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G K Pavlath

Publications and source records attributed to G K Pavlath.

33 records · Page 2Linked to original sources
PubMed1993

Cytoskeletal active drugs modulate signal transduction in the protein kinase C pathway.

The cytoskeletal network of cells is postulated to play a role in the signal transduction pathways of growth promoting stimuli. We show here that cytoskeletal active drugs modulate the mitogenic signal transduction pathway of the tumor promoter TPA in 3T3-L1 cells. Compounds which act on microtubules (vinblastine sulfate) and microfilaments (cytochalasin B) have opposite effects on DNA synthesis. Vinblastine sulfate leads to stimulation, whereas cytochalasin B causes potent inhibition of DNA synthesis in response to TPA. These drugs are cell cycle specific and apparently exert their regulatory action distal to activation of protein kinase C by TPA. The expression of four genes necessary for DNA synthesis in response to tumor promoters was examined: two nuclear proto-oncogenes (c-myc and c-fos), a transcription factor (c-jun/AP-1) and a key enzyme involved in polyamine synthesis (ornithine decarboxylase). c-jun mRNA levels are not modulated during the action of cytoskeletal disrupting drugs on TPA-mediated mitogenesis, whereas c-myc and c-fos mRNA levels are similarly enhanced. Expression of ornithine decarboxylase mRNA and protein is increased by vinblastine sulfate but decreased by cytochalasin B in TPA treated cells. These data indicate that changes in cytoskeletal organization may play a role in regulating the levels of an enzyme critical for DNA synthesis.

3T3 Cells↗
PubMed1992

Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation.

Gene delivery by transplantation of normal myoblasts has been proposed as a treatment of the primary defect, lack of the muscle protein dystrophin, that causes Duchenne muscular dystrophy (DMD), a lethal human muscle degenerative disorder. To test this possibility, we transplanted normal myoblasts from a father or an unaffected sibling into the muscle of eight boys with DMD, and assessed their production of dystrophin. Three patients with deletions in the dystrophin gene expressed normal dystrophin transcripts in muscle biopsy specimens taken from the transplant site one month after myoblast injection. Using the polymerase chain reaction we established that the dystrophin in these biopsies derived from donor myoblast DNA. These results show that transplanted myoblasts persist and produce dystrophin in muscle fibres of DMD patients.

Adult↗
PubMed1991

Systemic delivery of human growth hormone by injection of genetically engineered myoblasts.

A recombinant gene encoding human growth hormone (hGH) was stably introduced into cultured myoblasts with a retroviral vector. After injection of genetically engineered myoblasts into mouse muscle, hGH could be detected in serum for 3 months. The fate of injected myoblasts was assessed by coinfecting the cells with two retroviral vectors, one encoding hGH and the other encoding beta-galactosidase from Escherichia coli. These results provide evidence that myoblasts, which can fuse into preexisting multinucleated myofibers that are vascularized and innervated, may be advantageous as vehicles for systemic delivery of recombinant proteins.

Animals↗
PubMed1990

Steroids induce acetylcholine receptors on cultured human muscle: implications for myasthenia gravis.

Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. We show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. Our results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also to a direct effect on muscle. We propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.

Autoantibodies↗
PubMed1989

Localization of muscle gene products in nuclear domains.

The localization of gene products is central to the development of cell polarity and pattern specification during embryogenesis. To monitor the distribution of gene products encoded by different nuclei in the same cell in tissue culture, we fused cells of different species to form multinucleated non-dividing heterokaryons. In previous fusion studies, cell-surface antigens and organelles contributed by disparate cell types intermixed within minutes. Using heterokaryons produced with differentiated muscle cells, we demonstrate here that a muscle membrane component, the Golgi apparatus mediating its transport, and a sarcomeric myosin heavy chain are localized in the vicinity of the nuclei responsible for their synthesis. These results provide direct evidence that products (organelle, membrane and structural proteins) derived from individual nuclei can remain localized in myotubes, a finding with implications both for neuromuscular synapse formation and for the carrier state of Duchenne muscular dystrophy.

Animals↗
PubMed1989

In vivo aging of human fibroblasts does not alter nuclear plasticity in heterokaryons.

In vivo aging of human fibroblasts altered proliferative properties but not the potential for novel gene expression in response to muscle trans-acting factors. Heterokaryons produced by fusing fibroblasts with muscle cells permitted a dissociation of the effects of aging on cell division and other cell functions. Skin fibroblasts derived from fetal and adult stages of development were distinct cell types based on their doubling time, protein content, cell size, and specific binding of insulin and insulin-like growth factor I. Despite these differences in growth parameters, the two cell types were indistinguishable in heterokaryons. Muscle gene activation occurred in the absence of changes in chromatin structure requiring DNA replication. In addition, the time course, maximal efficiency, and effect of gene dosage on the expression of muscle gene products were similar for heterokaryons containing fetal and adult fibroblasts but distinct for heterokaryons containing keratinocytes. The difference between fibroblasts and keratinocytes in the time course of muscle gene expression is likely to reflect mechanisms of gene activation at the transcriptional level, since the kinetics of muscle protein accumulation paralleled that of muscle transcripts. These results indicate that nuclear plasticity is not altered in fibroblasts by in vivo aging.

Actins↗
PubMed1988

Isolation of human myoblasts with the fluorescence-activated cell sorter.

We have established procedures for the rapid and efficient purification of human myoblasts using the fluorescence-activated cell sorter. Our approach capitalizes on the specific reaction of monoclonal antibody 5.1H11 with a human muscle cell surface antigen. For each of the five samples analyzed, an enrichment of myoblasts to greater than 99% of the cell population was immediately achieved. Following 3 to 4 weeks of additional growth in vitro, sorted myoblast cultures remained 97% pure. Differentiation of the sorted myoblast cultures, assessed by creatine kinase activity and isozyme content, was comparable to that of pure myoblast cultures obtained by cloning, and was significantly greater than that of mixed fibroblast and myoblast cultures. An average of 10(4) viable myoblasts can be obtained per 0.1 g tissue, each with the potential to undergo approximately 40 cell divisions. Accordingly, if only two-thirds of this proliferative capacity is utilized, the potential yield approximates 10(12) myoblasts, equivalent to 1 kg of cells. Human myogenesis in vitro is no longer limited by cell number and is now amenable to molecular and biochemical analysis on a large scale.+

Abortion, Therapeutic↗
PubMed1988

Muscle cell components dictate hepatocyte gene expression and the distribution of the Golgi apparatus in heterokaryons.

Major changes in cytoarchitecture and gene expression were induced in short-term heterokaryons. When human hepatocytes were fused with mouse muscle cells, the hepatocyte Golgi apparatus changed from its usual polar location to a uniformly circumnuclear location typical of striated muscle. Human liver albumin ceased to be expressed, and expression of the human muscle cell-surface antigen 5.1H11 was induced without DNA replication or cell division. Coexpression of liver and muscle proteins was rarely observed. These novel findings provide insight into the regulation of gene expression and the targeting and localization of organelles with a central role in cell polarity, intracellular transport, and secretion.

Animals↗
PubMed1986

Expression of muscle genes in heterokaryons depends on gene dosage.

We report that gene dosage, or the ratio of nuclei from two cell types fused to form a heterokaryon, affects the time course of differentiation-specific gene expression. The rate of appearance of the human muscle antigen, 5.1H11, is significantly faster in heterokaryons with equal or near-equal numbers of mouse muscle and human fibroblast nuclei than in heterokaryons with increased numbers of nuclei from either cell type. By 4 d after fusion, a high frequency of gene expression is evident at all ratios and greater than 75% of heterokaryons express the antigen even when the nonmuscle nuclei greatly outnumber the muscle nuclei. The kinetic differences observed with different nuclear ratios suggest that the concentration of putative trans-acting factors significantly influences the rate of muscle gene expression: a threshold concentration is necessary, but an excess may be inhibitory.

Animals↗
PubMed1985

Plasticity of the differentiated state.

Heterokaryons provide a model system in which to examine how tissue-specific phenotypes arise and are maintained. When muscle cells are fused with nonmuscle cells, muscle gene expression is activated in the nonmuscle cell type. Gene expression was studied either at a single cell level with monoclonal antibodies or in mass cultures at a biochemical and molecular level. In all of the nonmuscle cell types tested, including representatives of different embryonic lineages, phenotypes, and developmental stages, muscle gene expression was induced. Differences among cell types in the kinetics, frequency, and gene dosage requirements for gene expression provide clues to the underlying regulatory mechanisms. These results show that the expression of genes in the nuclei of differentiated cells is remarkably plastic and susceptible to modulation by the cytoplasm. The isolation of the genes encoding the tissue-specific trans-acting regulators responsible for muscle gene activation should now be possible.

Aged↗
PubMed1983

Defective myoblasts identified in Duchenne muscular dystrophy.

A defect in the proliferative capacity of satellite cells, mononucleated precursors of mature muscle fibers, was found in clonal analyses of cells cultured from Duchenne muscular dystrophy (DMD) patients. The total yield of myoblasts per gram of muscle biopsy was decreased to 5% of normal. Of the DMD myoblast clones obtained, a large proportion contained a morphological class of flat distended cells that had an increased generation time and ceased to proliferate beyond 100-1,000 cells but could be induced to fuse and form myotubes. The altered muscle phenotype was detected in all cultures from DMD patients but was rarely found among myoblasts of controls. By age 14 yr, it comprised as man as 90% of DMD myoblasts. The remaining DMD myoblast clones, which initially grew well, had severely impaired proliferative capacity upon passage and further cultivation. Eventually all myoblasts from DMD muscle tissue exhibited defective growth potential. In contrast, the fibroblast yield and proliferative capacity from DMD samples did not differ from normal. Based on these findings, we propose a hypothesis for the etiology of DMD: Dividing myoblasts are required for muscle growth and maintenance, and the limited capacity of DMD myoblasts to grow is directly related to the progressive muscle degeneration characteristic of the disease.

Adolescent↗