Search PubMed⌕ Search

Biomedical subjects

J R Florini

Publications and source records attributed to J R Florini.

At least 55 records · Page 3Linked to original sources
PubMed1983

Polyamine depletion inhibits the differentiation of L6 myoblast cells.

Exposure to alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, inhibited the insulin induced differentiation of L6 myoblast cells. Differentiation was assessed by measuring creatine kinase activity and by determining the percentage of nuclei in myotubes. The levels of putrescine and spermidine increased in stimulated cultures prior to their differentiation and these increases were blocked by alpha-difluoromethylornithine. Provision of exogenous putrescine was able to reverse the inhibitory effect of the drug. The anti-differentiative effect is observed only if alpha-difluoromethylornithine is added within twenty-four hours of insulin stimulation. In the experimental protocol used, alpha-difluoromethylornithine was added as the cultures approached confluence and had no effect on their ultimate DNA content. Therefore, the effect of alpha-difluoromethylornithine on myoblast differentiation is not secondary to an effect on cellular proliferation. These results indicate that polyamines may be involved in the mediation of muscle cell differentiation.

Animals↗
PubMed1982

Effects of inhibitors of ornithine and S-adenosylmethionine decarboxylases on L6 myoblast proliferation.

The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C-DFMO uptake in the presence and absence of these compounds demonstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S-adenosyl-methionine-decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.

Adenosylmethionine Decarboxylase↗
PubMed1982

Inhibition of myoblast differentiation in vitro by a protein isolated from liver cell medium.

We have recently discovered that cells of Coon's Buffalo rat liver (BRL) line secrete a protein which is a potent inhibitor of skeletal myoblast differentiation in vitro. Using ion exchange and molecular exclusion chromatography, we have prepared this protein, which we designate "differentiation inhibitor" (DI), from the materials secreted by BRL cells maintained in serum-free medium. It is a relatively heat-stable protein which is inactivated by treatment with trypsin and mercaptoethanol and has an apparent molecular weight in the range 30,000--36,000. It exhibits no detectable mitogenic or lectin activity and differs from previously reported inhibitors of myoblast differentiation in several respects. It is active in all skeletal myoblast systems tested (Yaffe's L6 line as well as primary cultures of rat, chick, and Japanese quail myoblasts), and it blocks fusion, elevation of creatine kinase, and increased binding of alpha-bungarotoxin. Parallel fractionation of fetal bovine serum (FBS) and chick embryo extract (CEE) yields a peak of activity which similarly inhibits myoblast differentiation. We suggest that the differentiation inhibitor from BRL cells may correspond to the differentiation-inhibiting component(s) of FBS and CEE, and we call attention to the possibility that such a substance could play a role in embryonic growth of myoblasts and in satellite cell formation.

Animals↗
PubMed1982

Hormonal stimulation of myoblast differentiation in the absence of DNA synthesis.

The role of DNA synthesis in the final stages of muscle cell differentiation has been a subject of controversy for more than a decade. In an attempt to resolve disagreements over the necessity for a unique (or "quantal") mitosis just prior to the conversion of proliferating myoblasts to form postmitotic myotubes, we have studied the effects of insulin and somatomedin on the stimulation of myoblast differentiation with or without DNA synthesis. Under conditions in which at least 95% of [3H]thymidine incorporation was blocked by cytosine arabinoside, there was a 5- to 10-fold increase in the extent of differentiation (determined as fusion or creatine kinase elevation) on addition of insulin or multiplication-stimulating activity. The effect of the hormones was on myoblast differentiation, not enzyme induction; insulin did not cause any increase in creatine kinase when it was added to performed myotubes. These studies were done using two different cell types, Yaffe's L6 cell line and Japanese quail myoblasts in serum-free media; we obtained similar results in both. Our results are not compatible with the view that a quantal mitosis is required at a late stage of muscle cell differentiation.

Animals↗
PubMed1981

Insulin acts as a somatomedin analog in stimulating myoblast growth in serum-free medium.

A serum-free medium that supports the proliferation of myoblasts (but not of fibroblasts) has been developed recently in this laboratory. It is composed of 10(-6) M insulin, 10(-7) M dexamethasone, and 10(-5) M fetuin, and is designated medium MM-1. The latter two components gave optimal stimulation at or near "physiological" concentrations, but insulin was required at levels far in excess of those found in serum. Accordingly, we have now investigated the possibility that insulin acts as a weak analog of the somatomedins, as has been suggested in other systems. We found that maximal growth rates were observed when 10(-6) M insulin was replaced by 0.5 to 1.0 microgram/ml multiplication stimulating activity (MSA), indicating that insulin serves a somatomedinlike function of MM-1. We also investigated the possibility that a contaminant of fetuin is responsible for its action in MM-1 but found no evidence to support this suggestion. We conclude that MM-1 is suitable for the study of muscle cell growth and differentiation under rather well-defined conditions, and that insulin probably is serving as a somatomedin analog in this medium.

Animals↗
PubMed1981

Effect of rat age on serum levels of growth hormone and somatomedins.

The somatomedins are a family of hormones which appear to mediate many of the anabolic actions of growth hormone; these processes often exhibit an age-associated deterioration in intact animals. We have demonstrated the validity of a radioreceptor assay for the determination of somatomedin levels in rat serum. In this assay, we measure displacement of 125I-labeled Multiplication Stimulating Activity (MSA) from receptors prepared from human placental membranes. Results with this procedure confirm and extend a previous report from this laboratory indicating a significant decrease in somatomedin levels during the latter part of the lifespan. Data are presented to eliminate possible artifactual explanations for the observed age-related changes. Furthermore, we find that the decrease in somatomedin levels can not be a simple result of an age-related decrease in basal levels of growth hormone in serum. We conclude that the decrease with age in circulating levels of the somatomedins is most probably attributable to a decrease in the activity of responsiveness of the tissues (most probably liver) which secrete somatomedins in response to stimulation by growth hormone.

Aging↗
PubMed1981

Regulation of growth hormone release: evidence against negative feedback in rat pituitary cells.

To determine if the anterior pituitary gland is the site of negative feedback inhibition of GH release, we studied the effect of GH and multiplication-stimulating activity (MSA), a member of the somatomedin family, on isolated rat anterior pituitary cells in primary culture. The effect of GH was examined in two ways: 1) by adding to the cultures human GH (10 ng/ml to 20 microgram/ml) which was biologically active in the rat but not cross-reactive in the rat GH (rGH) RIA, and 2) by comparing rGH secretion in cultures of different cell densities. No suppression of either basal or prostaglandin E1-stimulated rGH release was found. An enhancement observed in serum-free conditions at high human GH concentrations was interpreted as a nonspecific response to protein, because bovine serum albumin produced the same effect. When added in the presence of serum, MSA (1--500 ng/ml) had no effect on rGH secretion. In the absence of serum, there were 71% and 30% increases in the basal and prostaglandin E1-stimulated rates of hormone release, respectively, possibly attributable to a trophic effect of MSA. Six other hormones having structural or functional similarity to either GH or somatomedin also failed to inhibit rGH secretion. Our results do not support the hypothesis that GH or somatomedin exerts a negative feedback effect on GH release directly on the anterior pituitary gland. Most likely, the hypothalamus or a higher brain center is the site for such regulation.

Animals↗
PubMed1980

The effect of serum and multiplication stimulating activity on L6 myoblast growth: the lack of correlation with cyclic nucleotide changes.

The role of cAMP and cGMP in triggering proliferation of rat myoblasts was evaluated by: (1) measuring effects of mitogens on intracellular cyclic nucleotide levels, and (2) observing effects of agents which altered cyclic nucleotide levels on cell proliferation. Multiplication stimulating activity (MSA, 1 microgram/ml), a member of the somatomedin family, stimulated cell proliferation after 48 hr. It had little effect on cellular cyclic nucleotide levels, measured by radioimmunoassay. Horse serum (HS) and fetal bovine serum (FBS) stimulated cell proliferation approximately equally. Neither affected cAMP levels; FBS reduced cGMP to 33% of control values, but HS had no effect. Thus, there was no simple correlation between mitogenic action and cyclic nucleotide levels at any time from 5 min to 24 hr after addition of a purified mitogen or serum. Furthermore, agents which caused substantial changes in cyclic nucleotide levels had no effect on cell proliferation. Prostaglandin E1 (5 microM) elevated cAMP 440% without affecting cGMP levels or cell growth. A potent phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX, 0.1 mM), when added alone or in the presence of MSA, HS, or FBS, elevated cAMP 200% and cGMP 167%, but it had little effect on their mitogenic action. Lastly, a purified mitogen such as MSA, unlike serum, must be present for extended periods of time in order to stimulate cell proliferation. This makes it unlikely that a trigger mechanism functions in initiating cell division. We conclude that neither cAMP nor cGMP appear to be second messengers for the mitogenic action of MSA or serum on muscle cells.

Animals↗
PubMed1980

Effect of rat age on blood levels of somatomedin-like growth factors.

Age-related changes in serum levels of somatomedin-like growth factors were measured using a bioassay based on myoblast proliferation in culture. The correspondence of this assay to thymidine incorporation and sulfation factor assays for somatomedins was demonstrated. Comparisons involved individual serum samples from a total of 28 rats at each of three age ranges (young = 2 to 6 month, middle-aged = 12 to 16 month, and old = 24 to 28 month) in five separate experiments. A small (10%) decrease in levels of somatomedin-like activity was observed between young and middle-aged animals, with a greater (20 to 40%) subsequent decrease in old rats. Assays of mixed samples indicated that the age-related decreases in somatomedin-like activity could not be attributed to increases in circulating levels of a dissociable inhibitor of this activity. Paired comparisons of amino acid uptake by isolated omohyoideus muscles from rats of the specified ages suggested that there were no significant age-related changes in response of these muscles to a somatomedin analog.

Aging↗
PubMed1980

Relative effects of the somatomedins, multiplication-stimulating activity, and growth hormone on myoblasts and myotubes in culture.

Responses of muscle cells in culture to somatomedins A and B (SM-A and SM-B), Temin's multiplication-stimulating activity (MSA), and a number of GH preparations from various species have been determined at both the myoblast and myotube stages of differentiation. Proliferation and amino acid (AIB) uptake by myoblasts as well as AIB uptake by myotubes were equally responsive to SM-A and MSA, demonstrating that MSA serves as a useful surrogate for SM-A in studies on muscle cells. SM-B exhibited no activity in either system, and it did not enhance the actions of SM-A. A number of GH preparations were inactive in muscle cell cultures from various sources, but bovine GH stimulated AIB uptake in myotubes. Both the magnitude of the response and the sensitivity of myotubes to bovine GH increased with time after differentiation of the cells in vitro, but conditions could not be found to demonstrate an effect of GH at or near physiological levels of the hormone. On the basis of our observations and published studies on the effects of GH on isolated muscles, we suggest that the early growth of muscle is stimulated primarily by the somatomedins, and that a direct response to GH develops relatively late after the differentiation of skeletal muscle.

Aminoisobutyric Acids↗
PubMed1980

Dexamethasone effects on myoblast proliferation and differentiation.

Effects of glucocorticoids on myoblast proliferation and differentiation were determined in cell culture. Dexamethasone (1 X 10(-7) M) decreased myoblast doubling time by 7 h and increased cell densities as much as 800% after 8 days of exposure of the cells to dexamethasone. Stimulation of myoblast proliferation by glucocorticoids was dose dependent, with half-maximal activity exhibited at concentrations of 3 X 10(-9) M dexamethasone or 2.3 X 10(-8) M corticosterone. In differentiating cultures, the rate of accumulation and final levels of DNA and creatine phosphokinase were highly stimulated by dexamethasone. However, normalization of creatine phosphokinase levels (a marker for myoblast differentiation) per microgram DNA demonstrated a small (30%) decrease in differentiation in cultures exposed to glucocorticoids. These results indicate that glucocorticoids stimulate early myogenesis by increasing the number of myoblasts but do not stimulate the differentiation of these cells.

Animals↗
PubMed1979

A serum-free medium for the growth of muscle cells in culture.

Rates of cell proliferation essentially equal to those in 10% serum were obtained when Yaffe's L6 myoblasts were incubated in Ham's F-12 medium containing 10(-5) M fetuin, 10(-6) M insulin, and 10(-7) M dexamethasone; we have designated this mixture muscle medium-1 (MM-1). Addition of other growth factors and hormones in various combinations did not increase the proliferation of myoblasts above the rate in MM-1, and neither fetuin nor insulin could be replaced by other growth factors. All glucocorticoids tested (but no other steroid hormones) were active. Fetuins prepared by the rather different procedures of Pedersen, Deutsch, and Spiro were all active, and the active material was heat labile and nondialyzable; this is the first cell culture system in which highly purified Spiro fetuin has been found active. Primary rat myoblasts proliferated more rapidly that fibroblasts in parallel cultures when incubated in MM-1. This simple medium, composed of relatively inexpensive and readily available components, should be useful for the study of muscle cell growth and differentiation.

Adrenal Cortex Hormones↗
PubMed1978

Stimulation by glucocorticoids of myoblast growth at low cell densities.

Proliferation of rat myoblasts was stimulated by addition of 10(-7) M dexamethasone to cells plated at 25 cells/cm(2). In the presence of fetal calf serum and chick embryo extract, physiological levels of glucocorticoids caused a 3- to 7-fold increase in colony formation. In mass cultures, proliferation of myoblasts was also stimulated by dexamethasone, but fibroblast growth was inhibited. These opposite effects of glucocorticoids on myoblast and fibroblast growth suggest their use in cloning muscle cells from mixed cell populations.

Animals↗