The effect of biotin on cellular functions in HeLa cells

HeLa cells cultured in a biotin-deficient medium show reduced rate of protein synthesis, DNA synthesis and growth. Addition of exogenous biotin to the cells cultured in biotin-deficient medium results in enhanced protein synthesis, DNA synthesis and cell growth. Continuous protein synthesis is required for the increase in DNA synthesis observed upon the addition of exogenous biotin to the cells cultured in biotin-deficient medium. These results suggest that cells cultured in biotin-deficient medium are arrested in the G1 stage of cell cycle and this block is removed upon the addition of biotin to the deficient medium.     

The direct stimulating action of thyroliberin on the biosynthesis of total protein in cultured liver cells of rat fetuses]

In experiments using fetal rat liver cultured cells TRH was shown to stimulate total protein synthesis but not RNA synthesis during long incubation. Somatostatin affected neither protein synthesis nor RNA synthesis in cultured liver cells. Possible physiological role of peripheral TRH during perinatal period in the rat is discussed.     

高糖高胰岛素对去甲肾上腺素诱导的心肌细胞肥大的影响

目的:利用体外培养的乳鼠心肌细胞,观测高浓度胰岛素和高浓度葡萄糖作用下对去甲肾上腺素诱导的心肌细胞肥大的影响,并探讨其可能作用机制.方法:以培养的乳鼠心肌细胞为模型分组给药后,用显微镜目镜计数心肌细胞搏动的频率;用Lowrys法测心肌细胞的蛋白质含量;用消化分离法,利用计算机图象分析系统测心肌细胞的体积;用[3H]leucine标记法测定心肌细胞蛋白的合成.结果:与对照组相比,去甲肾上腺素(NE)组、高糖组、高胰岛素组心肌细胞蛋白含量、体积、蛋白合成均有明显增加,而与高糖加NE组和高糖高胰岛素组相比较,高糖高胰岛素加NE组心肌细胞蛋白含量、体积、蛋白合成增加则更为显著.结论:单纯高浓度胰岛素培养可促进心肌细胞肥大,同时用高糖高胰岛素联合培养,可使去甲肾上腺素诱导的心肌细胞肥大作用进一步增强.     

The mammalian iris-ciliary complex affects organization and synthesis of cytoskeletal proteins of organ and tissue cultured lens epithelial cells.

A water soluble growth inhibitor was isolated from the mammalian ocular iris-ciliary complex. The molecular weight of this protein is 10 kD or lower as determined by ultrafiltration fractionation. The iris-ciliary (IC) complex water soluble protein(s) significantly inhibits synthesis of lower molecular weight proteins of the epithelial cells of the organ cultured mammalian ocular lens. It was also found that this inhibitory effect of IC is mediated via the structural organization of the lens. Monolayer cultures of the lens epithelial cells exposed to IC did not manifest any inhibition of their protein synthesis. Moreover, these tissue cultured lens epithelial (TCLE) cells showed a significant increase in their protein synthetic activities in response to the presence of IC factors in the culture medium. It is postulated that the IC activity is modulated via either the lens capsule, an extracellular matrix, or due to the specific organization of the intact lens. The specific effects of IC on the cytoskeletal organization and synthesis in the organ cultured lens epithelial (OCLE) and TCLE cells were also examined. Both groups, treated with IC factors, manifested significant alterations in their protein synthetic activities and cytoskeletal architecture. The 3H-leucine incorporation experiments showed that alpha-actin and alpha-tubulin synthesis is partially inhibited by IC factors in OCLE cells but vimentin synthesis is not, whereas in TCLE cells all of them showed increased synthesis in response to IC factors. Turnover rates of these proteins in both OCLE and TCLE cells were also computed. The immunofluorescence and microscopic evaluation of OCLE and TCLE cells exposed to IC factors illustrated significant alteration in the cytoarchitecture of the filaments. We demonstrate that an inhibitor(s) molecule of 10 kD or lower size isolated from IC inhibited protein synthesis of OCLE cells and stimulated protein synthesis in TCLE cells. The IC factor also affects the synthesis and organization of cytoskeletal filaments of both the OCLE and TCLE cells.     

Decreased heme content and cessation of cell growth in cultured chick embryo fibroblasts in the presence of horse serum: Stimulation of heme synthesis and cell growth by iron

A new spectrofluorometric method for heme quantitation in cultured fibroblasts is described. The method includes: (1) heme extraction by methanol/sulfuric acid, (2) partial purification of heme by a microchromatographic method, and (3) treatment of the purified heme by oxalic acid followed by fluorometric quantitation. Using this method, heme concentration was determined in chick embryo fibroblasts cultured in a medium supplemented with either 7% fetal bovine serum (FBS) or 10% horse serum (HS). In the presence of FBS, cultured cells actively divided and cells contained 34–55 pmol heme/mg protein. In contrast, cultures maintained in HS proliferated at a slower rate and contained 23–25 pmol heme/mg protein. The addition of 40 μM FeSO₄ to cultures maintained in the presence of HS stimulated cell proliferation, and the cellular heme concentration increased to 37–51 pmol/ mg protein. These findings suggest that the cessation of growth in the presence of HS may be due to decreased heme content in the cells and that the stimulation of cell growth by iron is mediated by its stimulation of heme synthesis.     

Follicle cell regulation of protein synthesis and developmental competence in sheep oocytes

The developmental capacity of sheep oocytes cultured outside the follicle was greatly increased by the presence of high concentrations of gonadotrophins (10 micrograms/ml) in the medium. However, even under these conditions, the developmental capacity of the oocytes was only half that of oocytes cultured within the intact follicle. The presence of the cumulus was essential for development; nearly all denuded oocytes failed to undergo cleavage. Maturational changes in the oocyte involving increased amino acid uptake increased incorporation and specific changes in protein synthesis were inhibited by the follicle cells; this suppression was alleviated by gonadotrophic hormones. The cumulus cells suppressed amino acid incorporation and, to some extent, the changes in protein synthesis. However, the suppression of amino acid uptake required the presence of the whole follicle. Patterns of protein synthesis by oocytes cultured outside the follicle differed from those in oocytes cultured within the follicle, irrespective of the presence of the cumulus or gonadotrophins. Analysis of single oocytes cultured outside the follicle showed that the protein profiles varied markedly even under identical culture conditions.     

Calcium-mediated regulation of the low density lipoprotein receptor and intracellular cholesterol synthesis in human epidermal keratinocytes

Unlike cells cultured under physiological Ca2+ concentrations (1-2 mM), keratinocytes cultured in media containing Ca2+ in low concentrations (less than 0.1 mM) do not stratify. The latter cells also differ with respect to several features of the regulation of cholesterol synthesis. In keratinocytes cultured in medium containing high Ca2+ concentrations (1.6 mM) and fetal calf serum, the rate of cholesterol synthesis was 20-30 times higher than in keratinocytes exposed to a low Ca2+ concentration. The rate of cholesterol synthesis did not change when high-calcium cells were deprived of extracellular sources of cholesterol but increased (8-10 fold) in deprived low-calcium cells. Furthermore, the addition of low density lipoprotein (LDL) reduced cholesterol synthesis markedly in low-calcium cells but had no effect on high-calcium cells. Finally, in keratinocytes cultured at low calcium concentrations the association and degradation of 125I-LDL was 20-30 times higher than in keratinocytes cultured under high-calcium conditions. Switching of the cells from the low-calcium to the high-calcium medium resulted in the induction of terminal differentiation within 15 hours and was accompanied by increased cholesterol and protein synthesis, increased competence of cells to form cornified envelopes, and reduced association of 125I-LDL. A gradual increase of the extracellular Ca2+ concentration was accompanied by a corresponding increase of cholesterol and protein synthesis and a decrease of the response of intracellular cholesterol synthesis to changes in the extracellular concentrations of lipoprotein. Various morphological techniques showed virtually no binding and internalization of LDL by keratinocytes cultured at the high-calcium level, whereas both were observed at the low-calcium level. Once internalized, the LDL was delivered to dense bodies representing lysosomes. It is concluded that in human epidermal keratinocytes, the expression of the LDL receptor and the endogenous synthesis of cholesterol are regulated by the conditions determined by the differentiation stage of the cells.     

In vitro induction of luteinizing hormone synthesis by estrogen in organ-cultured pituitary glands of the Japanese eel,Anguilla japonica

An organ culture method for pituitary glands isolated from immature Japanese eels (Anguilla japonica) was developed. This method could conserve the histological features of the pituitary glands for at least 21 days. The ability to synthesize gonadotropic hormone (GTH) in cultured eel pituitary glands was examined by detecting luteinizing hormone (LH) beta protein immunohistochemically. In a basal medium (Leibovitz L-15), LH beta-immunoreactive cells were very scarce, but after addition of estradiol-17beta (E2) a large number of immunoreactive cells appeared, particularly in the proximal pars distalis. The stimulatory effects of E2 on LH beta synthesis were dose (1-100 ng/ml)- and time (1.5-7 days)-dependent. Thus, in contrast with previous reports of the lack of a direct effect of E2 on GTH synthesis in primary cultured eel pituitary cells, the present results clearly indicate that E2 can stimulate GTH synthesis in immature eel pituitary glands. This organ culture method is useful to examine the actions of steroids and also other endocrine factors on the eel pituitary gland.     

Stimulation of guanylate cyclase and RNA polymerase II activities in HeLa cells and fibroblasts by biotin

HeLa cells cultured in a biotin-deficient medium showed reduced rates of protein synthesis, DNA synthesis and growth. Continuous synthesis is required for the increase in DNA synthesis observed upon addition of biotin to cells cultured in biotin-deficient medium. The addition of biotin to the biotin-deficient culture medium increased the activity of guanylate cyclase in both HeLa cells and fibroblasts. Both cell types cultured in biotin deficient medium showed reduced activity of RNA Polymerase II. The exogenous addition of biotin to the biotin-deficient cell cultures also resulted in increased activity of RNA Polymerase II in HeLa cells and fibroblasts. The maximal response was observed in 4 hours. Significant increase in enzyme activity was observed at 10^–8 M biotin in the culture medium. The growth promoting effect of biotin seems to involve stimulations of cellular guanylate cyclase and RNA Polymerase II activity.     

A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.     

The role of calcium ions for the expression of ricin toxicity in cultured macrophages

Ricin toxin, which consists of two distinct polypeptide moieties, A and B chains, is cytotoxic to the cultured macrophage cell line, J774A.1. Ricin is a protein synthesis inhibitor, and incubating macrophages for 4 hours with ricin (1 pM to 10 nM) in standard medium containing calcium and magnesium inhibited ³H-leucine incorporation into protein (97%, at 1 nM ricin). However, in Ca²⁺-free medium, protein synthesis was inhibited only 19%. EGTA pretreatment (to deplete intracellular calcium) also partly protected cells from protein synthesis inhibition, in spite of added calcium (2 mM) in the incubation medium. Decreased toxicity in the absence of extracellular calcium resulted from decreased toxin binding. Adding or deleting Mg²⁺ did not affect protein synthesis or binding of ¹²⁵I-ricin in cultured macrophages. We conclude that calcium is required for ricin to exert its inhibitory effect on protein synthesis in cultured macrophages.     

Quantitative Analysis of Protein Synthesis Altered by Estrogen in Cultured Xenopus Liver Parenchymal Cell

Using the primary culture system of male Xenopus laevis hepatocytes consisting of more than 95% parenchymal cells, the effect of estradiol-17 β (10⁻⁶M) on protein synthesis was quantitatively analyzed by ³H-leucine incorporation kinetics and the estimation of specific radioactivity of newly synthesized secretory protein. The cells in a well defined culture revealed high plating efficiency and very low DNA synthetic activity. The cultured cells could synthesize several secretory proteins containing serum albumin. The pattern of secreted proteins in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) did not alter with culture time but secretion rate of protein increased for 7 days, starting on the third day following inoculation. Estradiol added to the culture media extensively induced the synthesis of yolk precursor protein vitellogenin which accounted for 40–50% of the overall secretory protein synthesis and 20–30% of the total protein synthesis on day 7 of estradiol treatment. Ultimately, the total protein synthesis and secretory protein synthesis were stimulated 1.2–1.3 fold and 2.0–2.2 fold, respectively, over those of the control cells cultured in the absence of estradiol. These results indicated that the stimulation of protein synthesis was largely due to vitellogenin production. Such an estradiol-dependent stimulation of protein synthesis was also detected in the low molecular weight protein(s). On the other hand, albumin synthesis was evidently reduced by estradiol. Thus, estradiol had two different effects on protein synthesis.
The results obtained in this study will be discussed in relation to the findings o in vivo experiments.     

Rac1 links integrin-mediated adhesion to the control of lactational differentiation in mammary epithelia

The expression of tissue-specific genes during mammary gland differentiation relies on the coincidence of two distinct signaling events: the continued engagement of beta1 integrins with the extracellular matrix (ECM) and a hormonal stimulus from prolactin (Prl). How the integrin and Prl receptor (PrlR) systems integrate to regulate milk protein gene synthesis is unknown. In this study, we identify Rac1 as a key link. Dominant-negative Rac1 prevents Prl-induced synthesis of the milk protein beta-casein in primary mammary epithelial cells cultured as three-dimensional acini on basement membrane. Conversely, activated Rac1 rescues the defective beta-casein synthesis that occurs under conditions not normally permissive for mammary differentiation, either in beta1 integrin-null cells or in wild-type cells cultured on collagen. Rac1 is required downstream of integrins for activation of the PrlR/Stat5 signaling cascade. Cdc42 is also necessary for milk protein synthesis but functions via a distinct mechanism to Rac1. This study identifies the integration of signals provided by ECM and hormones as a novel role for Rho family guanosine triphosphatases.     

Involucrin synthesis is correlated with cell size in human epidermal cultures

Late in terminal differentiation, human epidermal keratinocytes form an insoluble protein envelope on the cytoplasmic side of the plasma membrane. Involucrin, a soluble protein precursor of the envelope, is synthesized at an earlier stage of differentiation, both in the natural epithelium and in cultured keratinocytes. Because keratinocytes are known to enlarge during differentiation, we looked for a correlation between involucrin synthesis and cell size, using antiserum raised against the purified protein. We found that virtually no cultured epidermal keratinocytes with a diameter less than or equal to 14 micrometer contained involucrin, but most cells greater than 17 micrometer did. Using density gradient centrifugation, we were able to isolate a population of small cells containing almost no involucrin, as judged by immunodiffusion, PAGE, and immunoprecipitation. Large cells possessed translatable mRNA for involucrin, whereas small cells did not. We conclude that when cultured keratinocytes reach a certain size (approximately 14 micrometer in diameter) the specific mRNA for involucrin begins to accumulate and synthesis of the protein begins.     

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.     

Platelet-derived growth factor AA homodimer stimulates protein synthesis rather than DNA synthesis in vascular smooth muscle cells from spontaneously hypertensive rats but not from normotensive rats.

Platelet-derived growth factor (PDGF) AB and BB isoforms were potent mitogens for cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). PDGF-AA promotes protein synthesis in a dose-dependent manner in SHR cells, whereas DNA synthesis was stimulated only slightly. However, this isoform did not activate either DNA or protein synthesis in WKY cells. PDGF-AA stimulated tyrosine phosphorylation of its receptor protein and phospholipase C-gamma 1 in SHR cell but not in WKY cells. These results indicate that vascular smooth muscle cell of SHR is uniquely responsive to PDGF-AA, presumably due to abnormality in receptor expression, in its hypertrophic response.     

Actions of insulin and vitamin A on Sertoli cells.

The synthesis of androgen-binding protein by cultured Sertoli cells is increased by insulin, retinol, follitropin and testosterone. Only follitropin will stimulate an increase in cyclic AMP in these cells, yet each agent individually increased the synthesis of androgen-binding protein in short-term cultures. For long-term culture of Sertoli cells follitropin, testosterone, insulin, and retinol appeared to act synergistically to prolong the ability of the cells to secrete androgen-binding protein. These are the first reported results which suggest an action of insulin and retinol directly on Sertoli cells even though the importance of both factors in male reproduction is known.     

Thermotolerance in cultured hepatoma cells: cell viability, cell morphology, protein synthesis, and heat-shock proteins

Heat treatment at 42 degrees C of cultured Reuber H35 rat hepatoma cells induced both a rapid decrease of the rate of protein synthesis and the rounding up of the cells. Reincubation at 37 degrees C resulted in a gradual flattening of the cells, resumption of protein synthesis, and the synthesis of heat-shock proteins. During the recovery period cells developed a resistance toward a treatment which otherwise should lead to heat-induced cell death. Thermotolerance measured in terms of cell survival was paralleled by thermal resistance of protein synthesis and the cellular ability to refrain from rounding up under heat stress.