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 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.     

Brain factor fromGalleria mellonella (Lepidoptera) stimulating silk gland activity

Brain extracts from day 1–4 last instar larvae ofGalleria mellonella (Lepidoptera) stimulate RNA synthesis in cultured silk glands from day 3 last instar larvae. When the fibroin-synthesizing posterior parts of silk glands were incubated for 3 h in vitro in the presence of brain extract (0.1 brain equivalent), [³H]-uridine incorporation into RNA was stimulated more than twofold. The stimulating effect of brain extract showed a dose response relationship. It is suggested that the heat-resistant and protease-sensitive brain factor is a peptide.     

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 the calmodulin inhibitor R24571 (calmidazolium) on rat embryos cultured in vitro

Summary The possible effects of inhibition of the calcium-binding protein, calmodulin, on mammalian morphogenesis have been investigated by culturing rat embryos in vitro from 9 1/2 to 11 1/2 days of development in the presence of R24571 (calmidazolium), a specific inhibitor of calmoldulin. Embryos cultured in 10^–2 mM R24571 for 48 h show inhibited development and exhibit a range of morphogenetic abnormalities including assymetry and neural tube defects. Embryos exposed to R24571 for the first 24 h of a 48 h culture are more severely affected than embryos exposed to R24571 for the last 24 h.Acknowledgments. We wish to thank Mr C. Veltkamp and Mr B. lewis for their expert help with the scanning electron microscopy and photography.     

Analysis of translation products synthesized in isolated rat hepatocytes treated with diethylnitrosamine

Summary Isolated rat hepatocytes were labeled with³⁵S-methionine in the presence of 25 mM diethylnitrosamine (DENA). The intrinsically labeled proteins were analyzed by one-and two-dimensional gel electrophoresis and the fluorographic patterns were compared with those obtained from untreated hepatocytes. The results of short term experiments (2 h) show that, in the presence of 25 mM DENA, protein synthesis is inhibited by 50%. This reduction encompasses all protein species without selective inhibition of certain proteins.This work was supported by CNR (Project Control of Neoplastic Growth) grant No. 810132696 and partially by AIRC.     

Role of extracellular matrix molecules in the development of the sodium current in quail mesencephalic neural crest cells

The occurrence of the voltage-dependent sodium current has been studied in developing neurons from quail mesencephalic neural crest on different substrates, using the whole-cell patch clamp technique. Explants from 9–12 somite embryos were cultured on dishes coated with type I collagen, fibronectin, laminin or on plastic dishes in a chemically defined medium. After 18 h of culture the sodium current was observed in 70% of the neurons tested, and at 24 h some of these neurons were able to generate an action potential. After 18–25 h cells grown on fibronectinor collagen I-coated dishes showed a significantly higher occurrence of the sodium current (83% and 84% respectively) as compared to cells grown on uncoated plastic dishes (51%). Moreover, in the presence of fibronectin, the current density of the sodium current was more than doubled in comparison with cells grown on other substrates.     

The effect of 5-bromodeoxyuridine on mouse embryos during neurulation in vitro

Summary Mouse embryos explanted at various stages during neurulation were cultured for 20–28 h in the presence of 25–900 g/ml of 5-bromodeoxyuridine (BUdR). BUdR strongly inhibited closure of the cranial neural tube, which was found to be stage-dependent. When mouse embryos were exposed to BUdR after development of the concave curvature in the neuroepithelium of the midbrain to the upper hindbrain regions, they became insensitive to the drug-induced open cranial neural tube. Histological observations showed that BUdR interfered with interkinetic migration and cytokinesis of the neuroepithelial cells. These cellular abnormalities were not dependent on the morphological development of the cranial neural folds. The³H-BUdR experiment confirmed that the label was mostly incorporated into the DNA fraction.Acknowledgment. This work was supported by Grant-in-Aids for scientific research No. 557469 and 58480391 from the Ministry of Education, Japan.     

Presence of lectin-like substances in the basement membrane of the human kidney glomerulus]

Crude extracts from the human glomerular basement membranes solubilized by pepsin or bacterial collagenase agglutinate normal or transformed human cells. Cytoagglutination is inhibited by N-acetyl-osamines. These properties are reminiscent of lectins. When agglutinated cells are incubated for an additional 20 hrs. period in minimal serum free medium but in presence of these basement membrane extracts, they attach to the glass and spread out.     

pEGFPN1-dnEGFR对胃癌细胞的化疗增敏作用

目的研究人 EGFR显性负性突变体真核表达载体(pEGFPN1 dnEGFR)对人胃癌细胞株 SGC 7901和 NCI N87化疗敏感性的影响,并探讨其可能机制.方法 MTT法测定奥沙利铂对稳定转染 pEGFPN1 dnEGFR和 pEGFP N1载体的两种胃癌细胞的量效反应.奥沙利铂作用各组细胞24h后,RT PCR检测各组细胞中 Caspase 3和 CyclinD1的 mRNA表达情况;Westernblot检测各组细胞中 Caspase 3和 CyclinD1蛋白表达情况.结果转染 pEGFPN1 dnEGFR后,两种胃癌细胞对奥沙利铂的敏感性增加,奥沙利铂对 pEGFPN1 dnEGFR转染组细胞的增殖抑制率(VI)与对照组相比有显著提高(P<0.05).RT PCR显示 pEGFPN1 dnEGFR转染组细胞 CyclinD1mRNA表达较对照组下降,而 Caspase 3mRNA表达较对照组升高(P<0.05);Westernblot显示 pEGFPN1 dnEG FR转染组细胞 CyclinD1蛋白表达较对照组下降,而 Caspase 3蛋白表达较对照组升高(P<0.05).结论 EGFR显性负性突变体能提高胃癌细胞对化疗药物奥沙利铂的敏感性,其机制可能与 Caspase 3和 CyclinD1有关     

Phosphorylation of cockroach antennal polypeptides: effects of second messengers and pheromonal blend

In insect antennal extracts, Schleicher et al.¹ showed that protein kinase C (PKC) inhibitors abolish the transience of pheromone-induced rapid inositol trisphosphate responses, which suggests that pheromonal signals act on phosphorylation of specific proteins. To confirm this hypothesis, we studied the effects of second messengers and a pheromonal blend on phosphorylation of antennal proteins in the cockroachPeriplaneta americana. Proteins from adult male antennae were phosphorylated in vitro in the presence of [³²P] triphosphate, then separated by SDS-polyacrylamide gel electrophoresis. Numerous phosphopolypeptides were visualized. The presence of Ca⁺⁺/calmodulin in the incubation medium resulted in increased phosphorylation of polypeptides with molecular weights of 38, 48, 51, 54 and 58 kDa. Stimulation of PKC by addition of Ca⁺⁺ phosphatidylserine (PS)/phorbol myristate acetate (PMA) resulted in the appearance of three phosphopolypeptides of 36, 70 and 120 kDa. In the presence of cyclic adenosine monophosphate, two new major polypeptides of 46 and 42 kDa appeared; the latter polypeptide also appeared in the presence of cyclic guanosine monophosphate. Comparison with polypeptide composition of tissue from the cerci, leg, brain and fat body showed that the 36 and 48 kDa polypeptides were specific to antennae, whereas the 120 kDa polypeptide was also present in the adult brain. When antennae are subjected to pheromonal stimulation for 16 seconds prior to homogenization, in vitro phosphorylation of the 120, 70, 64 and 38 kDa polypeptides was inhibited, whereas phosphorylation of the 58, 54, 51 and 48 kDa polypeptides was strongly stimulated. It is noteworthy that a 107 kDa polypeptide was observed only after pheromonal stimulation by Ca⁺⁺/PS/PMA. Our findings suggest that Ca⁺⁺-and PKC-dependent protein phosphorylation systems play an important role in the transduction of pheromonal signals in antennae of male cockroachP. americana. We speculate that specific phosphoproteins may modulate sensitivity and signal amplification during the olfactory transduction process.     

Colchicine induced interchanges in chillies (Capsicum annuum L.)

Summary Seeds ofCapsicum annuum L. cultivar cerasiformies were treated with 0.4 and 0.2% aqueous colchicine solution for 24 and 72 h respectively. Tetraploids were not realized; instead, interchange heterozygosity was observed in several plants in 0.4% treatment. The interchanges varied from 1 to 3 per plant. It is presumed that colchicine has induced chromosome breaks.     

Hyperthermia-induced apoptosis and the inhibition of DNA laddering by zinc supplementation and withdrawal of calcium and magnesium in suspension culture of tobacco cells

In the present paper we report examination of stereotypic hallmarks of apoptosis in heat-treated tobacco cells. Hyperthermia (44 °C, 4 h) caused apoptosis in 53.6% of cells when assayed 24 h after heat treatment. The induction of apoptosis by heat treatment was confirmed by flow cytometric assay. Cytological observations revealed condensation of the cytoplasm and nucleus, as well as nuclear collapse. DNA ladders were observed in DNA extracted from heat-treated cells, whereas DNA from control cells remained undegraded. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay revealed that 51.8% of the heat-treated cells (44 °C, 4 h) show positive reaction after a 24-h recovery. When cells were cultured in a medium supplemented with 0.4–5.0 mM ZnSO₄, internucleosomal DNA fragmentation induced by heat shock was completely negated. Strikingly, when cells were cultured in Ca²⁺ and/or Mg²⁺ free medium for 44 h followed by heat treatment, DNA laddering was not observed. The results suggest hyperthermia-induced apoptosis and a correlation between the regula tion of endonucleases and heat shock signal in apoptotic tobacco cells. Received 17 September 1998; received after revision 4 January 1999; accepted 4 January 1999     

Chronic sensory denervation reduces thrombin-stimulated endothelin release from aortic endothelial cells

The long-term (trophic) influence of perivascular nerves on the endothelium was investigated by measuring changes in thrombin-stimulated release of the potent vasoconstrictor, endothelin, after selective chronic denervation. Rat pups were treated with either guanethidine or capsaicin to destroy sympathetic or sensory nerves, respectively. The abdominal aortas from the rats at three months of age (5 pooled per experiment) were incubated with 4U thrombin/ml in medium for 24 h at 37°C, and the amount of endothelin released from the preparation determined by immunoassay. After neonatal sensory denervation there was a significant reduction in the thrombinstimulated release of endothelin compared to the controls (0.012±0.012 (4) compared to 0.063±0.012 (6), pmol/cm²/24 h, p<0.02). There was no change in endothelin release after sympathetic denervation. In summary, sensory nerves play a trophic role in the expression of endothelin in endothelial cells of the intima.     

The antioxidant function of Bcl-2 preserves cytoskeletal stability of cells with defective respiratory complex I

Human thyroid carcinoma XTC.UC1 cells harbor a homoplasmic frameshift mutation in the MT-ND1 subunit of respiratory complex I. When forced to use exclusively oxidative phosphorylation for energy production by inhibiting glycolysis, these cells triggered a caspase-independent cell death pathway, which was associated to a significant imbalance in glutathione homeostasis and a cleavage of the actin cytoskeleton. Overexpression of the anti-apoptotic Bcl-2 protein significantly increased the level of endogenous reduced glutathione, thus preventing its oxidation after the metabolic stress. Furthermore, Bcl-2 completely inhibited actin cleavage and increased cell adhesion, but was unable to improve cellular viability. Similar effects were obtained when XTC.UC1 cells were incubated with exogenous glutathione. We hence propose that Bcl-2 can safeguard cytoskeletal stability through an antioxidant function. Received 28 May 2008; received after revision 8 July 2008; accepted 29 July 2008     

Analysis of translation products synthesized in isolated rat hepatocytes treated with diethylnitrosamine

Isolated rat hepatocytes were labeled with 35S-methionine in the presence of 25 mM diethylnitrosamine (DENA). The intrinsically labeled proteins were analyzed by one- and two-dimensional gel electrophoresis and the fluorographic patterns were compared with those obtained from untreated hepatocytes. The results of short term experiments (2 h) show that, in the presence of 25 mM DENA, protein synthesis is inhibited by 50%. This reduction encompasses all protein species without selective inhibition of certain proteins.     

Development of rhythmic melatonin synthesis in cultured pineal glands and pineal cells isolated from chick embryo

The chick pineal gland exhibits circadian rhythms in melatonin synthesis under in vivo and in vitro conditions. A daily rhythm of melatonin production was first detectable in pineal glands isolated from chick embryos at embryonic day 16 and incubated under a LD cycle. All pineal glands isolated from 17-day-old and older embryos were rhythmic while no gland isolated at embryonic day 14 and 15 exhibited a daily rhythm in melatonin synthesis. Melatonin production in static cultures of embryonic pineal cells was rhythmic over 48 h if the cells were kept under a LD cycle. When embryonic pineal cells were incubated in constant darkness the rhythm in melatonin production was damped within 48 h. These results suggest that chick pineal cells from embryonic day 16 onwards are photosensitive but that the endogenous component of the melatonin rhythm is not completely developed at that age. A soluble analogue of cAMP stimulated and norepinephrine inhibited melatonin synthesis in cultured embryonic pineal cells. These findings indicate that the stimulatory and inhibitory pathways controlling melatonin synthesis in the mature pineal gland are effective in pineal cells isolated from chick embryos at least 2 days before hatching.     

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.     

A new substrate for cultures of dissociated primary rat brain

Different substrates were used to coat plastic petri dishes for the cultivation of dissociated fetal rat brain cells. Only on surfaces which were coated with a mixture of serum and non-reconstituted collagen, did the majority of the inoculated cells attach singly or as aggregates within 24 h. The attachment of the cells was followed by the outgrowth of cellular processes either from single cells or from aggregates in the same time period. This did not occur on collagen or serum treated or on regular plastic dishes. Under the latter conditions a similar outgrowth was observed only after 3–5 days.