Transforming growth factor-beta isoform expression in insulin-like growth factor stimulated myogenesis

Transforming growth factor betas (TGF-b?s) are the defining members of a superfamily of small proteins that are involved in the regulation of development and morphogenesis in a wide array of systems. Previous studies have demonstrated that TGF-b?s both inhibit and, under specialized conditions, induce the differentiation of myoblasts. TGF-b?s have been shown to be secreted by mouse C2C12 myoblast cultures undergoing differentiation. Insulin-like growth factors (IGFs) have also been shown to be secreted by myoblasts and to induce myogenesis. This study characterizes the effects of IGF treatment on the expression and secretion of TGF-b?s in the IGF-sensitive L6A1 myoblast line. IGF downregulated the expression of TGF-b?3 in a concentration-dependent manner at 24 and 48 hours; TGF-b?1 was not sensitive to IGF treatment at 24 hours but was downregulated by IGFs at 48 hours. This downregulation was mediated by the type I IGF receptor and modulated by IGF binding proteins secreted by the myoblasts. Some reexpression of TGF-b?1 and TGF-b?3 mRNAs was observed after extensive morphological differentiation had occurred. These results support the hypothesis that IGFs act through the IGF type I receptor as part of a concerted mechanism to modulate expression of the TGF-b? genes, as part of a coordinated set of changes associated with terminal myogenic differentiation. © 1995 Wiley-Liss, Inc.     

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.     

Influence of Edaphic Factors and Previous Crop on Pratylenchus spp. Population Densities in Potato

Root and soil samples from commercial potato fields were assayed for nematodes in 1983 and 1984. Pratylenchus spp. population densities in Suffolk County, New York, were consistently, though not always statistically, higher in potato fields that had been planted to rye or wheat rather than potatoes during the previous growing season. Regardless of the previous crop, population densities in the two potato production areas in Suffolk County differed significantly: population densities on the south fork were 1.9-5.5 times higher than those on the north fork. Species prevalence differed significantly on the two forks but was not related to the previous year''s crop. P. penetrans and P. crenatus were found primarily on the north and south forks, respectively. Differences in species distribution were associated with differences in soil types. P. crenatus was usually found on loams and silt loams, but P. penetrans was found more frequently on sandy soils.     

The role of polyamines in somatomedin-stimulated differentiation of L6 myoblasts

The somatomedins are potent stimulators of proliferation and differentiation of cultured myoblasts. In studies on the mechanism(s) of these actions, we have measured the activities of ornithine decarboxylase (ODC), an enzyme associated with rapid cell proliferation, and creatine kinase (CK), a biochemical marker for muscle differentiation, after treatment of L6 myoblast cultures with Multiplication Stimulating Activity (MSA), a member of the somatomedin family of insulinlike growth factors. ODC levels reached a peak 24 hours after MSA addition (before any detectable differentiation of the myoblasts) and then decreased as differentiation commenced and CK activity increased. Addition of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, caused a dramatic decrease in differentiation. Measurement of 3H-thymidine incorporation, DNA content, and cell number established that the effect of DFMO on differentiation was not a simple consequence of its antiproliferative actions. Cellular levels of putrescine and spermidine (but not spermine) decreased substantially following addition of DFMO to the cultures. The inhibitory effects of DFMO were abolished upon addition of exogenous polyamines to the medium. However, addition of polyamines in the absence of MSA or DFMO did not mimic the stimulation of differentiation by MSA. We conclude that polyamines play an essential role in the stimulation of L6 myoblast differentiation by somatomedins, but they are not sufficient to effect this stimulation.     

Aqueous-phase quantitative NMR determination of amino acid enantiomer ratio by 13C-NMR using chiral neodymium shift reagent

A neodymium-(S)-PDTA (PDTA = N,N,N′,N′-tetrakis[(hydroxycarbonyl)methyl]-1,2-diaminopropane) complex was found exceptionally useful in the quantitative determination of enantiomer ratios of water-soluble natural amino acids by ¹³C-NMR. The method is demonstrated on mixtures of l- and d-enantiomers of various amino acids. The interactions of the chiral shift reagent with the amino acid molecules were rationalized by molecular orbital calculations.     

Effect of sucrose gradient composition on resolution of RNA species

Resolution of RNA samples on sucrose gradients was strikingly dependent upon the sucrose concentration in the gradients. Linear 10–70% gradients separated peaks which were barely discernible as shoulders in 10–20% gradients. The resolution obtained in steep sucrose gradients was comparable to that observed upon electrophoresis in acrylamide/agarose gels.