ABSTRACT
Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which exerts a variety of effects on many cell types including neuronal cells, and is a potent mitogen for myoblasts. At concentrations of 0.1-0.3 ng/ml, the peptide stimulates a maximal increase in cell number. LIF initiates a prolonged proliferative response lasting up to 13 days, when myoblasts are exposed to it in culture. LIF expression can be detected in vivo during development of limb muscle and in adult regenerating skeletal muscle tissue. Here, we studied the levels of expression of alpha-bungarotoxin-binding sites as a measure of acetylcholine receptors (AChRs), myosin light chain pattern and rate of myotube formation in fused, control and LIF-treated muscle cultures derived from mouse hind-limb muscles. We found that both the level of expression of AChRs and myosin light chain pattern, are normal, following exposure of the cells to LIF. There was no difference in the rate of myotube formation between LIF-stimulated and control myoblasts over a 10-fold concentration range (0.3-3.0 ng/ml) as determined by nuclei counts. Taken together, these results suggest that LIF, in combination with other cytokines, may act in vivo to stimulate rapid growth, without significant differentiation, during the early phases of myogenesis.
Subject(s)
Growth Inhibitors/pharmacology , Interleukin-6 , Lymphokines/pharmacology , Muscle Development , Muscles/drug effects , Animals , Cell Differentiation/drug effects , Electrophoresis , Leukemia Inhibitory Factor , Mice , Mice, Inbred C57BL , Muscles/cytology , Myosin Light Chains/metabolism , Receptors, Cholinergic/metabolismABSTRACT
Leukemia inhibitory factor (LIF) is a member of the cytokine family of growth factors. It has been shown to exert a variety of actions on a diverse range of cell types, including neuronal, bone, and hemopoietic cells (Hilton, 1992, Trends Biochem. Sci., 17:72-76). In many of these cell types, studies have indicated the presence of specific receptors for LIF (Godard et al., 1982, J. Biol. Chem., 267: 3214-3222; Hilton et al., Proc. Natl. Acad. Sci. USA, 85:5971-5975; Hilton and Nicola, 1992, J. Biol. Chem., 267:10238-10247.). The mechanism by which these receptors act is believed to involve tyrosine phosphorylation and the signal transducing receptor component gp130. We have previously shown that LIF is capable of inducing both human and murine myoblasts to proliferate in culture (Austin et al., 1992, J. Neurol. Sci., 112:185-191). We now report that LIF binds specifically to receptors on the surface of myoblasts, with an equilibrium dissociation constant of 400 pM and the number of receptors per cell varies with cell density. Binding competition studies showed that LIF binding to these receptor sites was not competed for by a number of other growth factors which stimulate myoblast proliferation including basic fibroblast growth factor (bFGF), transforming growth factor-alpha (TGF alpha), insulin-like growth factor 1 (IGF-1), and interleukin-6 (IL-6). There was a time and concentration-dependent down-regulation of receptor numbers following preincubation of myoblasts with LIF. The processing of these receptors subsequent to binding, involves as a first step, internalization and degradation by the myoblast. LIF appeared to stimulate myoblast proliferation rather than cell survival.
Subject(s)
Growth Inhibitors/metabolism , Interleukin-6 , Lymphokines/metabolism , Muscle, Skeletal/metabolism , Animals , Autoradiography , Binding, Competitive , Bromodeoxyuridine/metabolism , Cell Nucleus/metabolism , Cells, Cultured , Embryo, Mammalian/cytology , Leukemia Inhibitory Factor , Leukemia Inhibitory Factor Receptor alpha Subunit , Mice , Mice, Inbred C57BL , Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Receptors, Cytokine/metabolism , Receptors, OSM-LIFABSTRACT
It has been shown previously that leukaemia inhibitory factor (LIF) and transforming growth factor-alpha (TGF-alpha) stimulate proliferation of primary cultures of murine myoblasts. We now show that human myoblasts respond in a similar manner to LIF and TGF-alpha. These responses occur over a range of growth conditions. There are total additive effects in both human and murine myoblasts between LIF and TGF-alpha and LIF and fibroblast growth factor-beta (FGF-beta), but not between LIF and interleukin-6 (IL-6) or insulin-like growth factor 1 (IGF-1). The LIF response is initiated by a short exposure to the cytokine and is maintained for prolonged periods in its absence.
Subject(s)
Cytokines/pharmacology , Growth Inhibitors/pharmacology , Interleukin-6 , Lymphokines/pharmacology , Muscles/cytology , Animals , Antibodies, Monoclonal/pharmacology , Cell Division/drug effects , Humans , Hybridomas/immunology , Leukemia Inhibitory Factor , Mice , Mice, Inbred C57BL , Muscles/drug effects , Platelet-Derived Growth Factor/pharmacology , Transforming Growth Factor alpha/pharmacologyABSTRACT
A rapid isotonic method for fractionation of nuclei from rat brain is described. This procedure is based on the use of discontinuous colloidal silica gel (Percoll) gradients. We start from a 63,000-g purified nuclear pellet (fraction P3) isolated from gray matter and white matter separately. This is followed by fractionation of fraction P3 in an initial differential centrifugation step on five-step Percoll gradients producing six nuclear fractions designated 1, 2, 3 (gray matter) and 4, 5, 6 (white matter). Fractions 2, 4, and 5 obtained from this centrifugation are heterogeneous. These fractions are subfractionated further under isopycnic conditions using five-step Percoll gradients to yield subfractions 2b, 4b, and 5c. Three methods were used to characterize the nuclear types. First, light and electron microscopic examination was used to identify the nuclei in each preparation and to assess the purity of each preparation. Second, the activities of RNA polymerase I and II were monitored. Third, the protein/DNA ratios of the nuclear fractions were determined. Fraction 1 was enriched in neuronal nuclei; fractions 2b and 4b in astrocytic nuclei; and fractions 3, 5c, and 6 in nuclei of oligodendrocytes. RNA polymerase I and II activity was highest in fraction 1, which also displayed the highest protein/DNA ratio. Electron microscopy showed that the various classes of nuclei are congruent to 90% pure. Therefore, the procedure described here is suitable for obtaining highly purified neuronal and three types of glial nuclei from rat brain.
Subject(s)
Brain/ultrastructure , Cell Nucleus/ultrastructure , Centrifugation, Density Gradient , Povidone , Silicon Dioxide , Animals , Brain/metabolism , Cell Nucleus/metabolism , DNA/analysis , DNA/metabolism , DNA-Directed RNA Polymerases/metabolism , Detergents/pharmacology , Electrophoresis, Polyacrylamide Gel , Female , Microscopy, Electron , Nerve Tissue Proteins/metabolism , Rats , Rats, Inbred StrainsABSTRACT
Hyaluronic acid differs from other glycosaminoglycans in its lack of covalently linked peptide, absence of sulphate groups, and the exceptional size of its single-chain polymers. These differences can be related to its distinct physical and functional properties, and may be pertinent to its greater abundance in early tissue development. In mature animals, the turnover of hyaluronic acid in tissues is reflected at least partly in the blood stream. The metabolism of circulating hyaluronic acid was therefore studied in fetal sheep after intravenous injection of [3H]acetylhyaluronic acid. Between 95% and 99% was removed within 6 min. Plasma radioactivity decayed by first-order kinetics, with a half-life between 0.8 and 1.25 min. The rate of elimination did not vary materially with hyaluronic acid fractions of widely disparate average Mr or with fetal age between 70 and 120 days. 3H2O was detected in plasma within 8-10 min. Labelled material found in urine from 10 min onward included polymers greater than or equal to 70,000 Mr, which indicates that urine may be a source of hyaluronic acid in amniotic fluid. Elimination from the plasma took place mainly in the liver, where labelled material was largely recovered in small metabolic residues as early as 28 min after injection. These were shown by high pressure liquid chromatography (h.p.l.c.) to include water, acetate, N-acetylglucosamine and a fraction tentatively identified as N-acetylglucosamine 1-phosphate. Tritium radioactivity was detected in hepatic lipids but not those of the spleen. Estimated plasma turnover was in the order of 10 micrograms/min per kg body weight. This is about 3-10 times that in adult animals and is consistent with an increased inflow of hyaluronic acid generated during the maturation of developing tissues.
Subject(s)
Fetal Blood/metabolism , Hyaluronic Acid/blood , Amniotic Fluid/metabolism , Animals , Body Fluids/metabolism , Half-Life , Hyaluronic Acid/urine , Kinetics , Liver/embryology , Liver/metabolism , Sheep , Tissue Distribution , Trachea/embryology , Trachea/metabolism , TritiumABSTRACT
Afferent lymph vessels entering popliteal lymph nodes of sheep were infused with [3H]acetyl-labelled hyaluronan of high Mr (4.3 x 10(6)-5.5 x 10(6)) and low Mr (1.5 x 10(5)). Analysis of efferent lymph and of residues in the nodes showed that hyaluronan presented by this route is taken up and degraded by lymphatic tissue. Labelled residues isolated in node extracts by gel chromatography and h.p.l.c. included N-acetylglucosamine, acetate, water and a fraction provisionally identified as N-acetylglucosamine 6-phosphate. Between 48 and 75% of the infused material was unrecovered, and had been presumably eliminated through the bloodstream as diffusible residues. Rates of degradation reached as high as 43 micrograms/h in a node of 2 g wt. infused with 56 micrograms/h. Some HA passed into efferent lymph and some was detected in the nodes, but fractions of Mr greater than 1 x 10(6) were not found in either. It is concluded that the amounts and Mr values of hyaluronan released from the tissues into peripheral lymph can be significantly underestimated by analysis of efferent lymph, i.e. lymph that has passed through lymph nodes. A substantial role in the normal metabolic turnover of at least one major constituent of intercellular matrix and connective tissue may now be added to the established functions of the lymphatic system.
Subject(s)
Hyaluronic Acid/metabolism , Lymphoid Tissue/metabolism , Adsorption , Animals , Chromatography, Gel , Chromatography, High Pressure Liquid , Female , Hyaluronic Acid/pharmacokinetics , Lymph/metabolism , Lymph Nodes/metabolism , Molecular Weight , Polyvinyls , Sheep , TritiumABSTRACT
The red blood cells and the low-density lipoproteins in hypertriglyceridemia have a lower ratio of unesterified cholesterol to phospholipid than normal. The low-density lipoproteins are also smaller and more dense in hypertriglyceridemia, and contain only 45% of the normal unesterified cholesterol mass. The phase behavior of the lipids shows that normal red cells and low-density lipoproteins are close to saturation with cholesterol, whereas in hypertriglyceridemia less cholesterol is present. Because newly secreted triacylglycerol-rich lipoproteins are poor in cholesterol, their excess production and transport in hypertriglyceridemia may prevent maintenance of the normal cholesterol content of blood cells and low-density lipoproteins. Partitioning of cholesterol into triacylglycerol-rich lipoproteins is able to account for significant fluxes of unesterified cholesterol in the plasma compartment.
Subject(s)
Cholesterol/blood , Erythrocytes/metabolism , Hyperlipoproteinemia Type IV/blood , Lipoproteins, LDL/blood , Humans , Phospholipids/bloodABSTRACT
These experiments were designed to test the hypothesis that the vitamin A fat-storage cell removes chylomicron remnant cholesterol from hepatic portal venous blood; A modified Ficoll density gradient ultracentrifugation procedure was used to isolate from rat liver cellular fractions that were enriched in vitamin A. In rats fed a normal diet and in rats fed excess vitamin A isolated hepatocytes were fractionated 15 min after the intravenous injection of chylomicrons labelled in vivo with radioactive cholesterol. The results showed that cholesterol radioactivity was not concentrated in the vitamin A enriched cellular fractions, so it was concluded that the vitamin A fat-storage cell is not implicated in clearance of chylomicron remnants by the liver.