Interaction of merosin (laminin 2) with very late activation antigen-6 is necessary for the survival of CD4+ CD8+ immature thymocytes.

The laminin alpha2-chain is a component of merosin, a member of the laminin family molecules, which is mainly expressed in the basement membranes of striated muscle. It is known that laminin alpha2 gene (lama2) null mutant mice (dy3k/dy3k) exhibit congenital muscular dystrophy (CMD). Because the laminin alpha2-chain is also expressed in the thymus, the role of merosin in the thymus was examined. In association with the onset of muscular dystrophy, CD4+ CD8+ double-positive (DP) thymocytes disappear by apoptotic cell death, while CD4+ CD8- or CD4- CD8+ thymocytes remain. In order to study the mechanisms leading to the selective death of DP cells in the absence of merosin, the role of the interaction between very late activation antigen-6 (VLA-6), a candidate merosin ligand in the thymus, and merosin was examined. The in vitro survival of thymocytes from normal mice was maintained by the addition of either anti-VLA-6 monoclonal antibodies (mAbs) or merosin. Furthermore, when the normal thymocytes were cultured on thymic epithelial cell lines, viable DP cell recoveries on wild-type epithelial cells were better than on cells from null mutant mice. The results suggest that DP cells are more sensitive to an uncharacterized apoptotic death signal, and that survival is supported by the interaction between VLA-6 and merosin.

Immune response to adenovirus-delivered antigens upregulates utrophin and results in mitigation of muscle pathology in mdx mice.

The upregulation of endogenous utrophin in skeletal muscle may lead to a new approach to the treatment of Duchenne muscular dystrophy (DMD). We found that injection of an E1, E3-deleted adenovirus vector expressing beta-galactosidase (beta-Gal) or green fluorescent protein (GFP) into the skeletal muscle of neonatal dystrophin-deficient mdx mice alleviated dystrophic pathology. In the adenovirus-infected muscles, an evaluation of sarcolemma stability showed low permeability and immunohistochemistry revealed utrophin upregulation at the extrasynaptic sarcolemma of mature muscle fibers. This utrophin upregulation was concomitant with endomysial cellular infiltration from a host immune reaction. There was no evidence of active muscle regeneration. In normal C57BL/10 mice, utrophin was also upregulated in adenovirus-injected skeletal muscles, where upregulated utrophin often coexisted with dystrophin. FK506 and anti-CD4 antibody administration decreased utrophin expression in adenovirus-injected mdx muscles and prevented the dystrophic phenotype from being mitigated, suggesting that an immune reaction is involved in utrophin upregulation. This is the first report demonstrating the improvement of the dystrophic phenotype as a result of the acquired overexpression of endogenous utrophin. Our findings provide an important clue to understanding the mechanism of utrophin expression and the development of an effective treatment for DMD.

Identification of EPS8 as a Dvl1-associated molecule.

Dishevelled (Dsh) is involved in both Wingless (Wg) and Frizzled (Fz) signaling pathways. To further determine the function of Dsh, we have performed yeast two-hybrid screening and isolated several genes encoding the molecules associated with the PDZ domain of Dvl1, one of the murine Dsh homologs. During the screening, we found that EPS8, which is a substrate for activated EGF receptor (EGFR), specifically interacted with Dvl1. This interaction was also confirmed in vitro. Through transfection studies, we observed the mutual action between Dvl1 and EPS8. Dvl1 was hyperphosphorylated in the presence of EPS8, whereas the tyrosine phosphorylation of EPS8 by activated EGFR was inhibited in the presence of Dvl1. Immunohistochemistry showed that Dvl1 and EPS8 expression overlap in particular tissues during organogenesis. These results indicate that interaction between Dvl1 and receptor tyrosine kinase signal plays certain roles in developmental events.

alpha1-syntrophin gene disruption results in the absence of neuronal-type nitric-oxide synthase at the sarcolemma but does not induce muscle degeneration.

alpha1-Syntrophin is a member of the family of dystrophin-associated proteins and is strongly expressed in the sarcolemma and the neuromuscular junctions. All three syntrophin isoforms have a PDZ domain that appears to participate in protein-protein interactions at the plasma membrane. alpha1-Syntrophin has additionally been shown to associate with neuronal nitric-oxide synthase (nNOS) through PDZ domains in vitro. These observations suggest that alpha1-syntrophin may work as a modular adaptor protein that can link nNOS or other signaling enzyme to the sarcolemmal dystrophin complex. In the sarcolemma, nNOS regulates the homeostasis of reactive free radical species and may contribute to the oxidative damage to muscle protein in muscle disease such as Duchenne muscular dystrophy. In this study, we generated alpha1-syntrophin knock-out mice to clarify the interaction between alpha1-syntrophin and nNOS in the skeletal muscle. We observed that nNOS, normally expressed in the sarcolemma, was largely absent from the sarcolemma, but considerably remained in the cytosol of the knock-out mice. Even though the distribution of nNOS was altered, the knock-out mice displayed no gross histological changes in the skeletal muscle. We also discovered that muscle contractile properties have not been influenced in the knock-out mice.

Effective restoration of dystrophin-associated proteins in vivo by adenovirus-mediated transfer of truncated dystrophin cDNAs.

A series of truncated dystrophin cDNAs (3.1-4.2 kbp) containing only three, three, two or one rod repeats with hinge 1 and 4 (named deltaDysAX2, AX11, AH3, M3, respectively) or no rod repeat retaining either hinge 1 or 4 (named deltaDysH1, H4, respectively) were constructed. These cDNAs were introduced into skeletal muscle of adult mdx mice using the adenovirus vector with a strong CAG promoter. deltaDysAX2, AX11, AH3 and deltaDysM3 expressed themselves successfully and recovered dystrophin-associated proteins effectively. Especially 3.7 kbp cDNA for deltaDysM3 offers the possibility of an approach utilizing newly developed virus vectors, such as an adeno-associated virus vector, toward gene therapy of Duchenne muscular dystrophy.

Laminin alpha2 chain-null mutant mice by targeted disruption of the Lama2 gene: a new model of merosin (laminin 2)-deficient congenital muscular dystrophy.

Using the gene targeting technique, we have generated a new mouse model of congenital muscular dystrophy (CMD), a null mutant for the laminin alpha2 chain. These homozygous mice, designated dy3Kldy3K, are characterized by growth retardation and severe muscular dystrophic symptoms and die by 5 weeks of age. Light microscopy revealed that muscle fiber degeneration in these mice begins no later than postnatal day 9. In degenerating muscles, considerable amounts of TUNEL positive nuclei were detected as well as DNA laddering, suggesting increased apoptotic cell death was involved in the process of muscle fiber degeneration.

[Introduction of rod-deleted dystrophin cDNA, delta DysM3, into mdx skeletal muscle using adenovirus vector].

The 6.3 kb mini-dystrophin cDNA, has been successfully introduced in dystrophic mdx mouse and phenotypes of muscular dystrophy has been considerably improved. We generate a dystrophin cDNA construct deleted more for rod domain of minidystrophin, resulting only one rod repeat with two hinge segments, 3.7 kb delta DysM3. Recombinant adenovirus vector, Ax delta DysM3 has been made using COS-TPC method. 125 kDa delta DysM3 was expressed in the cultured skeletal muscle cells, when Ax delta DysM3 was infected. delta DysM3 can effectively accumulate in sarcolemma and considerably recover of dystrophin-associated proteins when transferred into skeletal muscle of mdx mouse. This small-sized rod-deleted dystrophin can be incorporated into adeno-associated virus vector, which was recently proven to be good tool to carry the gene into skeletal muscle.

Myogenic regulatory factors can activate TATA-containing promoter elements via an E-box independent mechanism.

We have studied the effect of several myogenic regulatory factors on the activity of the promoter for a mouse gene encoding a skeletal myosin heavy chain (MyHC) expressed in adult (type IIB) muscle fibers. Co-transfection of myogenic factors is necessary for activity of the IIB promoter in mouse C2 myotubes in culture but not in quail myotubes in culture. Although this promoter contains one E-box within the first 192 base pairs upstream of the transcriptional start site, mutations in this motif demonstrate that it is not required for the transactivation effect of the myogenic factors. Analysis of other mutants suggests that the MEF2 and MHox DNA-binding factor binds to an evolutionarily conserved AT-rich motif. In addition, the IIB promoter appears to require the conserved TATA motif (CTATAAAAG) in order to be activated by the AT-rich sequences. The IIB promoter constructs produce RNA transcripts which begin at the natural site of transcriptional initiation in quail myotubes and in mouse C2 myotubes after co-transfection with myogenic factors; a second, minor, start site is also used in the co-transfected C2 myotubes. Results obtained after transfection of the mouse IIB promoter constructs in quail myotube cultures suggest that the overexpression of myogenic factors in C2 cultures does not result in an environment in which the control of IIB MyHC promoter activity is aberrant. Therefore, either the myogenic factors themselves, or other proteins induced by them, seem to interact directly with the basal transcription seem to interact directly with the basal transcription machinery to allow muscle-specific gene expression.

Promoter elements of the mouse complement C4 gene critical for transcription activation and start site location.

We have explored the template and factor requirements for transcription of the gene encoding the murine complement component C4, expressed predominantly but not exclusively in liver and mononuclear phagocytes. Competition experiments in transcription assays with liver nuclear extracts show that the regions upstream of the transcription initiation site are largely dispensable for obtaining basal levels of accurately initiated transcription. Activated transcription, however, depends on three upstream regulatory factors, two of which interact with target sites seemingly related to NF-1 (region -112/-87) and USF (region -85/-64), respectively. A third upstream regulatory factor has been detected by the surprising finding that double-stranded oligomers covering sequences proximal to the cap site (position -48 to -7) stimulate transcription from the C4 promoter specifically. Results of nucleotide deletions and site-directed mutations argue that the C4 initiator, that is, the most critical element for basal and accurate transcription of the gene, overlaps the cap site and extends into the transcribed sequences (-1 to +12). Immediately downstream of this region lies a last regulatory element (within the +5 to +43 boundaries) indispensable for high levels of transcription. These data assume wider interest because the C4 promoter does not contain TATA or CAAT boxes and does not feature any of the elements characteristic of the TATA-less genes so far reported.

E-box activator of the C4 promoter is related to but distinct from the transcription factor upstream stimulating factor.

The murine C4 promoter contains a motif (E-C4) active in transcriptional activation whose structure complies with the E-box consensus sequence recognized by the helix-loop-helix transcription factors. This site is found also in human and rat C4 promoters and has the structure (-75) CACGTG (-70) characteristic of the class B subset of b-helix-loop-helix-zipper proteins. We have challenged the hypothesis that the protein factor responsible for the E-C4-mediated transcriptional activation is identical to one of the previously characterized nuclear factors. The molecular mass of the E-C4 factor is slightly bigger than that of Hela upstream stimulating factor (USF) (43/44 kDa). Moreover, the nucleotides immediately adjoining the E-C4 core sequence contribute to the distinctive fine specificity of the E-C4 factor. Optimized USF and MYC DNA-binding sites, which differ in the nucleotides bordering the hexanucleotide box displace the E-C4 factor in competition assays but with lesser efficiency than the E-C4 site itself. Finally, the E-C4 factor fails to exhibit the heat resistance characteristic of USF proteins. The results show that the E-C4 transcription factor has DNA binding properties overlapping those of other helix-loop-helix proteins but is structurally distinct from the factors so far described. Although C4 is not the first liver gene endowed with an E-box-mediated activation, it affords the first example where such activation takes place in the context of a TATA-less promoter, and is functionally linked to an initiator element-dependent transcription.

The androgen-dependent C4-Slp gene is driven by a constitutively competent promoter.

The androgen-dependent liver protein Slp, together with its constitutively expressed closely related isoform C4, provides a model to address the question of which minimal alteration in DNA can shut off the expression of a gene in a manner reversible by testosterone or by trans-acting mutations. Previous work indicated that sequences located at -1.9, -0.45, and -0.25 kb from the transcription start site of the C4-Slp gene played a critical role in determining its unusual functional divergence from C4. Now, using quantitatively and qualitatively controlled transfection assays in HepG2 human hepatoma cells and mouse L fibroblasts, we have observed that the C4-Slp promoter is fully effective and unhindered by upstream sequences and that the C4 promoter has a consistent albeit modest superiority. The determinant of this nearly 2-fold difference does not coincide with the sites highlighted in previous studies but lies within the most cap-site-proximal nucleotides, at positions -189 to +48. We have also established conditions for cell-free transcription of C4 and C4-Slp from plasmid and cosmid templates by using nuclear extracts from rat and mouse liver of both sexes as well as from L cells. At variance with the rat alpha 2u-globulin gene, C4-Slp transcription in vitro does not require male factors, for it is expressed as efficiently as C4 by all nuclear extracts. Further, the minimal promoter sequences required to direct accurate initiation extend not farther than the most proximal 19 nucleotides. Because L cells efficiently express transfected cosmids covering the whole C4 gene or C4/C4-Slp recombinants, as well as plasmids carrying the C4-Slp promoter, but fail to express the full C4-Slp gene, we favor a model in which the expression of the gene is modulated intragenically.

Preparation and evaluation of Eudragit gels. II: In vitro release of salicylic acid, sodium salicylate, and ketoprofen from Eudragit L and S organogels.

The in vitro dissolution characteristic of salicylic acid, sodium salicylate, and ketoprofen from Eudragit L and S organogels was investigated by the rotation disk method. The dissolution pattern of salicylic acid and erosion of Eudragit L polymer from the organogels followed apparent zero-order kinetics, providing strong evidence for a surface erosion mechanism and negligible diffusional release of salicylic acid. On the other hand, the dissolution of salicylic acid from Eudragit S organogels was a linear function of the square root of time. The apparent dissolution rate of salicylic acid from Eudragit S organogels increased with increasing temperature from 32 to 42 degrees C and agitation rate from 50 to 200 rpm. A linear relationship was obtained between the logarithm of apparent dissolution rate constants and the reciprocal of absolute temperatures. The activation energy for release of salicylic acid from Eudragit S organogels was in the range of 2.99 to 5.57 kcal/mol. From various experimental results, it was concluded that the release process of salicylic acid from Eudragit S organogels was diffusion controlled through the organogels matrix.

Isolation and characterization of a novel human gene expressed specifically in the cells of hematopoietic lineage.

A novel cDNA clone designated as HS1, which show an expression pattern limited to human hematopoietic cells, was isolated. About 2kb mRNA of the clone was accumulated in all the mature and immature lymphoid and myeloid cell lines tested, and two of three erythroblastoid cell lines, but not in any cell lines of non-hematopoietic tissues. The same mRNA was also detected in normal lymphoid and myeloid tissues and peripheral blood lymphocytes, granulocytes and macrophages, but again not in non-hematopoietic tissues. Nucleotide sequence of the HS1 predicts a protein of 486 amino acids (Mr 53,931). N-terminal half of the protein retains unique repeating motifs, each of which shows a significant homology with the helix-turn-helix DNA-binding motif of several proteins reported previously. C-terminal half of the protein retains a region conserved between non-receptor tyrosine kinases (src family), phospholipase C(PLC)-148 and the crk oncogene product. A unique feature of HS1 suggests that the protein may be involved in signal transduction and regulation of gene expression.