Search PubMed⌕ Search

Biomedical subjects

E Engvall

Publications and source records attributed to E Engvall.

At least 19 recordsLinked to original sources
PubMed2001

An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy.

Congenital muscular dystrophy is a heterogeneous and severe, progressive muscle-wasting disease that frequently leads to death in early childhood. Most cases of congenital muscular dystrophy are caused by mutations in LAMA2, the gene encoding the alpha2 chain of the main laminin isoforms expressed by muscle fibres. Muscle fibre deterioration in this disease is thought to be caused by the failure to form the primary laminin scaffold, which is necessary for basement membrane structure, and the missing interaction between muscle basement membrane and the dystrophin-glycoprotein complex (DGC) or the integrins. With the aim to restore muscle function in a mouse model for this disease, we have designed a minigene of agrin, a protein known for its role in the formation of the neuromuscular junction. Here we show that this mini-agrin-which binds to basement membrane and to alpha-dystroglycan, a member of the DGC-amends muscle pathology by a mechanism that includes agrin-mediated stabilization of alpha-dystroglycan and the laminin alpha5 chain. Our data provides in vivo evidence that a non-homologous protein in combination with rational protein design can be used to devise therapeutic tools that may restore muscle function in human muscular dystrophies.

Agrin↗
PubMed2001

Laminin alpha 2 (merosin)-deficient muscular dystrophy and demyelinating neuropathy in two cats.

We report laminin alpha 2 (merosin) deficiency associated with muscular dystrophy and demyelinating neuropathy in two cats. The cats developed progressive muscle weakness, and atrophy. Either hypotonia or contractures resulted in recumbency, necessitating euthanasia. Muscle biopsies showed dystrophic changes including marked endomysial fibrosis, myofiber necrosis, variability of fiber size, and perimysial lipid accumulation. Immunohistochemistry showed that laminin alpha 2 chain was absent or reduced, while dystrophin and all the components of the dystrophin-associated glycoprotein complex were present and normal. One cat was examined in detail. Motor nerve conduction velocity (MNCV) was decreased, and ultrastructurally the peripheral nerves showed Schwann cell degeneration and demyelination. Brain imaging was not performed, but white matter changes were not apparent in the brain at necropsy. The disease in these cats is similar to primary or secondary merosin (laminin alpha 2)-deficient congenital muscular dystrophy (CMD) in humans and to dystrophia muscularis in mice.

Animals↗
PubMed2001

C7, a novel nucleolar protein, is the mouse homologue of the Drosophila late puff product L82 and an isoform of human OXR1.

The C7 gene was identified in a project aimed to characterize differential gene expression upon attachment of cells to extracellular matrix proteins in vitro. C7 is the homologue of Drosophila L82, a late puff gene (Stowers et al. (1999) Dev. Biol. 213, 116-130) and human OXR1, a gene, which protects cells against oxidation (Volkert et al. (2000) Proc. Natl. Acad. Sci. USA 97, 14530-14535). All are transcribed into multiple splice forms with a common 3' domain. Additional members of this novel gene family are found in a number of eukaryotic species. In the mouse, the C7 gene is highly and broadly expressed during development in at least 4 splice forms, 3 of which were sequenced. In the adult, the C7 gene is most highly expressed in brain and testis. Antibodies to recombinant C7 protein localized to nucleoli in a variety of cell types, suggesting that C7 may be involved in the formation or function of this important organelle.

Amino Acid Sequence↗
PubMed2001

Tetranectin in slow intra- and extrafusal chicken muscle fibers.

Tetranectin is a C-type lectin that occurs in the mammalian musculoskeletal system. In the present report we describe the first studies on an avian tetranectin. A full-length chicken tetranectin cDNA was isolated. Comparison of the deduced amino acid sequence of chicken tetranectin with mouse and human tetranectin showed an identity of 67 and 68%, respectively. Northern blot analysis demonstrated broad expression of chicken tetranectin mRNA, which was first detected on embryonic day 4. Tetranectin protein was detected in chicken serum and egg yolk. Since muscle is one of few tissues in which tetranectin protein is retained, we examined the distribution of tetranectin in various muscle types in chicken. Myofibers strongly positive for tetranectin were observed in several muscles including m. tibialis ant. and m. sartorius (from embryonic day 10 to adult). Using antibodies to fast and slow myosin heavy chains (MHC) and double immunostaining techniques, we found that tetranectin was restricted to slow (type I) muscle fibers. Similarly only slow intrafusal fibers accumulated tetranectin. The pattern of immunostaining in chickens differs markedly from that seen in mouse muscles, indicating that tetranectin performs a role in muscle that is not associated with a hitherto recognized muscle type or function.

Adaptation, Physiological↗
PubMed2001

Mice with a targeted deletion of the tetranectin gene exhibit a spinal deformity.

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.

Animals↗
PubMed2001

Skeletal muscle cell hypertrophy induced by inhibitors of metalloproteases; myostatin as a potential mediator.

Cell growth and differentiation are controlled in many tissues by paracrine factors, which often require proteolytic processing for activation. Metalloproteases of the metzincin family, such as matrix metalloproteases and ADAMs, recently have been shown to be involved in the shedding of growth factors, cytokines, and receptors. In the present study, we show that hydroxamate-based inhibitors of metalloproteases (HIMPs), such as TAPI and BB-3103, increase the fusion of C(2)C(12) myoblasts and provoke myotube hypertrophy. HIMPs did not seem to effect hypertrophy via proteins that have previously been shown to regulate muscle growth in vitro, such as insulin-like growth factor-I, calcineurin, and tumor necrosis factor-alpha. Instead, the proteolytic maturation of myostatin (growth differentiation factor-8) seemed to be reduced in C(2)C(12) cells treated with HIMPs, as suggested by the presence of nonprocessed myostatin precursor only in hypertrophic myotubes. Myostatin is a known negative regulator of skeletal muscle growth, belonging to the transforming growth factor-beta/bone morphogenetic protein superfamily. These results indicate that metalloproteases are involved in the regulation of skeletal muscle growth and differentiation, that the proteolytic maturation of myostatin in C(2)C(12) cells may be directly or indirectly linked to the activity of some unidentified HIMP-sensitive metalloproteases, and that the lack of myostatin processing on HIMP treatment may be a mediator of myotube hypertrophy in this in vitro model.

Blotting, Western↗
PubMed2000

Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion.

ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin and metalloprotease") family. ADAM12 has been implicated in muscle cell differentiation and fusion, but its precise function remains unknown. Here, we show that ADAM12 is dramatically up-regulated in regenerated, newly formed fibers in vivo. In C2C12 cells, ADAM12 is expressed at low levels in undifferentiated myoblasts and is transiently up-regulated at the onset of differentiation when myoblasts fuse into multinucleated myotubes, whereas other ADAMs, such as ADAMs 9, 10, 15, 17, and 19, are expressed at all stages of differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence in the membrane-proximal region of ADAM12 cytoplasmic tail; a second binding site was identified in the membrane-distal region. Co-immunoprecipitation experiments confirm the in vivo association of ADAM12 cytoplasmic domain with alpha-actinin-2. Overexpression of the entire cytosolic ADAM12 tail acted in a dominant negative fashion by inhibiting fusion of C2C12 cells, whereas expression of a cytosolic ADAM12 lacking the major alpha-actinin-2 binding site had no effect on cell fusion. Our results suggest that interaction of ADAM12 with alpha-actinin-2 is important for ADAM12 function.

ADAM Proteins↗
PubMed2000

Identification of homozygous and heterozygous dy2J mice by PCR.

The dystrophia muscularis dy2J/dy2J mouse is an animal model for one form of human congenital muscular dystrophy. A point mutation in the gene coding for the laminin-2 alpha 2 chain leads to the expression of a truncated, partially functional protein. We developed a simple assay for the detection of the dy2J allele, which contains a new NdeI restriction site. Genomic DNA was prepared from animals of known status and amplified by PCR. The digestion of the PCR product with the restriction enzyme resulted in characteristic profiles. Then, this technique was used to identify heterozygous mice among unaffected animals of unknown status. Subsequently, the heterozygous genotype of these mice was confirmed by the birth of dystrophic offspring after mating. This technique allows the detection of the dy2J allele in heterozygous and homozygous animals at any age.

Alleles↗
PubMed1999

Sarcoglycan isoforms in skeletal muscle.

The heterotetrameric sarcoglycan complex, composed of alpha-, beta-, gamma-, and delta-sarcoglycans, is an important component of the dystrophin-associated glycoprotein assembly in striated muscle. Mutations in any of the four genes encoding sarcoglycans cause a deficiency in all sarcoglycans in the sarcolemma and produce one of four types of limb-girdle muscular dystrophy. A fifth widely expressed sarcoglycan, epsilon-sarcoglycan, has been recently described. epsilon-Sarcoglycan is homologous to alpha-sarcoglycan, but whether it associates with the other sarcoglycans in muscle is not known. In this study, we use wild type and alpha-sarcoglycan-deficient mice to analyze the localization and association of sarcoglycans in skeletal muscle in vivo. The amounts of beta-, gamma-, and delta-sarcoglycans are reduced in alpha-sarcoglycan mutants, whereas the amount of epsilon-sarcoglycan is unchanged. We show here that epsilon-sarcoglycan is complexed with beta-, gamma-, and delta-sarcoglycans in both wild type and alpha-sarcoglycan mutant mice. We also use C2C12 myocytes to study the temporal expression and organization of sarcoglycan complexes during muscle cell differentiation in vitro. In C2C12 cells, alpha- and epsilon-sarcoglycans form separate complexes with beta-, gamma-, and delta-sarcoglycans. Both types of complexes are expressed at the cell surface and presumed to be functional. These results suggest that epsilon-sarcoglycan serves a function similar to that of alpha-sarcoglycan and that residual beta-, gamma-, and delta-sarcoglycan seen in mutant mice and alpha-sarcoglycan-deficient patients is due to its association with epsilon-sarcoglycan.

Animals↗
PubMed1999

Blood platelets contain and secrete laminin-8 (alpha4beta1gamma1) and adhere to laminin-8 via alpha6beta1 integrin.

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin gamma1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to gamma1 and beta1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin beta1 antibody-Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to beta1 and gamma1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin alpha4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20-35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to beta1 and alpha6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (alpha4beta1gamma1) and that the cells adhere to the protein by using alpha6beta1 integrin.

Amino Acid Sequence↗
PubMed1999

Pulsed-field gel electrophoresis and distribution of the genes zag and fnz in isolates of Streptococcus equi.

Streptococcus equi subsp. equi and subsp. zooepidemicus are important pathogens of the equine respiratory tract. Isolates of both subspecies were examined by pulsed-field gel electrophoresis (PFGE). With the exception of eight isolates, a unique band pattern was displayed for each of the 48 subsp. zooepidemicus isolates tested. A method to distinguish isolates of the genetically very homogeneous subsp. equi has hitherto not been available, although several methods have been tested. By the use of PFGE, 50 isolates of subsp. equi could be divided into eleven groups, each with a unique pulsotype. In addition, the recently characterised genes encoding the cell-wall proteins ZAG and FNZ of S. equi subsp. zooepidemicus strain ZV were shown by Southern blots to be present in all 98 tested isolates, including the type strains of the two subspecies. Binding assays showed that the expression of the two genes clearly differentiate between the two subspecies.

Adhesins, Bacterial↗
PubMed1999

Activation of the lama2 gene in muscle regeneration: abortive regeneration in laminin alpha2-deficiency.

Mutations in laminin alpha2, a subunit of the basement membrane protein laminin-2/merosin, cause merosin-deficient congenital muscular dystrophy. To gain insight into the molecular mechanism of disease, we generated and used a mutant mouse, dyW, in which the lacZ gene was inserted into the lama2 gene so that beta-galactosidase would be expressed in place of laminin alpha2. Heterozygous and homozygous mutant mice are normal at birth, but homozygous mice develop muscular dystrophy at 2 to 3 weeks of age. The lama2/lacZ gene was highly expressed in muscle in the early stages of embryonic myogenesis, but was down-regulated at later stages in both heterozygous and homozygous mice. No beta-galactosidase activity was detected in skeletal muscle after birth in adult heterozygous mice. In contrast, high beta-galactosidase activity was detected in postnatal homozygous mice. Induction of injury in heterozygous mice resulted in intense reexpression of beta-galactosidase in the injured muscle early in regeneration, with a decline in enzyme activity as repair of the tissue progressed. Although the initial response to injury was similar in heterozygous and homozygous mice with abundant beta-galactosidase-positive, mononucleated cells in the injured area, repair was rarely completed in the homozygous mice, evidently caused by excessive death of cells associated with immature myofibers. The defect in muscle repair was very efficiently corrected in homozygous dyW mice expressing a human LAMA2 transgene in skeletal muscle. The data show the importance of laminin alpha2 in muscle regeneration and suggest that a major contributor to disease in muscular dystrophy is abortive regeneration.

Animals↗
PubMed1998

Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro.

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.

Animals↗
PubMed1998

Merosin-deficient congenital muscular dystrophy. Partial genetic correction in two mouse models.

Humans and mice with deficiency of the alpha2 subunit of the basement membrane protein laminin-2/merosin suffer from merosin-deficient congenital muscular dystrophy (MCMD). We have expressed a human laminin alpha2 chain transgene under the regulation of a muscle-specific creatine kinase promoter in mice with complete or partial deficiency of merosin. The transgene restores the synthesis and localization of merosin in skeletal muscle, and greatly improves muscle morphology and integrity and the health and longevity of the mice. However, the transgenic mice share with the nontransgenic dystrophic mice a progressive lameness of hind legs, suggestive of a nerve defect. These results indicate that the absence of merosin in tissues other than the muscle, such as nervous tissue, is a critical component of MCMD. Future gene therapies of human MCMD, and perhaps of other forms of muscular dystrophy, may require restoration of the defective gene product in multiple tissues.

Animals↗
PubMed1998

Human ADAM 12 (meltrin alpha) is an active metalloprotease.

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins with structural homology to snake venom metalloproteases. We recently described the cloning and sequencing of human ADAM 12 (meltrin alpha). In this report we provide evidence that the metalloprotease domain of ADAM 12 is catalytically active. We used the trapping mechanism of alpha2-macroglobulin to assay for protease activity of wild-type and mutant ADAM 12 proteins produced in a COS cell transfection system. We found that ADAM 12 is synthesized as a zymogen, with the prodomain maintaining the metalloprotease in a latent form, probably by means of a cysteine switch. The zymogen could be activated chemically by alkylation with N-ethylmaleimide. Cleavage of the prodomain at a site for a furin-like endopeptidase resulted in an ADAM 12 protein with proteolytic activity. The protease activity was sensitive to inhibition by 1,10-phenanthroline and could be eliminated by mutation of the critical glutamate residue at the active site. The demonstration that the ADAM 12 metalloprotease domain is functional may have important implications for future studies that explore the role of ADAM 12 protein in development and disease.

ADAM Proteins↗
PubMed1998

Disruption of the lama2 gene in embryonic stem cells: laminin alpha 2 is necessary for sustenance of mature muscle cells.

Mutations in the gene coding for the alpha 2 chain of laminin-2 and -4 (merosin) cause a severe form of congenital muscular dystrophy in humans and mice. To establish a defined model for in vitro and in vivo studies of the role of laminin alpha 2/merosin in development and cell and tissue function, we generated several lines of mutant embryonic stem (ES) cell with disruption of the laminin alpha 2 chain gene. We find that homozygous mutant ES cells differentiate normally in vitro, giving rise to cardiomyocytes, myotubes, and smooth muscle cells in addition to many other cell types. However, the myotubes that are formed are unstable. They detach, collapse, and degenerate, a process which is initiated at the appearance of the mature, contractile phenotype of the cells. We propose that the detachment and death of contracting myotubes in vitro has its counterpart in vivo and that contraction-induced myofiber damage, along with the lack of survival cues provided by laminin alpha 2/merosin, is a significant contribution to muscle degeneration in merosin-deficient muscular dystrophy.

Animals↗
PubMed1998

A novel, secreted form of human ADAM 12 (meltrin alpha) provokes myogenesis in vivo.

The ADAM (A Disintegrin And Metalloprotease) family of cell-surface proteins may have an important role in cellular interactions and in modulating cellular responses. In this report we describe a novel, secreted form of human ADAM 12 (meltrin alpha), designated ADAM 12-S (S for short), and a larger, membrane-bound form designated ADAM 12-L (L for long form). These two forms arise by alternative splicing of a single gene located on chromosome 10q26. Northern blotting demonstrated that mRNAs of both forms are abundant in human term placenta and are also present in some tumor cell lines. The ADAM 12-L transcript can also be detected in normal human adult skeletal, cardiac, and smooth muscle. Human A204 embryonal rhabdomyosarcoma cells that do not differentiate into muscle cells and do not express any form of ADAM 12 were stably transfected with an ADAM 12-S minigene encoding the disintegrin domain, the cysteine-rich domain, and the unique 34 amino acid carboxyl terminus. Nude mouse tumors derived from these transfected cells contained ectopic muscle cells of apparent mouse origin as shown by species-specific markers. These results may have potential applications in the development of muscle-directed gene and cell therapies.

ADAM Proteins↗