Journal of Cellular Physiology

doi: 10.1002/jcp.1041190301pmid: N/A

Lim, Ramon; Miller, Joyce F.

doi: 10.1002/jcp.1041190302pmid: 6725414

10.1002/jcp.1041190302.abs A procedure for the bulk isolation of glia maturation factor (GMF) in high yield and high purity from bovine brains is outlined. The method involves extraction by homogenization and centrifugation, followed by ammonium sulfate precipitation and column chromatography with diethylaminoethyl (DEAE) Sephacel, Sephadex G‐75, and hydroxylapatite. The method results in a 10,000‐fold purification, a purity exceeding that of previously published procedures, and enables us to handle as much as 2.8 kg brain tissue or eight brains/week. The ability to mass‐produce GMF with this method greatly facilitates its biological studies, further purification, and chemical characterization. The isolated GMF shows a molecular weight of 13,000 on Bio‐gel P‐30 column and an isoelectric point of about 5.4 on isoelectric focusing. The isolated GMF is heat labile and susceptible to papain and ficin but relatively resistant to trypsin, neuraminidase, and endoglycosidase.

Nishikawa, Yoshihisa

doi: 10.1002/jcp.1041190303pmid: 6725415

10.1002/jcp.1041190303.abs Protein glycosylation mutants in the mouse mammary carcinoma cell line FM3A were selected for ability to withstand exposure to (2‐3H)mannose at 39°C. G258, one of the mutant cells isolated, has been characterized. G258 cells were temperature‐sensitive for cell growth. Moreover, G258 cells showed temperature sensitivity for (3H)mannose incorporation into the TCA‐insoluble fraction. To study the biochemical basis of the defect in glycoprotein biosynthesis, the formation of lipid‐linked saccharides was examined. The results showed that the formation of lipid‐linked oligosaccharides was severely inhibited in G258 cells at 39°C. At 33°C, G258 cells synthesized Glc3Man9GlcNAc2‐PP‐Dol, the fully assembled lipid‐linked oligosaccharides, but at 39°C, G258 cells were able to synthesize merely the smaller lipid‐linked oligosaccharides (approximately up to Man3GlcNAc2‐PP‐Dol), but were unable to synthesize the larger lipid‐linked oligosaccharides.

Lin, Qixiong; Blaisdell, Joyce; O'Keefe, Edward; Earp, H. Shelton

doi: 10.1002/jcp.1041190304pmid: 6327730

10.1002/jcp.1041190304.abs Hydrocortisone and dexamethasone produced a time‐dependent increase (125I)epidermal growth factor ((125I)EGF) binding in primary cultures of isolated rat hepatocytes. Maximally effective doses of glucocorticoids resulted in a 70–100% increase in binding. The effect was similar when hepatocytes were maintained on collagen‐coated plates or directly on culture dishes. The glucocorticoid‐mediated increase in (125I)EGF binding could be detected after 4 h exposure to glucocorticoid and was substantial by 8 h. The major effect of glucocorticoid appeared to be to increase the number of EGF receptors. While insulin (100 nM) had no effect on basal (125I)EGF binding, it significantly inhibited the increase produced by the glucocorticoid. Since the inhibitory effect of insulin was only observed when insulin was added with the inducing glucocorticoid, insulin appears to inhibit an early hydrocortisone‐mediated event.

Ikehara, Toshitaka; Yamaguchi, Hisao; Hosokawa, Keiko; Sakai, Tetsuhiro; Miyamoto, Hiroshi

doi: 10.1002/jcp.1041190305pmid: 6725416

10.1002/jcp.1041190305.abs The effect of changes in energy metabolism on Rb+ influx was studied in HeLa cells. Irrespective of whether ATP production was controlled by varying the activity of glycolysis or of oxidative metabolism on addition of certain combinations of glucose, carbonylcyanide m‐chlorophenylhydrazone, monoiodoacetic acid, and quercetin, Rb+ influx changed as a linear function of the ATP content, which varied in a wide range up to the normal level (15–20 nmol/mg protein or 3–4 mM). The difference between results obtained by these procedures was not significant. As the intracellular Na+ content varied at different ATP contents, the Na+ content was adjusted to similar levels by chilling the cells with varying ATP contents. However, a linear relation was still observed. A similar dependence was also obtained for cytoplasmic ATP, which would be more closely connected with the Na, K‐pump than total ATP. The ratio of ouabain‐sensitive Rb+ influx to the corresponding part of lactate production was nearly 2 in the presence of 2 mM glucose. From these results it is concluded that (1) active Rb+ influx, which is chiefly maintained by energy generated through glycolysis, can also be supported by oxidative metabolism; (2) Rb+ influx is regulated linearly as a function of the cellular ATP content up to the control level; but does not increase if ATP is raised still further; and (3) 2 Rb+ ions move concomitantly at the expense of one ATP molecule.

Vandenburgh, Herman H.

doi: 10.1002/jcp.1041190306pmid: 6327731

10.1002/jcp.1041190306.abs Serum stimulates embryonic avian skeletal muscle growth in vitro and the growth‐related processes of amino acid transport and protein synthesis. Serum also stimulates myotube Na pump activity (measured as ouabain‐sensitive rubidium‐86 uptake) for at least 2 h after serum addition. Serum‐stimulated growth depends on this Na pump activity since ouabain added at the same time as serum totally inhibits the growth responses. The relationship of myotube growth, Na pump activity, and transmembrane potential was studied to determine whether serum‐stimulated Na pump activation and growth are coupled by long‐term membrane hyperpolarization. When myotube amino acid transport and protein synthesis are prestimulated by serum, ouabain was found to have little inhibitory effect, indicating that the already stimulated growth‐related processes are not tightly coupled to continued Na pump activity. Serum‐stimulated protein synthesis is tightly coupled to Na pump activity, but only during the first 5–10 min after serum addition. When myotube transmembrane potentials were measured using the lipophilic cation tetraphenylphosphonium, serum at concentrations that stimulate myotube growth and Na pump activity was found to have little effect on the cell's transmembrane potential. Furthermore, partial depolarization of the myotubes with 12‐ to 55‐mM extracellular potassium does not prevent serum stimulation of myotube growth. Monensin was found to hyperpolarize the myotubes, but causes myotube atrophy. These results indicate that although Na pump activity is associated with initiation of serum‐stimulated myotube growth, continued Na pump activity is not essential, and there is little relationship between myotube growth and the myotube's transmembrane potential.

Buss, Janice E.; Chouvet, Claire; Gill, Gordon N.

doi: 10.1002/jcp.1041190307pmid: 6202705

10.1002/jcp.1041190307.abs Growth of a selected variant of A431 cells (clone 29) is stimulated by epidermal growth factor (EGF) in contrast to the growth inhibition caused by EGF in an unselected clone, A4318. Twelve phosphoproteins from each clone were compared to determine whether unique EGF‐dependent substrate phosphorylations might explain the cells' differing growth responses to EGF. Treatment of both clone 29 and A4318 cells with EGF increased phosphorylation of the EGF receptor/kinase and six cellular proteins identified on 2‐dimensional poly‐acrylamide gels. Four of these proteins (the EGF receptor/kinase and proteins of 36, 70, and 81 kd) contained phosphotyrosine in both clone 29 and A4318 cells, indicating that the same modification of several proteins occurred in cells which have totally different growth responses to EGF. Two proteins were identified whose phosphorylation was EGF dependent and which were unique to clone 29 cells; however, EGF increased phosphorylation of only serine residues in these proteins. This indicates that these proteins are not primary targets of the EGF‐dependent tyrosine‐specific protein kinase, but rather are substrates for serine‐specific kinase(s) activated as a consequence of EGF:receptor interaction. cAMP, which inhibited growth of both clones, was utilized to compare the effects of EGF when the growth response of both cell lines was similar. In the presence of cAMP, EGF increased A4318 cellular phosphotyrosine content and the phosphorylation of the same phosphotyrosine‐containing proteins of both clone 29 and A4318 cells. The in vivo activity of a second tyrosine‐specific protein kinase, p60v‐src in B77 Rous sarcoma virus (RSV)‐transformed newborn rat kidney (NRK) cells, was also unaffected by cAMP. Thus cAMP did not block the in vivo activity of two tyrosine‐specific kinases or the tyrosine phosphorylation of three specific protein substrates. A threshold model of tyrosine kinase activity is proposed as an alternative explanation for the differing growth responses to EGF.

Yamaoka, Kazuko; Hirai, Reiko; Tsugita, Akira; Mitsui, Hiromi

doi: 10.1002/jcp.1041190308pmid: 6327732

10.1002/jcp.1041190308.abs A new class of transforming growth factor (TGF), with chemical characteristics differing from previously reported TGFs, was isolated and purified from an avian sarcoma virus‐transformed rat cell line, 77N1. Purification steps were simple and consisted of ion‐exchange chromatography on diethylaminoethyl (DEAE)‐Sephacel, ammonium sulfate precipitation, Chromatofocusing, and DEAE‐Sephadex A‐25 chromatography. The purified TGF is a heat‐ and acidlabile protein with a molecular mass of 12,000 daltons and isoelectric point of 5.2–5.4. Because of the acid lability of this TGF, purification was carried out at neutral pH. The TGF induced DNA synthesis in growth‐arrested BALB 3T3 cells and promoted anchorage‐independent growth of nontransformed BALB 3T3 cells in soft agar; the latter activity is specific for the peptide growth factors, called TGFs, but it did not compete with epidermal growth factor (EGF) for binding to the EGF membrane receptors. The TGF activity was not potentiated by EGF. The purified preparation of the TGF stimulated BALB 3T3 cells to grow progressively in soft agar at a dose of 20 ng/ml.

Absher, Marlene; Cristofalo, Vincent J.

doi: 10.1002/jcp.1041190309pmid: 6725417

10.1002/jcp.1041190309.abs Hydrocortisone is a modulator of cell division and has been shown to prolong the replicative in vitro life span of human embryonic lung fibroblasts. Time lapse cinematography was used to analyze the proliferative behavior of individual cells in populations of fibroblasts exposed to hydrocortisone in young cultures during a single growth cycle and in aged cultures that had been continuousiy exposed to hydrocortisone. Results indicate that hydrocortisone causes a decrease in the interdivision time (IDT) of a portion of the cells in the population and this effect is augmented after continuous exposure to hydrocortisone. Hydrocortisone does not appear to increase the number of initial dividers in the population but increases growth rate in the early stages of the culture period. Analysis of mother‐daughter IDT pairs further suggests that hydrocortisone exerts its effects on IDT independently for a given cell.

Smith, J. A.; Winslow, D. P.; Rudland, P. S.

doi: 10.1002/jcp.1041190310pmid: 6609925

10.1002/jcp.1041190310.abs A number of single‐cell‐cloned cell lines have been used to examine the growth‐promoting effects of putative mammotrophic agents on the various cell types in normal and neoplastic rat mammary glands. A partially purified novel pituitary‐derived growth factor stimulates only cuboidal epithelial cells to divide whereas fibroblast growth factor (FGF) stimulates the growth of stromal and myoepithelial‐like cells. Epidermal growth factor (EGF) has a widespread but variable growth‐stimulating action, but prolactin and growth hormone are essentially inactive when added alone at a concentration of 5 μg/ml. Phosphoethanolamine stimulates the growth of one epithelial cell line and a derivative myoepithelial‐like cell line, but is inactive on the other cell lines tested. The use of defined cloned cell lines provides a direct and reproducible assay for the identification and purification of inducers of mammary growth.