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Biomedical subjects

R M Sklar

Publications and source records attributed to R M Sklar.

12 recordsLinked to original sources
PubMed1996

Stimulation of myogenic differentiation by a neuregulin, glial growth factor 2. Are neuregulins the long-sought muscle trophic factors secreted by nerves?

It has long been known that nerves stimulate growth and maintenance of skeletal muscles in ways not dependent on physical contacts, but numerous attempts to identify and characterize the myotrophic agent(s) secreted by nerves have been unsuccessful. We here suggest that products of the neuregulin gene may be these agents. The neuregulins are a family of proteins made by alternative splicing of a single transcript to give as many as 15 protein products. One member of this family, glial growth factor 2 (rhGGF2) is a very potent stimulator of myogenesis in L6A1 myoblasts, giving a maximal stimulation of cell fusion and creatine kinase elevation at a concentration of 1 ng/ml (18 pM). The stimulation of myogenesis is not rapid, but it is prolonged, continuing over a period of at least 6 days. The effects of rhGGF2 are additive with those of insulin-like growth factor I (IGF-I) or its analog R3-IGF-I, suggesting that the actions of these two myotrophic agents differ in at least one rate-limiting step. We have observed one possible difference; unlike the IGFs, rhGGF2 does not induce elevation of the steady state level of myogenin mRNA.

Animals↗
PubMed1993

Methylprednisolone selectively affects dystrophin expression in human muscle cultures.

Glucocorticoid therapy slows the progression of Duchenne muscular dystrophy. In muscle cultures, the addition of the glucocorticoid methylprednisolone increases myogenesis in most normal mixed and clonal cultures. Conversely, in some normal clonal and most dystrophic cultures, methylprednisolone inhibits fusion. However, in fusion-arrested normal and Becker muscular dystrophy cultures, dystrophin is expressed independently of fusion and of myosin heavy chain expression, and in some cases, expression is apparently enhanced by methylprednisolone. We suggest that dystrophin is a muscle-specific protein that does not require fusion for expression, and the methylprednisolone-induced enhancement of dystrophin expression may account for some of the clinical benefits of glucocorticoids in vivo.

Blotting, Western↗
PubMed1993

Expression of major histocompatibility complex antigens in cultures of clonally derived human myoblasts.

We examined class I and class II HLA antigen expression by flow cytometry in clonal cultures derived from normal human skeletal muscle biopsies. Both HLA class I and class II antigens were constitutively expressed in all clones studied. By altering the constituents of the culture medium, we could modulate the expression of HLA class II but not HLA class I antigens. We noted heterogeneity in the expression of HLA class II antigens among different clones; an increased expression was associated with an increased propensity of myoblasts to fuse. These findings indicate that normal aneurally cultured myoblasts may express both HLA class I and class II antigens, that this expression may be modulated by in vitro agents, and that the presence of these antigens may relate to the process of in vitro myoblast fusion.

Cell Adhesion Molecules, Neuronal↗
PubMed1993

Direct effects of cyclosporin A and cyclophosphamide on differentiation of normal human myoblasts in culture.

We examined the direct effects of the commonly used immunosuppressive agents cyclosporin A and cyclophosphamide on cultures of clonally derived aneural human myoblasts. When applied to cultures in doses reflecting the therapeutic dose in vivo, both cyclosporin A and cyclophosphamide had dose-related reproducible effects on myoblast fusion: fusion was enhanced by cyclophosphamide and inhibited by cyclosporin A. These findings indicate that immunosuppressive agents may have effects on muscle that are independent of their ability to regulate the immune system.

Cell Differentiation↗
PubMed1992

Differential glucocorticoid effects on the fusion of Duchenne/Becker and control muscle cultures: pharmacologic detection of accelerated aging in dystrophic muscle.

We report that the glucocorticoid methylprednisolone (Mepd) enhanced myogenesis in normal primary human muscle cultures, but inhibited myogenesis of most Duchenne/Becker muscle cultures. A decline in the magnitude of myogenic stimulation of Mepd correlated with age in a random group of control patients, including some with neurologic diseases other than Duchenne/Becker dystrophy. A case of Duchenne muscular dystrophy from an exceptionally young patient yielded a muscle culture that was myogenically stimulated by Mepd. These results suggest that continuous cycles of degeneration and regeneration of dystrophic muscle in vivo may result in a change of the glucocorticoid response of the muscle progenitor cells. The glucocorticoid effects suggest caution in the long-term clinical use of these agents for muscle disease such as Duchenne muscular dystrophy.

Adolescent↗
PubMed1991

Methylprednisolone increases dystrophin levels by inhibiting myotube death during myogenesis of normal human muscle in vitro.

The glucocorticoid methylprednisolone (Mepd) increased dystrophin and myosin heavy chain levels in differentiated cultures of cloned human myoblasts. Mepd increased the number of myotubes per area by preventing myotube death and detachment during myogenesis in vitro. Myotube death was the result of an endogenous process initiated early during myoblast fusion. It occurred between days 4 and 5 of differentiation (3 days after its initiation) and was inhibited by cycloheximide, indicating that a programmed death mechanism may be involved. Inhibition of myotube death accounted for the increased levels of muscle-specific proteins; the amount of dystrophin per myonucleus was the same with or without Mepd treatment. These effects of glucocorticoids on primary muscle cultures may bear on the recent observation that prednisone transiently enhances muscle function in Duchenne muscular dystrophy.

Adult↗
PubMed1991

Fos RNA accumulation in multiple sclerosis white matter tissue.

In order to better characterize the molecular events that accompany lesion development in multiple sclerosis (MS), we studied the accumulation of RNA specific to the nuclear proto-oncogenes c-fos and c-myb in post mortem white matter brain tissue. RNA was prepared from plaque and periplaque regions of 6 different MS brains, from "normal" white matter regions of 3 MS brains and from 6 normal control samples. Quantitation of specific RNA corresponding to each proto-oncogene was performed by Northern blot hybridization and by scanning densitometry. Results indicate a 2-fold increase in c-fos RNA in MS white matter, compared to control tissue. No c-myb signal was identified in any sample. In situ hybridization studies confirmed the selective upregulation of c-fos RNA levels in MS tissue, and suggested that glial cells and not inflammatory cells were responsible for the enhanced c-fos signal. These results suggest that persistent glial cell activation is present within chronic MS lesions irrespective of whether the lesions are active (e.g., inflammatory) or inactive.

Blotting, Northern↗
PubMed1990

Defective dystrophin in Duchenne and Becker dystrophy myotubes in cell culture.

We examined normal and dystrophic human myotubes in cell culture for expression of dystrophin, the protein product of the Duchenne muscular dystrophy locus. Dystrophin levels in developing myotubes detected by Western blotting increased after 24 hours and reached maximum levels after 10 days in fusion medium. We did not detect dystrophin in myotubes cultured from Duchenne myoblasts (7 cases). Myotubes from a Becker muscular dystrophy patient's biopsy produced a lower molecular weight (approximately 408 kd) dystrophin, which was the same size in a whole muscle preparation from the same biopsy. This 408-kd dystrophin was the expected size for this Becker patient whose DNA was deleted for exons 45-48 of the Duchenne gene. This cell culture system will allow a detailed analysis of the effects of potential pharmacologic agents on steady-state dystrophin levels.

Adult↗
PubMed1988

A simple method for DNA purification from peripheral blood.

A new, simple, and inexpensive method for the rapid isolation of DNA from whole blood is described. Cell nuclei are prepared by lysis of cytoplasmic membranes and DNA within the nuclear pellet is dispersed with guanidine isothiocyanate and precipitated with isopropanol. DNA prepared in this way restricts completely and results in low backgrounds of nonspecific hybridization after Southern analysis. The yields of DNA are similar to those obtained by more tedious traditional procedures. Numerous genomic DNA samples can be prepared from whole blood in 2 h, thus facilitating gene linkage or other molecular studies in which large numbers of individuals are required.

1-Propanol↗
PubMed1983

O6-Methylguanine removal by competent and incompetent human lymphoblastoid lines from the same male individual.

Two cell lines, one proficient (Mex+) and one deficient (Mex-) in the ability to remove O6-methylguanine, have been isolated by Epstein-Barr virus-mediated transformation of a single blood sample obtained from a normal human male. Extracts of untreated cells differ in their O6-methylguanine transferase (methyl acceptor protein) activity. Although both lines arise from the same individual, they show great difference in their sensitivities to the toxic action of N-methyl-N'-nitro-N-nitrosoguanidine. Chromosome counts of the strains reveal a modal number of 46 for both. Neither X-inactivation nor a gross abnormality in chromosome number can be the cause of the difference between the two lines.

Adenine↗