rich homology repeats. Entactin binds to laminin, collagen IV, fibrinogen, and fibronectin. In the parietal endoderm M1536-B3 cell line, the laminin-entactin complex is formed intracellularly and transported in membrane enclosed vesicles to the extracellular compartment. Transfection of human choriocarcinoma JAR cells, which do not synthesize entactin, with entactin cDNA results in the synthesis and insertion of entactin into the extracellular matrix where it becomes associated with laminin and collagen IV. Indirect immunofluorescent staining also reveals that entactin co-localizes with fibronectin in the extracellular matrix of the embryonal carcinoma-derived 4CQ cell line. These observations suggest that entactin plays an important role in the assembly and properties of diverse extracellular matrices. In addition, entactin binds to immobilized fibrinogen, and more specifically, to the A alpha and B beta chains. The binding of radiolabeled entactin to immobilized fibrinogen is not dependent on metal ions, and is inhibited by antibodies against either fibrinogen or entactin, soluble fibrinogen, and unlabeled entactin. This interaction combined with the chemotactic and phagocytic promoting activities of entactin may be important in hemostasis and would healing.
- Published
- 1993
- Full Text
- View/download PDF
41. Laminin B1 expression is required for laminin deposition into the extracellular matrix of PC12 cells.
- Author
-
Reing J, Durkin ME, and Chung AE
- Subjects
- Animals, Blotting, Northern, Blotting, Western, Clone Cells, DNA Probes, Extracellular Matrix ultrastructure, Fluorescent Antibody Technique, Laminin isolation & purification, Macromolecular Substances, Mice, PC12 Cells, Poly A isolation & purification, Poly A metabolism, RNA isolation & purification, RNA metabolism, RNA, Messenger, Transfection, Extracellular Matrix metabolism, Laminin genetics, Laminin metabolism
- Abstract
The extracellular matrix of rat pheochromocytoma PC12 cells was shown by indirect immunofluorescence to consist of a network of fibronectin. The matrix did not contain laminin. The cells synthesized messenger RNA for fibronectin, laminin B2, and s-laminin but not for entactin or the B1 and A chains of laminin. Laminin B2 but not laminin B1 was detectable in the culture medium and in cell lysates. A full-length cDNA clone for the B1 chain of laminin was constructed in the plasmid p-444, which contains the neomycin-resistance marker and human beta-actin promoter. PC12 cells were transfected with this recombinant plasmid, and stable neomycin-resistant clones were isolated and characterized. Clones that synthesized laminin B1 messenger RNA were found to deposit a laminin-containing matrix. In many of these clones the deposition of the fibronectin matrix was greatly diminished. The laminin matrix was predominantly localized in the intercellular spaces forming a honeycomb pattern. The morphology of the laminin-synthesizing transfected cells was markedly different from the parental cells. The cells grew in tight clusters that were resistant to dissociating agents. It is concluded that the B1 chain of laminin contains information that is required for the formation of a stable laminin-containing extracellular matrix network either by interaction with cell surface receptors or other extracellular matrix components. Furthermore, expression of the laminin B1 gene may be a central regulatory point in determining extracellular matrix composition during embryogenesis.
- Published
- 1992
42. Risk factors for injuries due to the 1990 earthquake in Luzon, Philippines.
- Author
-
Roces MC, White ME, Dayrit MM, and Durkin ME
- Subjects
- Case-Control Studies, Confidence Intervals, Demography, Disaster Planning, Emergency Medical Services, Humans, Odds Ratio, Philippines epidemiology, Risk Factors, Socioeconomic Factors, Disasters, Wounds and Injuries etiology
- Abstract
On 16 July 1990, an earthquake measuring 7.7 on the Richter scale struck the island of Luzon, Philippines. A case-control study was carried out to identify the risk factors for earthquake-related injuries and at the same time observations were made on the rescue efforts. Being hit by falling objects was the leading cause of injury (34%). Those injured during the tremor were more likely to have been inside buildings constructed of concrete or mixed materials (odds ratio, 2.6; 95% confidence interval (CI), 1.7-4.1) and to have been on the middle floors of multistorey buildings (odds ratio, 3.4; 95% CI, 2.2-5.5). Leaving a building during the earthquake was a protective behaviour (odds ratio, 0.3; 95% CI, 0.2-0.8). Of the 235 survivors who were trapped and rescued alive from the rubble, 99% were rescued within 48 hours of the impact of the tremor. These findings should prove useful in developing seismic safety codes. People should be taught proper evasive actions to take during earthquakes, and training in basic first aid and methods of rescue should be an integral part of community preparedness programmes.
- Published
- 1992
43. Gene mapping of mouse laminin A and B2 subunits using mouse-Chinese hamster somatic cell hybrids.
- Author
-
Kaye NW, Chung AE, Lalley PA, Durkin ME, Phillips SL, and Church RL
- Subjects
- Animals, Blotting, Southern, Hybrid Cells, Mice, Laminin genetics
- Abstract
Laminin is a multichain extracellular matrix glycoprotein found primarily in basement membranes. The molecule is made up of three subunits, designated as A, B1, and B2. Using an 850-base cDNA probe for the mouse laminin A-chain and a 1000-base cDNA probe for the mouse laminin B2 chain, we have screened mouse-Chinese hamster somatic cell hybrids in order to assign the genes for each of these polypeptides to their respective mouse chromosome. We have determined that the mouse laminin B2-chain gene is located on chromosome 1 (confirming this assignment) and the laminin A-chain gene is located on chromosome 17.
- Published
- 1990
- Full Text
- View/download PDF
44. Entactin: structure and function.
- Author
-
Chung AE and Durkin ME
- Subjects
- Amino Acid Sequence, Animals, Cell Adhesion, Gene Expression, Humans, Molecular Sequence Data, Protein Conformation, Basement Membrane physiology, Calcium-Binding Proteins physiology, Extracellular Matrix physiology, Extracellular Matrix Proteins physiology, Membrane Glycoproteins physiology
- Abstract
Entactin is an integral and ubiquitous component of the basement membrane. The amino acid sequences of the mouse and human molecules have been determined and exhibit 85% sequence identity. The molecule is organized into three structural domains, an N-terminal globule (I) is linked to a smaller C-terminal globule (III) by a rigid stalk (II) largely consisting of cysteine-rich EGF-like homology repeats and a cysteine-rich thyroglobulin homology repeat. The molecule binds calcium ions and supports cell adhesion. However, its major function may be the assembly of the basement membrane. The carboxyl globule binds tightly to one of the short arms of laminin at the inner rodlike segment. This same region is also believed to be responsible for the attachment of entactin to type IV collagen at approximately 80 nm from its carboxyl noncollagenous end. Entactin therefore could serve as a bridge between the two most abundant molecules in the basement membrane. Supporting evidence for this role has been obtained from transfection of human choriocarcinoma, JAR, cells with the entactin gene. JAR cells synthesize laminin and type IV collagen but not entactin. Transfection of entactin into the cells stimulated incorporation of laminin and type IV collagen along with entactin into the extracellular matrix and into structures resembling focal contacts. The calcium-binding activity of entactin may play a role in the matrix assembly process. The protease sensitivity of entactin suggests that it may be a target for proteolytic activity during tissue remodeling, metastasis, and other events requiring the turnover of the basement membrane.
- Published
- 1990
- Full Text
- View/download PDF
45. Control of laminin synthesis during differentiation of F9 embryonal carcinoma cells. A study using cDNA clones complementary to the mRNA species for the A, B1 and B2 subunits.
- Author
-
Durkin ME, Phillips SL, and Chung AE
- Subjects
- Animals, Bucladesine pharmacology, Cell Line, Cloning, Molecular, DNA analysis, Embryonal Carcinoma Stem Cells, Kinetics, Laminin genetics, Mice, Neoplastic Stem Cells, Nucleic Acid Hybridization, Plasmids, Protein Biosynthesis, RNA, Messenger genetics, Tretinoin pharmacology, Cell Differentiation, Laminin biosynthesis, RNA, Messenger analysis
- Abstract
Molecular clones complementary to the mRNA species for the A, B1 and B2 chains of murine laminin were identified by hybrid-selection and in vitro translation. Northern blot analysis demonstrated that the three clones, p59 (A), p2 (B1) and p16 (B2) hybridized to mRNA species 9.8, 6.0, and 8.0 kb in length, respectively. The three clones were used as probes to monitor the steady-state levels of laminin mRNA species during differentiation of F9 embryonal carcinoma cells induced by treatment with retinoic acid and dibutyryl cyclic AMP. The steady-state levels of the three mRNA species appeared to increase in a coordinate manner. Undetectable levels at the beginning of induction were followed by a dramatic increase in the levels of the three mRNA species between 48 and 72 h. The kinetics parallel the increase in laminin synthesis and the striking morphological changes previously reported.
- Published
- 1986
- Full Text
- View/download PDF
46. Primary structure of the mouse laminin B2 chain and comparison with laminin B1.
- Author
-
Durkin ME, Bartos BB, Liu SH, Phillips SL, and Chung AE
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Basement Membrane analysis, DNA analysis, Laminin genetics, Mice, Microscopy, Electron, Models, Molecular, Molecular Sequence Data, Molecular Weight, Laminin analysis
- Abstract
One of the major components of basement membranes is the glycoprotein laminin, made up of three disulfide-bonded subunits, the A, B1, and B2 chains. We have isolated and sequenced overlapping mouse laminin B2 chain cDNA clones covering 7562 base pairs. The deduced amino acid sequence predicts that the mature B2 chain consists of 1572 residues, has an unglycosylated molecular weight of 173,541, and possesses 14 potential N-linked glycosylation sites. Analysis of the predicted secondary structure shows the presence of six domains, two rich in alpha-helical structure, two composed of homologous cysteine-rich repeat units, and two globular regions. The organization of the molecule is very similar to that of the mouse laminin B1 chain, and significant sequence homology between the B1 and B2 chains was found in their two cysteine-rich domains and in their amino-terminal globular domains.
- Published
- 1988
- Full Text
- View/download PDF
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