Effects of butyrate and insulin and their interaction on the DNA synthesis of rumen epithelial cells in culture

Rumen epithelial cells (REC) were incubated in the presence of various concentrations of butyrate or insulin or with both of them, to obtain information on their effect on the DNA synthesis of cultured cells. The 24-h values of 3H-thymidine incorporation into cellular DNA were measured in the presence of butyrate, insulin or butyrate plus insulin. While butyrate reduced DNA synthesis, insulin produced an increase over the control. Combined butyrate plus insulin treatment influenced the incorporation of label in accordance with the relative proportion of these two substances.     

In vitro effects of methionine-enkephalin,somatostatin and insulin on cultured gonadal cells of the snailHelix aspersa

Isolated snail gonadal cells were cultured in the presence of synthetic neuropeptides in order to determine the subsequent effect of these substances on gonadal synthetic activities. Gonadal cells were incubated for 24 h in concentrations of methionine-enkephalin, somatostatin and insulin ranging from 10^–4 M to 10^–9 M, in medium 199 supplemented with 6% Ultroser G. Synthesis of DNA and protein by the cultured cells were simultaneously estimated by measuring incorporation of³H thymidine and³⁵S methionine. The rate of labelled precursor incorporation was measured using the liquid scintillation technique. All substances tested exerted a dose-dependent effect. The synthetic activity of the cultured cells was highest when the concentration of the peptides added to the medium approximated the physiological levels. Methionine-enkephalin, somatostatin and insulin at 2×10^–8 M significantly increased³H thymidine incorporation, by 62%, 69% and 69% respectively, and protein synthesis by 42%, 57% and 57%, respectively. In the case of juvenile gonadal cultured cells, a similar increase in³H and³⁵S incorporation was registered for a 10^–7 M peptide concentration. Both lower and higher peptide concentrations inhibited³H thymidine and³⁵S methionine incorporation. Pharmacological studies suggest the existence of methionine-enkephalin and somatostatin-like receptors on snail gonadal cells. These results indicate that our gonadal cell culture model provides a useful tool for the study of the neuroendocrinological control of the activity of snail gonadal cells.     

Effects of prolactin and growth hormone on DNA synthesis of rat mammary carcinomas in vitro

Summary Explants derived from mammary carcinomas of DMBA-treated female Sprague-Dawley rats were cultured for 5 days in Medium 199 containing insulin and corticosterone. The addition of ovine prolactin to the culture media resulted in a consistent significant increase in H³-thymidine incorporation into DNA. DNA synthesis of explants treated with either ovine or human growth hormone was intermediary to prolactin-treated cultures and control cultures. A combination of prolactin and human growth hormone often increased DNA synthesis above either hormone alone, suggesting a possible growth synergism between these peptides.Supported by NIH research grant No. CA-13777 and American Cancer Society research grant No. ET-59.NIH Research Career Development Awardee No. CA-35027.     

Relationship between DNA and cholesterol synthesis in kidney after refeeding

Summary DNA and cholesterol synthesis were investigated in the kidneys of fasted-refed rats. Refeeding resulted in an increase in kidney DNA synthesis, as measured by³H-thymidine incorporation, starting at 72 h. The increase in DNA synthesis was accompanied by a stimulation of cholesterol synthesis, as measured by¹⁴C-acetate incorporation into cholesterol.     

Untersuchungen über den Wirkungsmechanismus von Procarbazin (Natulan®)

Summary The cytotoxic methylhydrazine derivative procarbazine or Natulan® has a marked influence on the synthesis of deoxyribonucleic acid in Ehrlich ascites carcinoma cells. The incorporation of³H-thymidine into DNA — measured by autoradiographic methods — is inhibited.     

Effect of epidermal growth factor on growth and maturation of fetal and neonatal rat small intestine in organ culture

Summary Small intestinal explants from pre- and post-natal rats were incubated in an organ culture system in the absence and presence of epidermal growth factor (EGF). The rate of synthesis of small intestinal DNA and protein as well as the activity of lactase and alkaline phosphatase increased rapidly between 17 and 20-day gestational age, whereafter they declined. The maximal incorporation of³H-thymidine and¹⁴C-alanine into DNA and protein, respectively, was significantly stimulated by EGF (100 ng/ml). EGF had no effect on the activity of either lactase or alkaline phosphatase in the small intestinal explants.Acknowledgment. The project was supported by grants from the Veterans Administration Research Service. The authors wish to thank Dr. M. C. Geokas, Chief, Department of Medicine, Veterans Administration Medical Center, Martinez, CA, for providing us with excellent laboratory facilities and for his encouragement in this study.     

Thymidine H3 incorporation in the nurse cells of amphigonic and parthenogenetic ovaries ofMegoura viciae (Horn. Aph.)

Conclusions In the amphigonic females ofM. viciae an active nuclear incorporation of thymidine H³ occurs during growth which may be attributed to continuous endomitotic divisions. However, even when the nurse cells are functioning fully, and when the nuclei appear to have achieved maximum development, incorporation of the thymidine H³ continues, and, as was seen in other insects, does not affect all the nuclei. Incorporation would therefore seem to be due not to the continuance of endomitotic divisions but rather to the synthesis of metabolic DNA. On the other hand, even if one supposes that in the nurse cells of the amphigonic ovary endomitotic processes continue right up to the end of vitellogenesis of the amphigonic winter egg, this is quite out of the question so far as the parthenogenetic ovary is concerned. Diploid nurse cells are functioning continuously, since in a parthenogenetic ovary a great many ovocytes reach maturity one after the other, passing through all stages of development to produce the embryos. The nurse cells always retain, in such cases, their characteristic appearance right from the beginning of their differentiation³ and therefore thymidine H³ incorporation cannot be ascribed to continuous endomitotic divisions. It can therefore be assumed that the active synthesis which occurs in the nuclei does not concern genetically stable DNA but a metabolic DNA. The above results thus add new weight to the assumption by former authors that metabolic DNA may be synthesized in the nurse cells of amphigonic insects as well.

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Riassunto Nell'afideMegoura viciae le cellule nutrici diploidi dell'ovario partenogenetico e quelle poliploidi dell'ovario anfigonico si comportano in maniera analoga incorporando timidina H³ durante l'accrescimento ovocitario. Tale incorporazione viene attribuita alla sintesi di DNA metabolico.

     

Effects of isaxonine phosphate and analogs on fibroblast metabolism in culture

Summary Human skin fibroblasts in confluent cultures were incubated for 24 h in the presence of isaxonine phosphate (Nerfactor) and several related factors. The incorporation of¹⁴C-proline into secreted proteins and the release of collagen into the medium were inhibited. When the cells incubated for an additional period of 24 h after thorough washing, protein and collagen syntheses were found to be identical to those of controls, demonstrating that the inhibition of protein synthesis was independent of any toxic effect. When cells were incubated in the presence of both isaxonine and colchicine, the secretion of collagen was more inhibited than by colchicine alone, and proteins accumulated in the cells.     

Kinetics of DNA repair synthesis in guinea-pig pancreatic slices following in vitro exposure to N-methyl-N-nitrosourethane

Summary In vitro exposure of guinea-pig pancreatic slices to NMUT resulted in an increase in hydroxyurea-insensitive³H-TdR incorporation into DNA; this represents DNA repair synthesis following NMUT-induced DNA damage. The kinetics of this hydroxyurea-insensitive³H-TdR incorporation suggest that the NMUT-induced DNA damage is largely repaired within 2 hours.Environmental Health Programs, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 (USA). These studies were supported by National Cancer Institute (Bethesda, Md., USA). Contract No. N01-CP-23284. Technical assistance of MS.Mary Majdan is gratefully acknowledged.     

Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63 Ag8 cells

Summary Using the suspension cell line P3X63 Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (M_r 40 kD) and Semliki Forest virus core protein (M_r 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0°C. Subsequent resealing of the pores was completed after a 5-min incubation at 37°C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37°C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K⁺, Mg²⁺, and Ca²⁺) and resealing (in the presence of K⁺, Mg²⁺, and Ca²⁺), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.     

Effect of epidermal growth factor on growth and maturation of fetal and neonatal rat small intestine in organ culture

Small intestinal explants from pre- and post-natal rats were incubated in an organ culture system in the absence and presence of epidermal growth factor (EGF). The rate of synthesis of small intestinal DNA and protein as well as the activity of lactase and alkaline phosphatase increased rapidly between 17 and 20-day gestational age, whereafter they declined. The maximal incorporation of 3H-thymidine and 14C-alanine into DNA and protein, respectively, was significantly stimulated by EGF (100 ng/ml). EGF had no effect on the activity of either lactase or alkaline phosphatase in the small intestinal explants.     

DNS-Synthese,Blastentransformation und Mitosen in gemischten,homologen Granulozyten-Lymphozytenkulturen

Summary Lymphocyte stimulation in vitro could be obtained with allogeneic but not autochthonous or isogeneic granulocytes. Blastoid transformation, mitosis and activated DNA synthesis measured by H³-thymidine incorporation occurred simultaneously. The cytoimmunological relevance and further applications of the findings are discussed.

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Mit Unterstützung der schweizerischen Krebsliga.     

Mitogenic activity of selenoorganic compounds in human peripheral blood leukocytes

Summary A variety of organoselenium compounds were originally described as antiinflammatory, antioxidant or glutathione-peroxidase-like agents, and as inhibitors of prostaglandin and leukotriene synthesis. Recently, the compounds have also been found to be inducers of interferon gamma and tumor necrosis factor in human peripheral blood leukocytes (PBL). We evaluated the effects of bis [2-(N-phenylcarboxamido)phenyl] diselenide and Ebselen^®; 2-phenyl-1,2-benzisoselenazol-3(2H)one, on the incorporation of tritiated thymidine into the DNA of PBL cultured in vitro. Both compounds were mitogenic and this effect was correlated with the expression of interleukin 2 receptor in T-lymphocytes. Therefore, we suggest that the selenoorganic compounds may induce mitogenic cytokines.     

Mechanism of age-dependent involution in embryonic chick notochords

To study the possible mechanism of the age-dependent involution of the notochord, isolated mesenchymefree notochords of chick embryos were cultured in vitro and compared with their counterparts in vivo. Two different aspects were evaluated: (1) DNA synthesis measured by [³H]thymidine incorporation and visualized by autoradiography and (2) cell death quantified by counting the number of pyknotic nuclei. The results demonstrate that [³H]thymidine uptake by notochords shows an age-dependent decrease in vitro as well as in vivo. The number of [³H]thymidine-labelled notochord cells, however, is higher in vitro than in vivo. At the same time, there is an age-dependent increase in pyknosis in the notochord in vivo and in vitro. So, during the aging process, the number of both pyknotic nuclei and of [³H]thymidine-labelled nuclei suggest a high turnover of notochord cells in vitro. From these results, we can conclude that the process of involution in aging notochord seems to be controlled by a programmed intrinsic process, which might be influenced partially by the microenvironment in vivo.     

Increased assembly of cytoskeletal proteins associated with the transformation of human liver cells into fibroblast-like cells

A topoisomerase II inhibitor, novobiocin, and a deacetylase inhibitor, butyrate, synergistically transformed human liver cells into fibroblast-like cells. This morphological change was associated with an increased production of procollagen type III peptide and a simultaneous assembly of actin, tubulin, vimentin and cytokeratin. Novobiocin and butyrate had no marked effect on the phosphorylation state of cytokeratin proteins, but synergistically enhanced [³H]acetate uptake. From these results, it can be speculated that protein acetylation plays an important role in inducing the assembly of cytoskeletal proteins and the morphological transformation of human liver cells.     

Influence of progesterone on protein and RNA synthesis in cultured chick embryo liver cells

Summary Chick embryo liver primary cultures, when added with progesterone, exhibit, in comparison with the controls, a normal growth, a decline of both³H-uridine uptake and incorporation into total RNA, a decline of³H-leucine and¹⁴C serine incorporation into the proteins. Progesterone is not able to stimulate phosvitin synthesis induction.These studies were supported in part by the Italian CNR grants.     

Contrasting effects of RNA and protein synthesis blocking on natural and lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

Summary In this study, earlier observations^2,9 concerning the independence of both natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) from DNA synthesis have been confirmed. In addition, blocking of RNA synthesis by actinomycin D and of protein synthesis, reversibly by puromycin (PM) and irreversibly by emetine (EM) had different effects on NCMC and LDCC against³H-thymidine-prelabeled HEp-2 target cells. Similarly to the Con A-induced proliferation of lymphocytes, LDCC activity was also inhibited by blocking of RNA and protein synthesis. NCMC to HEp-2 target cells was not affected by blocking of RNA synthesis, while both PM and EM strongly enhanced NCMC activity.     

Production of insulin-like growth factor I (IGF-I) and IGF-binding proteins by rat intestinal stromal cells in vitro

To determine if intestinal stromal cells secrete diffusible factors such as insulin-like growth factors (IGFs) capable of regulating epithelial cell growth in vitro, stromal cells were isolated by enzymatic digestion of rat intestine. Incorporation of [³H]thymidine into DNA and [¹⁴C]leucine into protein of IEC-6 cells, a model intestinal epithelial cell line, was significantly increased (two- to threefold) when the IEC-6 cells were co-cultured with stromal cells, relative to IEC-6 cells grown alone. Medium conditioned by stromal cells stimulated DNA synthesis of IEC-6 cells in a dose-dependent manner. Analysis of the conditioned medium revealed that intestinal stromal cells secreted IGF-I, but little IGF-II, in addition to an M _r 32,000 IGF-binding protein (IGFBP-2) and an IGFBP having M _r∼ 24,000. We conclude that rat intestinal stromal cells secrete one or more diffusible factors, which may include IGF-I and IGFBPs, capable of stimulating proliferation of IEC-6 cells in vitro. Received 25 August 1997; received after revision 7 November 1997; accepted 20 November 1997     

Interaction of galectin-1 with caveolae induces mouse embryonic stem cell proliferation through the Src, ERas, Akt and mTOR signaling pathways

Galectins have the potential to provide a promising alternative for unveiling the complexity of embryonic stem (ES) cell self-renewal, although the mechanism by which galectins maintain ES cell self-renewal has yet to be identified. Galectin-1 increased [³H]-thymidine incorporation as well as cyclin expression and decreased p27^kip1 expression. Src and caveolin-1 phosphorylation was increased by galectin-1, and phospho-caveolin-1 was inhibited by PP2. In addition, inhibition of caveolin-1 by small interfering RNA and methyl-β-cyclodextrin (Mβ-CD) decreased galectin-1-induced cyclin expression and [³H]-thymidine incorporation. Galectin-1 caused Akt and mTOR phosphorylation, which is involved in cyclin expression. Galectin-1-induced phospho-Akt and -mTOR was inhibited by PP2, ERas siRNA, caveolin-1 siRNA and Mβ-CD. Furthermore, mTOR phosphorylation was decreased by LY294002 and Akt inhibitor. Galectin-1-induced increase in cyclin expression and decrease in p27^kip1 was blocked by Akt inhibitor and rapamycin. In conclusion, galectin-1 increased DNA synthesis in mouse ES cells via Src, caveolin-1 Akt, and mTOR signaling pathways. Received 30 October 2008; received after revision 18 February 2009; accepted 24 February 2009     

A differential effect of L-serine on the incorporation of3H-deoxycytidine and3H-thymidine into DNA of rat thymocytes

Summary The addition of L-serine to short-term cultures of rat thymocytes stimulated the incorporation of³H-deoxycytidine into DNA, but simultaneously depressed the incorporation of³H-thymidine into DNA.This investigation was supported by NIH grants No. CA-10291 and T12CA8021 from the National Cancer Institute.