The effects of amino acids and ammonium on the growth of plant cells in suspension culture

A suspension culture of soybean (Glycine max L.) was grown on a defined medium in which the nitrogen sources were nitrate (25 mM) and ammonium (2 mM). The cells did not grow on nitrate unless the medium was supplemented with ammonium or glutamine. The l- and d-isomers of 12 amino acids tested singly could not replace ammonium. Most amino acids (4 mM) inhibited growth when the cells were cultured on nitrate and ammonium. Cells from five other plants (Reseda luteoli L.; Triticum monococcum L.; flax, Linum usitatissimum L.; horseradish, Amoracia lapathifolia Gilib; Haplopappus gracilis L.) grew on the defined medium with nitrate (25 mM) as the sole nitrogen source. Higher cell yields were obtained when ammonium (2 mM) or glutamine also was present. Supplementing the defined medium with high concentrations of ammonium (20 mM) inhibited growth of soybean, Haplopappus, and wheat cells. Addition of citrate (5 mM) relieved the inhibitory effects of ammonium in soybean and wheat cells but not in the Haplopappus cells.     

The effects of epidermal growth factor on neural crest cells in tissue culture

Epidermal growth factor (EGF) stimulates the release of hyaluronic acid (HA) and chondroitin sulfate proteoglycan (CSPG) from quail trunk neural crest cultures in a dose-dependent fashion. It also promotes the expression of cell-associated heparan sulfate proteoglycan (HSPG) as detected by immunofluorescence and immunoprecipitation of the 3H-labeled proteoglycan. Furthermore, EGF stimulates [3H]thymidine incorporation into total cell DNA. These results raise the possibility that EGF or an analogous growth factor is involved in regulation of neural crest cell morphogenesis.     

Comparative effects of chemotherapeutic agents on the growth and survival of human adrenal carcinoma cells in culture.

Adrenocortical carcinoma is an uncommon malignancy that is usually fatal within a short time after diagnosis. We have investigated the effects on the growth and survival of SW-13 human adrenal carcinoma cells in culture of some currently used and some potentially new agents in the treatment of adrenal cancer. Established drugs tested were mitotane, cisplatin, etoposide, 5-fluorouracil, and suramin. Other agents studied included adenine arabinofuranoside, cytosine arabinofuranoside, 2-methoxyestradiol, and paclitaxel. The most potent chemotherapeutic agents in this system were paclitaxel and 2-methoxyestradiol, with EC (50) of 1.8x10 (-8) and 3.3x10 (-7) M, respectively. Cytosine arabinofuranoside and cisplatin both had the same EC (50) of 7.0x10 (-7) M, and etoposide 1.1x10 (-6) M. All the other agents tested required much higher doses for effect, including mitotane, the current most commonly used chemotherapy for adrenal cancer, with an EC (50) of 3.3x10 (-4) M. These data suggest that paclitaxel, 2-methoxyestradiol, and cytosine arabinofuranoside should be further evaluated for their potential in the chemotherapy of adrenal carcinoma.     

The various effects of fractionated oxidized low density lipoproteins on the growth of smooth muscle cells in culture

The effects of fractionated oxidized low density lipoproteins (oxidized LDL) on the growth of vascular smooth muscle cells (VSMC) and their relationship to the formation of lysophosphatidylcholine (lyso-PC) as well as the activation of protein kinase C (PKC) were studied. VSMC were isolated from porcine aorta by explant culture. LDL was isolated from porcine blood by sequential ultracentrifugation and oxidized LDL was obtained by incubating LDL with 5 µM CuSO₄ at 37° C for various lengths of time. Our results showed that LDL oxidized for 12 h and eluted from fast protein liquid chromatography at 43 min inhibited the growth of VSMC, and that LDL oxidized for longer than 48 h and eluted at 48 min stimulated the growth of VSMC. The formation of lyso-PC in the oxidized LDL correlated well with its stimulatory effect, suggesting that lyso-PC is responsible for the mitogenic effect of oxidized LDL. This stimulatory effect of oxidized LDL was inhibited by staurosporine, a PKC inhibitor. Treatment with oxidized LDL increased the activity of membrane PKC, but it decreased that of cytosolic PKC, suggesting the translocation of PKC from cytosol to the membrane in the presence of oxidized LDL. These results suggested that the oxidized LDL-stimulated VSMC growth was mediated by the formation of lyso-PC and the activation of PKC.     

The effects of various hormones on the surface morphology of testicular cells in culture.

The purpose of this investigation was to study the effects of various hormones on the surface morphology of 20-day-old rat testicular cells in culture. Aggregates containing primarily Sertoli cells and germinal cells were obtained by enzymatic digestion. The surface morphology of the cells composing these aggregates was characterized under various culture conditions using light microscopy and scanning electron microscopy. The cytoplasmic processes of Sertoli cells became highly branched and filamentous after being cultured in the presence of rat, human or ovine FSH. Identical branching and filamentation was observed when Sertoli cells were cultured in rat TSH. Finally, numerous large blebs were observed on the surfaces of germinal cells cultured in the presence of insulin. These results suggest that the branching and filamentation of Sertoli cell cytoplasm observed after FSH stimulation are not specific for that hormone.     

The pH-dependence of sugar-transport and glycolysis in cultured Ehrlich ascites-tumour cells.

1. pH-dependence of glycolysis has generally been ascribed to the effects of pH on the activities of glycolytic enzymes. The present study shows that sugar transport is pH-dependent in cultured Ehrlich ascites-tumour cells. 2. The rates of glucose consumption, of 3-O-methylglucose transport, and of 2-deoxyglucose transport and phosphorylation increased as linear functions of pH, as the pH of the cell culture medium was increased from 6.1 to 8.5. Transport of glucose, as measured in ATP-depleted cells, was pH-dependent to the same extent as transport of the non-metabolizable sugars. 3. Glucose consumption rates were about 8-fold higher at pH 8.5 than at pH 6.4. About 65-85% of glucose was converted into lactate. Sugar transport rates were 2.5-fold higher at pH 8.5 than at pH 6.3. 4. pH affected both simple diffusion and facilitated diffusion. pH effect was mainly on the Vmax. of 2-deoxyglucose uptake, and on the rapid-uptake phase of 3-O-methylglucose transport. 5. It was estimated that about 70% of the pH effect on the rates of glucose consumption may be due to the effect on sugar transport and the remainder to the effect on the activities of glycolytic enzymes.     

Ultrastructural effects of nerve growth factor on PC 12 pheochromocytoma cells in spinner culture

PC12 pheochromocytoma cells treated with nerve growth factor (NGF) for two weeks in spinner cultures quickly begin to form processes after plating on an appropriate substrate, while cells freshly exposed to NGF in monolayer culture initiate neurite outgrowth only after a lag period of several days. The present ultrastructural studies indicate that PC 12 cells treated with NGF in spinner cultures do not form neurites, but do form short extensions comparable to those which have been reported within the first two days of exposure to NGF in monolayer cultures. These extensions contain organelles believed to be required for locomotion and for transport of cytoskeletal and membrane components and neurotransmitters. They also form bulbous distensions in which numerous chromaffin-type granules accumulate. These findings suggest that NGF may affect cells in spinner cultures by promoting development or activation of axonal transport mechanisms, and that the existence of these mechanisms may contribute to the neurite outgrowth which the cells exhibit when plated. NGF-treated PC 12 cells in spinner cultures do not accumulate the agranular synaptic-like vesicles, which are typically found in comparably treated monolayer cultures and which have been hypothesized to be sites of acetylcholine storage. These and other data demonstrate that attachment to a substrate can selectively modulate the responses of PC 12 cells to NGF.     

The effects of dopamine on prolactin mRNA levels in rat pituitary cells in culture.

Dopamine is known to be the prolactin-release inhibiting factor, but the effects of dopamine itself on regulation of prolactin messenger RNA have been little studied because of the instability of dopamine. We have compared the effects of dopamine and bromocriptine on the levels of prolactin mRNA and on the rates of synthesis, storage, and release of prolactin in primary cultured rat pituitary cells. The cells were incubated for 72 h with no secretagogue (control group) or in the presence of either dopamine (10 mumol/L) plus ascorbic acid (100 mumol/L) or bromocriptine (0.1 mumol/L). Prolactin mRNA was measured in cell extracts by means of slot blots, and newly synthesized prolactin was measured in similar incubations by the addition of [3H]leucine, followed by gel electrophoresis. The levels of total prolactin were measured by radioimmunoassay. Prolactin mRNA was reduced to 78 +/- 9% (mean +/- SEM) of control levels in bromocriptine-treated cells and to 59 +/- 7% in dopamine-treated cells, demonstrating that dopamine stabilized by ascorbic acid was able to reduce the levels of prolactin mRNA in rat pituitary cells in culture. Dopamine may act at sites in addition to the dopaminergic D2 receptor, since the level of prolactin mRNA was reduced more by a supramaximal dose of dopamine than by a supramaximal dose of bromocriptine. The results of the [3H]prolactin and prolactin measurements suggested that availability of mRNA was not a major factor in controlling the rate of prolactin synthesis.     

The effects of a macrophage-derived cytotoxin on the growth and metabolism of target cells.

Peritoneal macrophages obtained from Sarcoma I (SaI)-immune CS7BL/6 mice release a heat-labile cytotoxin, specific macrophage cytotoxin (SMC), following a two hour interaction with appropriate target cells. Specific macrophage cytotoxin specifically inhibits A/Jax spleen cells from mitogenically responding to concanavalin A, whereas syngeneic CS7BL/6 spleen cells are unaffected. Treatment of target cells with SMC results in early alterations in RNA and DNA metabolism. The uptake and incorporation of ³H-uridine was found to be initially elevated while intracellular levels of ³H-thymidine became markedly reduced. Furthermore, the cytotoxic action of SMC was found to be rapidly accelerated and amplified by low levels of actinomycin-D.     

Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture

Primary cultures of neuronal and glial cells from 1-day-old neonatal rats contain high affinity receptors for insulin-like growth factor I (IGF-I). The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 3 nM for neuronal cells and 4 nM for glial cells. Unlabeled insulin was 20-50 times less potent. Apparent molecular mass of the alpha subunits of the IGF-I receptor was 125 kDa in neuronal and 135 kDa in glial cells. IGF-I induced autophosphorylation of the IGF-I receptor beta subunit in lectin-purified membrane preparations in a dose-dependent manner. The major phosphoamino acid of the beta subunit in both cell types was tyrosine in the IGF-I-stimulated state and serine in the basal state. Apparent molecular mass of the beta subunits of the IGF-I receptors was 91 kDa for neuronal and 95 kDa for glial cells. Tyrosine kinase activity of the IGF-I receptors was demonstrated by IGF-I-induced phosphorylation of the exogenous substrate poly(Glu, Tyr) 4:1 in both cell types. IGF-I had no effect on 2-deoxyglucose uptake in neuronal cells. In contrast, in glial cells, IGF-I stimulated 2-deoxyglucose uptake at very high doses, presumably acting via the insulin receptor. The effect of IGF-I as a neurotrophic growth factor in both neuronal and glial cells was demonstrated by its stimulation of [3H]thymidine incorporation. These findings suggest the IGF-I is an important growth factor in nervous tissue-derived cells.     

The study of rheological effects on vascular endothelial cells in culture

A number of cell culture studies have been reported on the influence of shear stress on vascular endothelial cells. Although through such studies much has been learned about the effect of an endothelial cell's hydrodynamic environment on its structure and function, the reports indicate significant differences in methodology. Using cell shape as an indicator of differences that might result from differing methodologies, an investigation of the influence of selected variables has been carried out. The results presented indicate that not only are such variables as the level of shear stress and the duration of exposure important, but also substrate, media composition, characteristics of the cell itself, and the nature of the flow, e.g. whether it is steady state or pulsatile.