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

Summary 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 of³H-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.     

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.     

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.     

Variations in DNA synthesis and the activities of acid and alkaline deoxyribonucleases during regeneration of planaria (Polycelis tenuis)

Variations of DNA synthesis were established for the first time during planarian regeneration by 32P phosphoric acid incorporation. A first peak of DNA synthesis occurred between the 10th and the 18th hour after sectioning (maximum at 12 hrs.). Subsequently, DNA synthesis increased again progressively after the 24th hour to a plateau between 48 and 72 hrs. after regeneration. Simultaneously, variations of alkaline and acid DNases were determined.     

Effects of isaxonine phosphate and analogs on fibroblast metabolism in culture

Human skin fibroblasts in confluent cultures were incubated for 24 h in the presence of isaxonine phosphate (Nerfactor) and several related factors. The incorporation of 14C-proline into secreted proteins and the release of collagen into the medium were inhibited. When the cells were 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.     

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.     

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.

     

Activity of soluble factors produced by Epstein-Barr virus-transformed human B lymphocytes on different cell lines

Summary Self-stimulatory growth factors, produced by a human Epstein-Barr virus (EBV)-positive lymphoblastoid B cell line, named BA-D10-4, have been tested for the capacity to induce DNA synthesis in various human and animal cell lines, including lymphoid, either EBV-positive or EBV-negative, and non-lymphoid cell lines. It has been found that BA-D10-4 cells produce growth factors which seem to be essential for their sustained proliferation in vitro, and which increase DNA synthesis in different primate lymphoid cells, independently of the presence of the EBV genome and of the lymphocyte lineage.     

Cellular control of the tick-borne virus antigen production in persistently infected cell culture.

The influence of inhibition or stimulation of cellular DNA synthesis on tick-borne virus antigen production in persistently infected cell culture was studied. Either mitomycin C or cytosine-arabinoside caused cessation of antigen-containing cell number increase. Stimulation of cellular DNA synthesis by growth medium change increased the level of antigen-containing cells. When HEp-2-Sof culture was synchronized, a correlation was observed between the entrance of cells into DNA synthesis phase and the increase of proportion of antigen-containing cells.     

Cellular control of the tick-borne virus antigen production in persistently infected cell culture

Summary The influence of inhibition or stimulation of cellular DNA synthesis on tick-borne virus antigen production in persistently infected cell culture was studied. Either mitomycin C or cytosine-arabinoside caused cessation of antigen-containing cell number increase. Stimulation of cellular DNA synthesis by growth medium change increased the level of antigen-containing cells. When HEp-2-Sof culture was synchronized, a correlation was observed between the entrance of cells into DNA synthesis phase and the increase of proportion of antigen-containing cells.     

Cell proliferation in the rat pituitary gland: a mechanism of control in prolactin cells.

We investigated the relationship between prolactin content and DNA replication in the anterior pituitary gland. Thymidine incorporation in pregnant rats is significantly lower than in virgin controls. This is accompanied by a decreased activity of DNA polymerase. Sulpiride administration to pregnant rats enhances thymidine incorporation to levels similar to virgin controls. The results indicate a negative feedback between prolactin content and DNA synthesis in the rat anterior pituitary gland.     

Effects of isoxazolyl-naphthoquinoneimines on growth and oxygen radical production in Trypanosoma cruzi and Crithidia fasciculata

Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis in Trypanosoma cruzi and stimulated O2 uptake and O2-. generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and O2-. generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H2O2 production were obtained with Crithidia fasciculata despite the presence of catalase in this organism.     

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.     

Activity of soluble factors produced by Epstein-Barr virus-transformed human B lymphocytes on different cell lines.

Self-stimulatory growth factors, produced by a human Epstein-Barr virus (EBV)-positive lymphoblastoid B cell line, named BA-D10-4, have been tested for the capacity to induce DNA synthesis in various human and animal cell lines, including lymphoid, either EBV-positive or EBV-negative, and non-lymphoid cell lines. It has been found that BA-D10-4 cells produce growth factors which seem to be essential for their sustained proliferation in vitro, and which increase DNA synthesis in different primate lymphoid cells, independently of the presence of the EBV genome and of the lymphocyte lineage.     

Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63Ag8 cells

Using the suspension cell line P3X63Ag8 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 (Mr 40 kD) and Semliki Forest virus core protein (Mr 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0 degrees C. Subsequent resealing of the pores was completed after a 5-min incubation at 37 degrees 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 degrees 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.     

Inhibitory action of serum from a Laron dwarf on normal cellular function

Glucose uptake and O2 consumption of confluent glial cells grown in culture were measured in the presence of serum-free buffer and compared with those measured in the presence of serum from a normal volunteer, from an hGH-deficient dwarf and from a Laron dwarf. Cellular glucose uptake and respiration in the absence or presence of insulin or hGH are inhibited by Laron serum.