Pyruvate-Induced changes in hepatoma cell morphology and macromolecular synthesis

Cells maintained in basal growth medium with 0.2–1.0% serum often require citric acid cycle intermediates for optimal viability. We have found that pyruvate added to minimal growth medium causes cellular flattening and formation of external processes accompanied by increaded DNA synthesis in cultured hepatoma cells (HTC cells). Cells were cultured in plalstic T-flasks (0.5, 1.0, or 2.0 × 10⁶ cells/flask) containing 5 ml medium (90% Eagle's Basal Medium (BME) and 10% Swim's S-77) with various concentrations of fetal calf serum (0.2,0.25, 0.5, 1.0, 2.0, 10%) and either pyruvate (50, 100, 250,500, 1,000μg/ml), or one of: dibutyryl cAMP (DBcAMP) or dibutyryl cGMP (DBcGMP) at 10^?3, 10^?4, or 10^?5 M. At 44–48 hr cultures were pulsed with tritiated thymidine, uridine, or lecucine. Cells became attached to the plastic surface within 24hr. Cells in medium with 0.25 to 2.0% serum had a rounded appearance. With added pyruvate, cellular flattening, process formation, and an increased adherence to the substratum was absorbed. By 48 hr, culture without pyruvate grew in rounded clusters; with pyruvate, cells formed extensive interconnecting processes that appeared loosely attached to the monolayer surface. At the cell densities tested, process formation was maximal with 250 to 500 μg/ml pyruvate. Cytochalasin B blocked flattening and process formation; EDTA (1 mg/ml) caused retraction of processes within 3 min, and a slow dissolution of these structures within cells was observed. DBcAMP or DBcGMP did not induce process formation. Flattening and process foormation in pyruvate-enriched cultures were accompanied by marked stimulation of DNA synthesis and smaller increases in RNA and protein synthesis. Cell number was not affected. These pyruvate-induced changes suggest that alterations in energy metabolism, or precursors that enhance viability and macromolecular synthesis in mammalian cell cultures, may exert marked effects on cellular morphology without corresponding changes in growth of neoplastic liver cells.     

Alterations in craniofacial growth induced by isotretinoin (13-cis-retinoic acid) in mouse whole embryo and primary mesenchymal cell culture

Recent evidence has demonstrated that 13-cis-retinoic acid (13-cis-RA, or isotretinoin) is responsible for various craniofacial malformations in the rodent and human embryo. Our studies have been directed toward understanding this effect using mouse whole embryo and primary cell cultures. In whole embryo culture, 13-cis-RA caused significant overall embryonic growth retardation, especially in the primary and secondary palatal processes. In embryos explanted on day 10 of gestation and exposed for 24 or 48 hr, the mesenchyme beneath the epithelium of the nasal and maxillary processes contained pyknotic nuclei as well as a dramatically reduced number of nuclei incorporating 3H-thymidine. The secondary palatal processes and the roof of the oral-nasal cavity had fewer mesenchymal cells than control embryos. The incorporation of 3H-thymidine into TCA-insoluble macromolecules was 30% less in the retinoid-treated heads. In primary cell cultures from day-12 mouse secondary palatal mesenchyme, subsequent cell growth was decreased at concentrations of 13-cis-RA greater than 1 X 10(-5) M. After a 40-hr treatment period, labeling indices in retinoid-treated cells were significantly lower than control values (25% compared with 40%). Retinoic acid also caused a significant, concentration-dependent decrease in 3H-thymidine incorporation. The inhibitory effect of 13-cis-RA on proliferation of oral-nasal mesenchymal cells appears to be related to the production of craniofacial malformations.     

Characterization of glutamine transport into resting and concanavalin A-stimulated peripheral human lymphocytes

Characteristics of glutamine transport, its substrate specificity, and its pattern of competitive and non-competitive inhibition in response to amino acid analogues were determined in peripheral human lymphocytes, incubated with or without concanavalin A (Con A). Maximum capacity of transport (Vmax) at 37 degrees C and 136.9 mM Na+ was 30 pmol/10(6) cells/30 seconds, while the apparent Km was 142 microM. In cells exposed to 10 mM histidine, asparagine, serine, or leucine transport of glutamine declined to 28%, 15%, 17%, and 21%, respectively, of the rates in controls. Inhibition by histidine (Ki = 0.58 mM) and serine (Ki = 0.25 mM) was competitive, by leucine was non-competitive (Ki = 0.64), while alpha-methylamino-isobutyric acid and 2-amino carboxy-bicyclo (2.2.1)-heptane had no effect. In cells cultured for 24 hours with or without 10 micrograms/ml Con A, the apparent Km was 70 microM vs. 89 microM and Vmax 73 vs. 26 pmol/10(6) cells/30 seconds. Sodium depletion (9.0 mM NaCl) greatly diminished glutamine transport in resting and stimulated cells. Inhibition of glutamine transport by serine was sodium sensitive, while inhibition by histidine and asparagine was not. Serine had no competitive effect in sodium-depleted media. The data demonstrate what appear to be two carrier systems for glutamine, sodium sensitive and sodium insensitive. It is suggested that glutamine transport into lymphocytes occurs via processes similar to System N and System ASC described in other cells, with System ASC as the sodium-sensitive component. Con A augments the capacity rather than the affinity of glutamine transporting systems.     

Negative regulation of hepatocyte growth factor gene expression in human lung fibroblasts and leukemic cells by transforming growth factor-beta 1 and glucocorticoids.

Hepatocyte growth factor (HGF), a mesenchymal-derived factor which regulates growth, motility, and morphogenesis of epithelial and endothelial cells, functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. We have now obtained evidence that transforming growth factor-beta 1 (TGF-beta 1) and glucocorticoids are negative regulators for HGF gene expression. When TGF-beta 1 or dexamethasone was added to cultures of MRC-5 human embryonic lung fibroblasts and HL-60 human promyelocytic leukemic cells, the amount of HGF secreted into the culture medium was inhibited to 30-40% of that of control cultures by 10 ng/ml TGF-beta 1 and to 40-50% by 10(-6) M dexamethasone. The inhibitory effect of TGF-beta 1 and dexamethasone on HGF synthesis in MRC-5 cells was additive, thereby suggesting that TGF-beta 1 and dexamethasone exert effects through distinct mechanisms. Hydrocortisone also inhibited HGF synthesis with the same potency as dexamethasone; however, testosterone, estriol, and beta-estradiol had no effect. The rate of HGF synthesis in MRC-5 cells, as measured by pulse labeling with [35S]methionine and subsequent immunoprecipitation, was suppressed to 30-40% of the control with 10 ng/ml TGF-beta 1, and to 30-45% by 10(-6) M dexamethasone. HGF mRNA levels in MRC-5 cells and HL-60 cells were dose-dependently suppressed by TGF-beta 1 and dexamethasone; 10 ng/ml TGF-beta 1 suppressed HGF mRNA levels to 32% and 35% of control culture, respectively, in MRC-5 cells and HL-60 cells, and 10(-6) M dexamethasone suppressed to 43% and 38%, respectively. Thus, TGF-beta 1 and glucocorticoids seem to inhibit HGF synthesis by suppressing the expression of the HGF gene. We propose that a negative regulation of HGF gene expression by TGF-beta 1 or glucocorticoids may be involved in physiological or pathological processes during tissue regeneration.     

Visualization of interstitial cells of Cajal in the mouse colon by vital staining

Interstitial cells of Cajal (ICCs) are believed to be a major element in generating the spontaneous rhythm of the gastrointestinal tract. A prominent problem in the study of these cells has been the difficulty in observing them in intact tissues. We used the lipophilic dye DiI to stain ICCs in the submucosal-circular muscle border of freshly dissected mouse colon. The placement of small DiI crystals in this area resulted in the labeling of ICC-like cells. Two main morphological cell types, viz., bipolar and multipolar, were noted. Bipolar cells had two primary processes emerging from the poles of an elongated soma. The mean length of these processes was 78.7 μm. These cells constituted 42.3% of the sample (n=105). Multipolar cells (54.3% of total) had a less elongated soma and extended 3–6 main processes whose mean length was 56.3 μm. These processes showed no preferred direction. The length of the primary processes of bipolar cells was 40% greater than that of multipolar cells (P<0.02). Three cells (2.9%) had only one primary process. The DiI stain could be converted into a stable electron-opaque product. Electron-microscopic observations showed that these cells had the typical appearance of ICCs reported in previous studies. This staining method should be useful for physiological investigations of ICCs in gastrointestinal tissues. Received: 16 September 1997 / Accepted: 11 November 1997     

Biosynthesis and Expression of Gangliosides During Differentiation of Chick Embryo Retina Cells In Vitro

Cells from neural retina from 7-day chick embryos were cultured on polylysine-coated dishes up to 7 days. The small, round-shaped cells at seeding differentiated progressively, and after 4 days in vitro the majority had enlarged bodies and abundant processes. The content of protein and DNA was essentially unchanged during the entire period of culture. The incorporation of radioactivity from [3H]glucosamine into gangliosides declined slightly, reaching about 65% of the initial values at the end of the culture period. The proliferating activity measured by the incorporation of [3H]thymidine into DNA decreased to 10% or less of the initial value after 3 days in vitro. Almost at the same chronological times as in ovo, the synthesis of GD3 and of a ganglioside partially identified as GT3 decreased from 70 and 19% of the total incorporation into gangliosides in the first 20 h of culture to about 7 and 5%, respectively, after 3 days in vitro. Conversely, the synthesis of GD1a increased from about 6% at the beginning to about 70% at the end of the culture times. Immunocytochemical analyses of the expression of gangliotetraosyl gangliosides in cultured cells showed that these gangliosides appeared in the bodies and processes of cells having neuronal morphology; very little immunostaining of the scarce flattened cells, probably Müller cells, was found. The results indicate that the changes in ganglioside metabolism, which lead to decreased synthesis of gangliosides lacking the galactosyl-N-acetyl-galactosaminyl disaccharide end and to increased synthesis of gangliotetraosyl gangliosides, occur in cells that in culture differentiate into neurons.     

A possible differentiation of anterior pituitary cells in collagen gels into neurons

Summary Dispersed cells from anterior pituitary glands of male rats were cultured embedded in collagen gels or on a plastic surface. After 6 or more days in culture, cells in collagen produced more prolactin than those on plastic. The cultures in collagen had fewer fibroblasts than those on plastic. Many cells cultured in collagen developed processes, and a few cells in every culture had long extended processes that sometimes branched and resembled those of neurons. About 60% of the cells in culture including cells with well developed processes bound the monoclonal antibody A2B5, an antibody that binds to neuronal cells in culture. Fibroblasts did not have detectable binding of A2B5. Some cells with short processes reacted with antiserum to prolactin or ACTH, but the cells with neuronlike processes did not. Collagen gels may provide a matrix in which cells from the anterior pituitary gland can differentiate into neuronlike cells.     

Zinc uptake into MCF-10A cells is inhibited by cholesterol depletion

The mechanism for cellular Zn uptake was investigated by depleting cell cholesterol levels, a treatment that disrupts lipid rafts/caveolae-dependent processes and inhibits coated-pit budding. Incubation of MCF-10A human breast epithelial cells with hydroxypropyl-beta-cyclodextrin significantly lowered cell cholesterol levels and significantly inhibited cellular zinc uptake measured at 10 min, but had no effect on 2-deoxyglucose uptake. Replacing potassium for sodium in the uptake buffer significantly stimulated Zn uptake by 20%. The effects of potassium depletion and chlorpromazine on Zn uptake were investigated to determine the contribution of coated-pit endocytosis. Potassium depletion following hypotonic shock significantly inhibited Zn uptake into MCF-10A cells approximately 15%. Chlorpromazine at 20 microg/ml inhibited uptake approximately 30%. The data support the hypothesis that Zn uptake into MCF-10A cells involves lipid rafts/caveolae. The relatively mild effects of potassium depletion and chlorpromazine suggest that a small portion of Zn uptake may require coated pit endocytosis.     

Effect of retinoic acid on the proliferation and phagocytic capability of murine macrophage-like cell lines

Retinoic acid (RA) exerted a variable degree of growth inhibitory activity on the macrophage-like cell lines P388D1, J774.2, WEHI-265, WEHI-3, and PU-5. Comparison of cell proliferation and clonal growth suggests that at concentrations of 10(-9)-10(-6) M the inhibitory activity stems from processes leading to elongation of cell cycle time and not from terminal differentiation processes. RA was shown to be a potent inducer of the development of high-phagocytic phenotypes (assessed by phagocytosis of heat-killed yeast cells) in the P388D1, J774.2, and WEHI-265 cell lines which differ substantially in their proliferative and adherence characteristics. The PU-5 and WEHI-3 cell lines were not induced by RA to express an enhanced phagocytic activity toward heat-killed yeast cells. The augmented phagocytic capability was dose dependent over a wide range of RA concentrations. In P388D1 cells, 2 X 10(-12) M RA already exerted significant phagocytosis augmentation effects, which progressively increased up to 2 X 10(-5) M RA, the highest concentration tested. Retinal, retinyl acetate, and retinol had similar effects to those of RA on both cell adherence and phagocytosis in P388D1 cells, albeit at concentrations four to six orders of magnitude higher. Optimal development of the high-phagocytic phenotype in P388D1, J774.2, and WEHI-265 cells required at least 96 hr of culture in the presence of RA; at 48 hr and 23 hr the effects were already substantial, whereas at 4 hr of exposure to RA no significant enhancement of phagocytosis could be detected. Thus both extended periods of culture in the presence of RA (more than two to three cell cycles) and high concentrations were needed for induction, in more than 90% of the cells, of the expression of a high-phagocytic phenotype. The reversion to a low-phagocytic phenotype upon removal of RA was also rather slow and required several cell cycles. In P388D1 cells RA also enhanced the phagocytosis of latex beads but had no effect on the phagocytosis of starch particles, or the extent of binding of immunoglobulin G-coated sheep red blood cells (SRBC). The expression of receptors for concanavalin A and for nonopsonized SRBC remarkably increased in RA-treated cells, as was the ability to perform Fc-receptor mediated erythrophagocytosis. Both P388D1 cells and WEHI-265 cells were induced by RA to express nitroblue tetrazolium reducing activity. The data suggest that RA induces profound changes in the functional capabilities of macrophage-like cell lines which are apparently not dependent on cessation of growth and terminal differentiation processes.     

A disintegrin and metalloproteinase 10 regulates antibody production and maintenance of lymphoid architecture

A disintegrin and metalloproteinase 10 (ADAM10) is a zinc-dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B cell-specific ADAM10-deficient mice (ADAM10(B-/-)). In this study, we show that ADAM10 levels are significantly enhanced on germinal center B cells. Moreover, ADAM10(B-/-) mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10(B-/-) displayed impaired germinal center formation, had fewer follicular Th cells, decreased follicular dendritic cell networks, and altered chemokine expression in draining lymph nodes (LNs). Interestingly, when spleen and LN structures from immunized mice were analyzed for B and T cell localization, tissues structure was aberrant in ADAM10(B-/-) mice. Importantly, when ADAM10-deficient B cells were stimulated in vitro, they produced comparable Ab as wild type B cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B cell activation, likely because of the decrease in follicular Th cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure after Ag challenge.     

Proliferation and differentiation potential of cryopreserved human skin-derived precursors

Objectives: Skin‐derived precursors are recognized to be a potentially autologous and accessible source of neural precursor cells for drug screening or cell‐based treatments, in many neurological disorders. Thus, it is necessary to investigate appropriate methods for cryopreservation of such human skin‐derived precursors (hSKPs). The aim of this study was to evaluate different cryopreservation techniques for retention of hSKPs to discover an optimized protocol. Materials and methods: We cryopreserved hSKPs treated with 0%, 10%, 20%, 30% and 40% foetal bovine serum (FBS) and three concentrations of dimethylsulphoxide (DMSO) 5%, 10% and 15%, with two different storage periods in liquid nitrogen (2 days: short‐term storage; and 2 months: long‐term storage). Then, we assessed survival and proliferation levels of the cells after freeze–thaw processes, by viability measurement and colony‐forming assay. For detecting hSKPs, we used immunocytochemistry and RT‐PCR assessments. Results: Our findings indicated that hSKPs cryopreserved in 5% DMSO without FBS, had better survival and proliferation potentials compared to other working formulations. With various concentrations of cryoprotectants over different time periods, hSKPs retained their differentiation potentiality and were able to differentiate into neurons (NFM and βΙΙΙ tubulin‐positive), glial cells (GFAP‐positive) and smooth muscle cells (SMA‐positive). Conclusions: Results revealed that in only 5% DMSO, hSKPs could be cryopreserved for long‐term storage with considerable survival and proliferation levels, without losing multipotency.     

An autoradiographic study of rabbit ovarian surface epithelium before and after ovulation

Morphologic studies suggest that the proliferative activity of the ovarian surface epithelium (OSE) may vary during the reproductive life cycle. To further investigate this phenomenon, rabbit ovaries obtained before and after induction of ovulation with human chorionic gonadotropin (hCG) were incubated in medium containing 3H-methylthymidine and processed for autoradiography. Before ovulation, the labeling index (LI) of OSE cells varied from 0.04% to 0.22%. Twelve hours after hCG, the maximal LI (9.02 +/- 0.38%) was seen in OSE cells adjacent to the ovulatory stigma. The LI remained elevated at Days 1 and 5 post-hCG in OSE cells overlying corpora lutea. At Day 12, numerous papillary processes were observed at the apex of each corpus luteum. The maximal LI (16.44 +/- 1.31%) had now shifted to the OSE cells covering these processes. Eighteen days after hCG stimulation, the LI of OSE cells near the corpora lutea had returned to preovulatory levels. A slight increase in the LI of OSE cells not associated with ovulatory sites was also observed after ovulation. This study shows that a significant fraction of OSE cells undergoes DNA synthesis throughout most of the postovulatory period.     

Quantification of ATP-producing and consuming processes of Ehrlich ascites tumour cells

ATP production of Ehrlich ascites tumour cells was estimated on the basis of their coupled respiration and lactate formation. ATP-consuming processes were assessed from the effects of selective inhibitors of RNA synthesis, protein synthesis and proteolysis, Na+/K+-ATPase on respiration. The extent of protein synthesis and proteolysis were also determined directly. From these values and those of the inhibition of respiration by selective inhibitors, a P/O ratio of 2.2 was calculated. About 75% of the total ATP consumption could be assigned to specific processes. The major ATP-consuming processes of tumour cells in an amino-acid-enriched medium, in which they are in an approximate steady state, are protein synthesis with about 30% of total ATP consumption, and Na+/K+-ATPase with about 20%, while RNA synthesis, ATP-dependent proteolysis and Ca2+-ATPase contribute about 10% each. In an amino-acid-free glucose medium, protein synthesis is reduced to a third, with a corresponding decrease of respiration, whereas the rate of the other ATP-consuming processes is unchanged.     

Cell properties after repeated transplantation of spontaneously and of SV40 virus transformed mouse cell lines. I. Growth in culture.

"Spontaneously" or SV40 virus transformed AL/N mouse cell lines were passed repeatedly through syngeneic mice. Cell lines were re-established in culture from minced pieces of tumors in the presence of concentrated fetal calf serum or from tumor cells dispersed by trypsin. The aim of this study was to compare the two cell lines in regard to the selection processes which operate during such procedures by characterization of the resulting cell lines. Measurements of growth in tissue culture on substratum showed no significant difference between any of the transformed cell lines. The SV40 transformed cells and its derivative cells had a low anchorage requirement for growth. The greatest anchorage requirement for growth was in the normal untransformed cells and in the derivative cells from the "spontaneously" transformed cells which were established from minced tumors. The spontaneously transformed cells and all derivative cells had high tumorigenicity (TD50 is less than 10-2). The SV40 transformed cells had no observable tumorigenicity (TD50 is greater than 10-8), except when injected into irradiated mice (TD50 = 1-5 X 10-5 in the immunocompetent mice, 5 X 10-4 in the irradiated mice). The SV40 transformed derivative cells maintained their SV40 specific T antigen and their susceptibility to lysis by specific antiserum.     

Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture.

1. Adenosine, a potent vasodilator, is transported very efficiently by pig aortic endothelium in monolayer culture (approx. 50pmol/min per 10(6) cells at 2 micrometer). Uptake proceeds by diffusion at high (millimolar) substrate concentrations, and by two discrete transport processes (Km approx. 3 micrometer and 250 micrometer) at lower concentrations. Over 90% of the adenosine taken up at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides (mainly ATP). 2. The high-affinity process is selectively inhibited by dipyridamole and by nitrobenzylthioinosine. Adenine preferentially inhibits the lower-affinity process, papapaverine inhibits both transport processes, and inosine has no significant effect. 3. Pig aortic smooth-muscle cells in culture show no high-affinity transport system for adenosine; uptake is much slower at low concentrations than that by endothelium (approx. 5pmol/min per 10(6) cells at 2 micrometer). Over 80% of the incorporated adenosine at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides. 4. The uptake of adenosine by smooth-muscle cells is powerfully inhibited by adenine, but dipyridamole is much less potent than in endothelium. 5. We conclude that endothelial cells are mainly responsible for the removal of circulating adenosine.     

Histochemical studies of the differentiation of microglial cells in the cerebral hemispheres of chick embryos and chicks

Using histochemical procedures to reveal the presence of nucleoside diphosphatase (NDPase), thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase), we investigated the appearance, distribution and ultrastructure of amoeboid and microglial cells in the cerebral hemispheres of chick embryos and young chicks, in order to elucidate the relationship between these two cell populations. On day 6 of incubation, a few round cells exhibiting NDPase, TPPase and AcPase activity were first detected in the thin mantle layer of the cerebral hemisphere. In the corpus striatum, these round cells increased rapidly in abundance until day 13 of incubation, after which their numbers gradually decreased, so that, on day 19 of incubation, they had entirely disappeared. Between day 10 and day 17 or 18 of incubation, round cells were located mainly in the zone of the mantle layer closest to the lumen. On day 10 of incubation, NDPase-, TPPase- and AcPase-positive cells that had a few short cytoplasmic processes (poorly ramified cells) were detected in the intermediate and basal zones of mantle layer. They increased in abundance until day 17 or 18 of incubation and thereafter rapidly decreased in number. Round and poorly ramified cells exhibited NDPase activity on their plasma membranes and in their cytoplasmic vacuoles, with TPPase and AcPase activity being localized within their vacuoles. On day 19 of incubation, NDPase- and TPPase-positive cells with long, well-ramified cytoplasmic processes (well-ramified cells) were observed in the corpus striatum, these being mainly localized in the basal zone. After hatching, these cells increased rapidly in abundance and were distributed throughout the corpus striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological and functional differentiation of cultured vascular smooth-muscle cells

Summary In numerous investigations using cultured smooth-muscle cells, investigators have consistently added 10–20% fetal calf serum (FCS) to the medium to maintain viable cells. In the present study we utilized an optical technique to investigate whether smooth-muscle cells, cultured with or without FCS, maintain their contractile activity in vitro. With such optical measurement, we were able to detect signals due to spontaneous contractions, in muscle cells cultured in FCS-free medium for up to 8 days, and, for the first time, were also able to observe the conduction of these cell contractions.The ultrastructural characteristics of cultured smooth-muscle cells during contractile activity, were also examined by electron microscopy. The cells were mature and well-differentiated, and were packed with numerous myofilaments. They had developed long cell processes, and were linked to one another by gap junctions.These observations indicated that the smooth-muscle cells, cultured without FCS for 7 to 8 days, were morphologically mature and maintained their contractile activity, whereas the cells cultured in FCS-containing medium showed no detectable signs of contractile activity.     

The effect of serum,EGF, PGF2α and insulin on S6 phosphorylation and the initiation of protein and DNA synthesis

To test the connection between S6 phosphorylation and the activation of protein and DNA synthesis, we compared the effects of serum, epidermal growth factor (EGF), prostaglandin F_2α (PGF_2α) and insulin (which is not mitogenic in these cells). Increasing concentrations of serum or EGF produced roughly parallel effects on all three processes, though the maximum response elicited by EGF (10^?9 M) was only a portion of that caused by saturating levels of serum (7.5% to 10%). PGF_2α (8.5 × 10^?7 M) alone acted similarly to EGF (10^?9 M) and with EGF produced a synergistic effect on all three processes. Insulin (10^?9 M) alone stimulated both S6 phosphorylation and protein synthesis to approximately the same level as EGF or PGF_2α, but had no effect on initiation of DNA synthesis. Thus neither stimulation of S6 phosphorylation nor activation of protein synthesis is sufficient for initiation of DNA synthesis. The requirement for S6 phosphorylation could not be dissociated from the activation of protein synthesis. Ribosomes containing the most highly phosphorylated forms of S6 appear to have a selective advantage in entering polysomes.     

Spontaneous neurite outgrowth and vasoactive intestinal peptide-Iike immunoreactivity of cultures of human paraganglioma cells from the glomus jugulare

Summary The chief cells of paraganglionic tissues have morphological and functional similarities to adrenal chromaffin cells, and both cell types are derived from the neural crest. In the present investigation cells from two glomus jugulare paragangliomas were studied in culture. Approximately 50% of the cells from one tumor, and 7% from the other spontaneously formed neurite-like processes. Numerous granular and agranular synaptic-like vesicles also appeared in the process-forming cells. In contrast to findings with normal and neoplastic adrenal chromaffin cells, addition of nerve growth factor (NGF) to the culture medium had no major effects on proportion of cells with processes. Dexamethasone caused only a small decrease in process length. Culturing of the tumors also appeared to promote production of material with VIP-like immunoreactivity. It is concluded that the phenotype of paraganglioma as well as pheochromocytoma cells may be altered in vitro. Responsiveness to specific factors such as NGF or steroids, however, may vary for related tumor cell types in different anatomic locations.     

Two forms of cerebellar glial cells interact differently with neurons in vitro

Specific interactions between neurons and glia dissociated from early postnatal mouse cerebellar tissue were studied in vitro by indirect immunocytochemical staining with antisera raised against purified glial filament protein, galactocerebroside, and the NILE glycoprotein. Two forms of cells were stained with antisera raised against purified glial filament protein. The first, characterized by a cell body 9 microns diam and processes 130-150 microns long, usually had two to three neurons associated with them and resembled Bergmann glia. The second had a slightly larger cell body with markedly shorter arms among which were nestled several dozen neuronal cells, and resembled astrocytes of the granular layer. Staining with monoclonal antisera raised against purified galactocerebroside revealed the presence of immature oligodendroglia in the cultures. These glial cells constituted approximately 2% of the total cell population in the cultures and, in contrast to astroglia, did not form specific contacts with neurons. Staining with two neuronal markers, antisera raised against purified NILE glycoprotein and tetanus toxin, revealed that most cells associated with presumed astroglia were small neurons (5-8 microns). After 1-2 d in culture, some stained neurons had very fine, short processes. Nearly all of the processes greater than 10-20 micron long were glial in origin. Electron microscopy also demonstrated the presence of two forms of astroglia in the cultures, each with a different organizing influence on cerebellar neurons. Most neurons associated with astroglia were granule neurons, although a few larger neurons sometimes associated with them. Time-lapse video microscopy revealed extensive cell migration (approximately 10 microns/h) along the arms of Bergmann-like astroglia. In contrast, cells did not migrate along the arms of astrocyte-like astroglia, but remained stationary at or near branch points. Growth cone activity, pulsating movements of cell perikarya, and ruffling of the membranes of glial and neuronal processes were also seen.