Stimulation of heparan sulfate proteoglycan synthesis and secretion during G1 phase induced by growth factors and PMA

Fetal calf serum (FCS) and PMA (phorbol 12-myristate-13-acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n-butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8-bromo adenosine 3′:5′-cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([³H]-thymidine and Br-dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre-treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G₁ phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G₁ phase. J. Cell. Biochem. 70:563–572, 1998. © 1998 Wiley-Liss, Inc.     

Role of nuclear proteins on [3H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Insulin-like Growth Factor I Regulation of Swarm Rat Chondrosarcoma Chondrocytes in Culture

Insulin-like growth factor I (IGF-I) is anabolic for chondrocytes and is thought to be important in regulating such normal cartilaginous tissues as the epiphyseal growth plate. In the present studies, we have investigated the role of IGF-I in the regulation of neoplastic cartilage. Chondrocytes cultured from a transplantable rat chondrosarcoma were analyzed for responsiveness to IGF-I with respect to DNA and glycosaminoglycan synthesis as determined by labeling with radioactive thymidine and sulfate, respectively. Stimulation of [³H]thymidine and [³⁵S]sulfate incorporation by IGF-I was two to four times that in serum-free controls, with half-maximal stimulation at 1 × 10^-9M. The efficacy of IGF-I was approximately one-half of that of serum in stimulating [³H]thymidine incorporation and was comparable to that of serum for [³⁵S]sulfate incorporation. When Swarm rat chondrosarcoma chondrocytes were cultured in the presence of IGF-I and exposed to graded concentrations of anti-IGF-I antibody, [³H]thymidine incorporation and [³⁵S]sulfate incorporation were attenuated in a dose-dependent fashion to 29 and 25% of antibody-free controls, respectively. Nonspecific antibody not raised against IGF-I was not inhibitory. These observations suggest that the majority of IGF-I action on these cells is susceptible to immunoinhibition. To estimate the contribution of IGF-I to the regulation of these cells by serum, Swarm rat chondrosarcoma chondrocytes were cultured with graded concentrations of either calf serum or fetal calf serum in the presence of anti-IGF-I antibody, nonspecific antibody, or no other additives. Specific antibody attenuated the effect of calf serum on both [³H]thymidine and [³⁵S]sulfate incorporation with overall inhibition of 52% (P < 0.01) and 48% (P < 0.001), respectively. Nonspecific antibody superimposed small, variably stimulatory or inhibitory effects on those of calf serum. When chondrosarcoma chondrocytes were incubated with fetal calf serum, anti-IGF-I antibody exerted a minimal inhibitory effect, reducing both [³H]thymidine and [³⁵S]sulfate incorporation by less than 25%. The immunoinhibition of both pre- and postnatal serum could be overcome in a dose-dependent fashion by increasing serum concentrations. These results suggest that the factors influencing Swarm rat chondrosarcoma chondrocytes may be developmentally regulated and that the contribution of IGF-I to the action of serum increases between fetal and postnatal life. These data support the hypothesis that chondrosarcoma is a somatomedin-responsive neoplasm and suggest that this tumor may be susceptible to interventions directed toward mechanisms that block insulin-like growth factor action.     

Stimulation of bone formation by prostaglandin E2

We examined the effect of prostaglandin E₂ (PGE₂), in the presence or absence of cortisol, on bone formation in 21-day fetal rat calvaria maintained in organ culture for 24 to 96 h. [³H]Thymidine and [³H] proline incorporation were used to assess DNA and collagen synthesis, respectively. Changes in dry weight and DNA content were assessed after 96 h.PGE₂ (10⁻⁷ M) stimulated both DNA and collagen synthesis in calvaria. The effect on DNA synthesis was early (24 h), transient and limited to the periosteum. Collagen synthesis was stimulated at a later time (96 h), predominantly in the central bone. Cortisol (10⁻⁷ M) inhibited DNA and collagen synthesis. The addition of PGE₂ reversed the inhibitory effects of cortisol on DNA synthesis and content and increased collage synthesis in central bone to levels above control untreated cultures.We conclude that PGE₂ has stimulatory effects on bone formation and can reverse the inhibitory effects of cortisol. Hence the effects of cortisol may be mediated in part by their ability to reduce the endogenous production of prostaglandins.     

Incorporation of exogenous ganglioside GM1 into neuroblastoma membranes: Inhibition by calcium ion and dependence upon membrane protein

Since exogenous gangliosides are known to promote neuritogenesis, the incorporation of exogenous GM1 into neuroblastoma membranes was examined. Neuro-2A cells, synchronized in the G1/G0 phase, were suspended in HEPES buffered saline containing 10^–4 M [³H]GM1, and membrane incorporation was measured as radioactivity remaining with the cell pellet following incubation with serum-containing medium and trypsin. Calcium ion (0.01 to 10 mM) reduced incorporation of exogenous GM1, due to its interaction with GM1 micelles in solution. When cells were treated with proteases prior to incubation with GM1, the inhibitory effect of Ca²⁺ was lost and total incorporation into membranes was lowered by approximately one order of magnitude. Pretreatment of cells with 0.05% trypsin resulted in an inhibition of GM1 incorporation within 5 minutes. When trypsinized cells were resuspended in complete growth medium, the cells recovered the ability to incorporate GM1 with time, and this paralleled labeling of cellular protein with [³H]leucine. The role of membrane protein in the incorporation of exogenous GM1 could not be explained by the lytic release of cytosolic transfer proteins nor the artifactual coating of the cell surface by serum proteins. These results suggest that the incorporation of exogenous gangliosides into cellular membrane lipid bilayers cannot be fully explained by considerations of lipophilicity alone, and leads us to propose that initial recognition by membrane protein(s) is necessary.Abbreviations used GM1 H³NeuAc-GgOse₄Cer - HBS HEPES buffered saline - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum     

Prostaglandins E1 and E2 stimulate the proliferation of pulmonary artery smooth muscle cells

Prostaglandins E₁ and E₂ are thought to be inhibitors of the growth of systemic vascular smooth muscle cells (SMC). However, their effect on the proliferation of SMC from the pulmonary artery (PA) has not been described and was the subject of this investigation. Cultures of bovine PA SMC were exposed to PGE₁ and PGE₂ under various conditions and their growth was assessed. PGE₁ and PGE₂ did not inhibit the growth of PA SMC in 10% fetal calf serum (FCS), but instead caused a dose dependent (10 ng - 1 μg/ml) increase in [³H]-thymidine incorporation when added to cultures containing 0.5% FCS; the highest doses resulted in 95% and 75% increases in [³H]-thymidine uptake at 24 hours with PGE₁ and PGE₂ respectively. This was accompanied by a modest increase in actual cell numbers (e.g., 20% with 1 μg/ml PGE₁). Furthermore, PGE₁ could mimic insulin-like growth factor (IGF-1) by potentiating the stimulation of SMC growth by fibroblast growth factor, suggesting that PGE₁ may act as a progression factor in the growth cycle of these cells. There was, however, no effect of PGE₁ on the proliferation of bovine aortic SMC. We conclude that, contrary to most reported effects on systemic SMC, PGE₁ and PGE₂ do not inhibit the proliferation of PA SMC but rather stimulate it.     

Additive effect of oestradiol-17 beta and serum on synthesis of deoxyribonucleic acid in guinea-pig endometrial cells in culture.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Transforming growth factor β1 induces mitogenesis in fetal rat brown adipocytes

The presence of transforming growth factor β₁ (TGF-β₁) for 24 or 48 h stimulated DNA synthesis, the percentage of cells in the S + G₂/M phases of the cell cycle, and cell number, as compared to quiescent cells. The mitogenic capacity of TGF-β₁ (1 pM) was similar to that shown by 10% fetal calf serum (FCS). TGF-β₁ for 48 h increased by 5-fold the percentage of cells containing (³H)thymidine-labeled nuclei as compared to quiescent cells. In addition, single fetal brown adipocytes, showing their typical multilocular fat droplets phenotype, become positive for (³H)thymidine-labeled nuclei in response to TGF-β₁. Moreover, TGF-β₁ induced the mRNA expression of a complete set of proliferation-related genes, such as c-fos (30 min), c-myc and β-actin (2 h), and H-ras, cdc2 kinase, and glucose 6-phosphate dehydrogenase (G6PD) at 4 and 8 h, as compared to quiescent cells. Concurrently, TGF-β₁ for 12 h increased the protein content of proliferating cellular nuclear antigen (PCNA) by 6-fold and p21-ras by 2-fold. Although our results demonstrate that TGF-β₁ induces the expression of very early genes related to cell proliferation, TGF-β₁ could be acting either as a mitogen or as a survival factor to induce proliferation in fetal brown adipocytes. © 1996 Wiley-Liss, Inc.     

DNA synthesis in cultured human fibroblasts: Regulation by 3′ : 5′-cyclic amp

The addition of serum to density-inhibited human fibroblast cultures induced a wave of DNA synthesis, measured as [³H] thymidine incorporation into acid-precipitable material, beginning after 8–12 hr and reaching maximum levels at 16–24 hr. Addition of dibutyryl-3′ : 5′-cyclic AMP (DBcAMP) together with serum inhibited [³H] thymidine incorporation by 75–95%. When DBcAMP was added for the first 4 hr of serum stimulation and then removed, the wave of DNA synthesis was not delayed. This suggested that serum could induce DNA synthesis even though cyclic AMP concentrations were maintained at high levels by DBcAMP during this initial period. These results are inconsistent with the hypothesis that it is the immediate transient reduction in 3′ : 5′-cyclic AMP concentration following the addition of serum that triggers DNA synthesis. By contrast, DBcAMP added 8 hr after serum inhibited [³H] thymidine incorporation to the same extent as DBcAMP added at the same time as serum. This indicated that a step essential for DNA synthesis and occurring late in G₁ was inhibited by high concentrations of 3′ : 5′-cyclic AMP.     

An Age-Wise Comparison of Human Airway Smooth Muscle Proliferative Capacity

We compared the proliferation of neonatal and adult airway smooth muscle cells (ASMC) with no/moderate lung disease, in glucose- (energy production by glycolysis) or glucose-free medium (ATP production from mitochondrial oxidative phosphorylations only), in response to 10% fetal calf serum (FCS) and PDGF-AA. In the presence of glucose, cell counts were significantly greater in neonatal vs. adult ASMC. Similarly, neonatal ASMC DNA synthesis in 10% FCS and PDGF-AA, and [Ca²⁺]i responses in the presence of histamine were significantly enhanced vs. adults. In glucose-free medium, cell proliferation was preserved in neonatal cells, unlike in adult cells, with concomitant increased porin (an indicator of mitochondrial activity) protein expression. Compared to adults, stimulated neonatal human ASMC are in a rapid and robust proliferative phase and have the capacity to respond disproportionately under abnormal environmental conditions, through increased mitochondrial biogenesis and altered calcium homeostasis.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Arachidonic acid metabolism in growth control of A549 human lung adenocarcinoma cells

The role of individual eicosanoids of the arachidonic acid (AA) cascade in the growth control of A549 human lung adenocarcinoma cells has been studied. Cyclooxygenase and lipoxygenase metabolites of [¹⁴C]AA incorporated were actively synthesized in the cultures of tumor cells with full confluence unaccomplished. In such cultures inhibitors of AA metabolism (indomethacin and esculetin) and also a lipoxygenase metabolite of AA, 15-hydroxyeicosatetraenoic acid (15-HETE), significantly suppressed the incorporation of [³H]thymidine and biosynthesis of prostaglandin E₂(PGE₂). Other lipoxygenase metabolites of AA (5-HETE and 12-HETE) had no effect on these parameters. The basic fibroblast growth factor (bFGF) had practically no affect on the growth of A549 cells and the PGE₂ production in cultures with 5% fetal calf serum (FCS); however, in the presence of 0.5% FCS this factor significantly increased the number of tumor cells. The growth-stimulating effect of bFGF was completely abolished by a cyclooxygenase inhibitor indomethacin. The data suggest a key role of PGE₂ in the growth control of A549 cells with an active synthesis of cyclooxygenase and lipoxygenase metabolites of AA, its importance in realization of the mitogenic effect of bFGF, and specific features of 15-HETE as a down-regulator of the PGE₂-dependent proliferation.     

[3H]Dopamine Labeling of D3 Dopaminergic Sites in Human, Rat, and Calf Brain

Abstract: The binding of [³H]dopamine to brain regions of calf, rat, and human was investigated. The calf caudate contained the highest density of [³H]dopamine binding sites, with a B_max value of 185 fmol/mg protein, whereas rat and human striatum contained one-third this number of sites. The K_D values for [³H]dopamine in all tissues were 2–3 nM. Dopaminergic catecholamines (dopamine, apomorphine, 6,7-dihydroxy-2-aminotetralin, and N-propylnorapomorphine) inhibited the binding of [³H]dopamine in all three species, at low concentrations, with IC₅₀ values of 1.5 to 6 nM. Neuroleptics, in contrast, inhibited the binding at high concentrations (with IC₅₀ values of 200 to 40,000 nM). The [³H]dopamine binding sites were saturable, heat-labile, and detectable only in dopamine-rich brain regions; these sites differed from D₂ dopamine sites (labeled by [³H]butyrophenone neuroleptics), and from D_l dopamine sites (labeled by [³H]thioxanthene neuroleptics) associated with the dopamine-stimulated adenylate cyclase. We have, therefore, called these high-affinity [³H]dopamine binding sites D₃ sites. [³H]Apomorphine and [³H]ADTN also appeared to label D₃ sites. These ligands however, were less selective than [³H]dopamine, and labeled sites other than D₃ as well. Assay conditions were important in determining the parameters of [³H]dopamine binding. The optimum conditions for selective labeling of the D₃ dopaminergic sites, using [³H]dopamine, required the presence of EDTA and ascorbate.     

Induced changes in the rates of uridine- 3 H uptake and incorporation during the G 1 and S periods of synchronized Chinese hamster cells

The rates of uridine-5-³H incorporation into RNA and the rates of uridine uptake into the acid-soluble pool during the cell cycle of V79 Chinese hamster cells were examined. Cells cultured on Eagle''s minimal essential medium supplemented with fetal calf serum, lactalbumin hydrolysate, glutamine, and trypsin displayed rates of incorporation and uptake which increased only slightly during G₁ and accelerated sharply as DNA synthesis commenced. In contrast, cells cultured on minimal essential medium supplemented only with calf serum exhibited rates of incorporation and uptake which increased linearly through both G₁ and S. The transition from one pattern to the other can be induced within 24 hr and is completely reversible. The nonlinear pattern exhibited by cells grown on the supplemented fetal calf serum medium can also be overcome with high exogenous uridine concentrations. In the presence of 200 µM uridine, these cells display a linear pattern of increase in rates of uridine incorporation and uptake. It is concluded that at lower uridine concentrations the pattern of increase in the rate of uridine incorporation into RNA during the cell cycle for a given population of cells is dependent upon the rate of uridine entry into the cell, and that this pattern is not rigidly determined but can be modified by culture conditions.     

[3H]Propyl β-Carboline-3-Carboxylate as a Selective Radioligand for the BZ1 Benzodiazepine Receptor Subclass

Abstract: Ethyl β-carboline-β-carboxylate (β-CCE) is a mixed-type inhibitor of [³H]flunitrazepam ([³H]FNM) binding to benzodiazepine receptors in noncerebellar regions of rat brain. These findings may represent the presence of either receptor multiplicity or negative cooperativity among benzodiazepine receptors. [³H]Propyl β-carboline-3-carboxylate ([³H]PrCC) has previously been shown to bind specifically to benzodiazepine receptors of rat cerebellum. In the present study we found no indication of the presence of true negative cooperativity among benzodiazepine receptors when [³H]PrCC was used as radioligand. However, we observed that [³H]PrCC labelled only 57% of [³H]FNM binding sites in rat hippocampus (B_max values) and 71% in rat cerebral cortex, whereas the number of receptors labelled by both ligands was equal in the cerebellum. Hofstee analyses of the shallow inhibition curves seen in hippocampus and cerebral cortex when [³H]FNM binding was inhibited by β-CCE indicate that β-CCE and some other β-carboline-3-carboxylate derivatives interact preferentially with a subclass of receptors, and that the percentage of this subclass is equivalent to the number of receptors labelled by [³H]PrCC. We conclude that [³H]PrCC at low concentration (0.3–0.4 × 10^-9 M) labels a subclass of benzodiazepine receptors, BZ₁, while another class, BZ₂ receptors, are not labelled by [³H]PrCC when filtration assays are used. By parallel determinations of the proportion between [³H]FNM and [³H]PrCC binding we calculated the percentage of BZ₁ receptors in several regions of rat, guinea pig and calf brain and in mouse forebrain. The values ranged from approximately 50% in hippocampus to 90% in the guinea pig pons.     

Mechanism of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes: III. Serum-bound and oxidized 2-mercaptoethanol are available for the augmentation

The fetal calf serum (FCS) that was incubated with 2-mercaptoethanol (2-ME) followed by the removal of free 2-ME could support the antibody response to sheep erythrocytes in vitro as effectively as native FCS plus 2-ME. The supporting activity of 2-ME-pulsed FCS was reversibly abrogated by the treatment with dithiothreitol followed by dialysis. In addition, iodoacetamidetreated FCS did not acquire the supportiveness by 2-ME pulsing. These observations suggest that the activity of 2-ME-pulsed FCS would be due to the mixed disulfide between 2-ME and FCS components. On the other hand, the disulfide form of 2-ME (2-ME_ox) could also augment the antibody response as effectively as fresh 2-ME (the reduced form). These derivatized forms of 2-ME as well as fresh 2-ME was found to stimulate the transport of [³⁵S]cystine into murine lymphocytes when the uptake was examined by the long-term experiments (24 hr). These stimulations were thought to be mediated by the formation of the mixed disulfide between 2-ME and cysteine because the lymphocytes promoted the reaction of [³⁵S]cystine with 2-ME_ox- or 2-ME-pulsed FCS to produce the mixed disulfide that had been shown to be taken up by the lymphocytes four to five times more rapidly than cystine. Therefore, it was suggested that 2-ME_ox and 2-ME-pulsed FCS could augment the antibody response in a similar fashion to 2-ME by stimulating the uptake of cystine, an essential amino acid.     

De novo purine and pyrimidine biosynthesis during thymocyte concanavalin A stimulation

Measurement of the incorporation of [¹⁴C]formate and [¹⁴C]HCO₃⁻ enable us to characterize the activity of the synthetic pathways of purine and pyrimidine nucleotides in vitro throughout the process of the conA stimulation of rat thymocyte populations enriched for immunocompetent cells by isopycnic centrifugation. Our results show that de novo synthesis compensates for the total absence of exogenous nitrogenous bases and nucleosides in the culture medium. The magnitude of the proliferative response in media supplemented with dialysed fetal calf serum (FCS) was found to be the same as that observed when complete FCS was used in the culture medium. The induction of de novo synthesis (1) contributes to the expansion of the free nucleotide pool (the quantity of ATP measured in the perchloric acid (PCA)-soluble material of a same number of cells is increased by a factor of 10); (2) supplies the nucleotides necessary for nucleic acid synthesis (the total number of cells is increased by a factor of 3 after 4 days culture period). The activity of the metabolic pathways involved appears to be solicited by the dynamic requirements for thymocyte stimulation. For each step in the cellular activation a steady state of adenylic nucleotide synthesis and condensation into nucleic acids is established.     

Metabolic Fate of [3H]1-β-d-Arabinofuranosyl-5-[(E)-2-bromovinyl]uracil in Herpes Simplex Virus Type 1-Infected Cells

The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [³H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [³H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [³H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [³H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [³H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [³H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P₁ digestion reduced 99% of the [³H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [³H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [³H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [³H]BV-araU is incorporated inside viral DNA strands in infected cells.     

Ionic responses and growth stimulation in rat astroglial cells: differential mechanisms of gangliosides and serum

Rat astroglial cells respond to fetal calf serum (FCS) and gangliosides, including GM1, by undergoing proliferation. Here, we show that addition of FCS but not GM1 causes an increase in Na+, K+-pump activity, as measured by ouabain-sensitive 86Rb+ influx. The increase of Na+, K+-pump activity by FCS was due to increased Na+ influx (measured with 22Na+). This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+/H+ exchange. Amiloride also blocked the FCS-stimulated incorporation of [3H]thymidine into DNA. Two defined polypeptide growth factors, epidermal growth factor and fibroblast growth factor were also able to elicit an amiloride-sensitive Na+ influx and an ouabain-sensitive K+ uptake in these astroglial cells, in the presence of FCS or insulin. Thus, GM1 differs from serum and growth factors in the mechanisms by which these agents stimulate the proliferation of the astroglial cells used here.