The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata

Summary Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25–2.0 Mrads is optimal for cell growth.     

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.     

The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata

Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25-2.0 Mrads is optimal for cell growth.     

Effect of an ecdysteroid on DNA synthesis,DNA polymerase α and thymidine kinase activities ofDrosophila melanogaster cells in vitro

Summary 10^–7 M 20-OH-ecdysone treatment of a diploidDrosophila clone results in an inhibition of 60% of the DNA synthesis from 18 h of treatment on. After 48 h of hormone treatment the thymidine kinase activity is 70% inhibited; concomitantly a 60–70% lowering of the acid-soluble specific activity is observed. In the meantime the DNA polymerase activity is reduced.     

Surface model of the collagen fibril as determined by two-stage plastic replica

Summary Two-stage plastic replicas of fresh and vacuum dried rat tail collagen were examined by electron microscopy. Microphotometer tracings made from the replica micrographs showed that the surface of the collagen has a periodic undulating topography which includes an approximately 20–40 Å deep depression located within the major band distance.This work was supported by the Hendricks Research Fund No. 93252 and the V. A. Research Service Project No. 098-147718-01.     

A short-term culture method for chromosome preparation from somatic tissues of adult mussel (Mytilus edulis)

In order to improve the efficiency of mussel chromosome preparation, a tissue culture procedure has been developed. Mantle and foot explants were grown in tubes in media composed of Eagle's Basal Medium supplemented either with salts or seawater, enriched with egg yolk, adjusted to pH 7.50, and containing penicillin and streptomycin. After 4 days of incubation at 18°C, antibiotics were renewed and after 6–7 days, cultures were ready for harvesting and preparation of microscopical slides. The cultures were a source of actively dividing cells and consistent metaphase spreads were obtained. Evidence from BrdU incorporation suggested that cells could undergo several rounds of replication. The chromsome spreads were good enough for karyotyping and to successfully silver stain the nucleolar organizer regions.     

Interleukin 1 modulates collagen accumulation by rat granulation tissue cells both in vivo and in vitro

Summary In in vivo studies 0.5 U human interleukin 1 (IL-1) was incoculated daily into a subcutaneously implanted viscose cellulose sponge. IL-1 significantly decreased the dry weight (7.8%) and the hydroxyproline content (24.2%) of granulomas. When the cultured rat granulation tissue cells were exposed to IL-1 (0.5–2.0 U/ml) their collagen production decreased to 80% of that in controls. No effect on cell proliferation was detected.     

The assessment of the rate of digestion of serum proteins in mosquito by gel-diffusion and immunoelectrophoresis

Zusammenfassung Die Verdauungsgeschwindigkeit von Serumproteinen im Darm von Mücken (Armigeres subalbatus) wurde mit Gel-Diffusion und Immunoelektrophorese bestimmt. Es konnten wenigstens 2 Bänder mit Gel-Diffusion in einer Mücke nachgewiesen werden, die menschliches Blut gesaugt hatte. Während das äussere Band nach 12–18 h verschwindet, konnte das innere noch nach 48–56 h als Albuminfraktion des menschlichen Serums bestimmt werden.     

Compensatory caspase activation in MPP+-induced cell death in dopaminergic neurons

Many have hypothesized that cell death in Parkinsons disease is via apoptosis and, specifically, by the mitochondrial-mediated apoptotic pathway. We tested this hypothesis using a mouse dopaminergic cell line of mesencephalic origin, MN9D, challenged with the Parkinsonism-causing neurotoxin MPP⁺ (1-methyl-4-phenylpyridinium ion). Apoptosis was the main mode of cell death when the cells were subjected to MPP⁺ treatment under serum-free conditions for 24 h. Caspase-3 and caspase-9, however, were not activated, thus indicating the existence of alternate or compensatory cell death pathway(s) in dopaminergic neuronal cells. Using caspase inhibitors, we demonstrated that these pathways involve caspase-2, –8, –6 and –7. A time-course study indicated that activation of caspase-2 and –8 occurred upstream of caspase-6 and caspase-7. Upon MPP⁺ challenge, the apoptosis-inducing factor was translocated from the mitochondria into the MN9D cytosol and nucleus. These results suggest the existence of alternative apoptotic pathways in dopaminergic neurons.Received 20 September 2004; received after revision 5 November 2004; accepted 22 November 2004     

Preferential neurotoxicity of pentobarbital on nerve and glial cells in culture

Summary The effect of pentobarbital was studied in a mixed population of nerve and glial cells dissociated from brains of 7-day chick embryos and maintained in culture. Pentobarbital-Na was added in various concentrations ranging from 5×10^–5 M to 1×10^–3 M. The neuronal density was monitored by counting of neurons, neuronal identity was established by staining for Nissl Bodies and acetylcholinesterase. Over a culture period of 3 weeks, it was found that the barbiturate exerts a preferential dose-dependent cytotoxic effect on neurons.The expert technical assistance of Miss A. Wolf is appreciated. This work was supported by grant 6-74-27, INSERM (Physiologie et Pathologie du Développement Nerveux).     

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.     

Inhibitory effect of direct current on cell division and cell proliferation

Summary Direct current (0.1 to 0.2 mA) fully inhibited cell division and cell proliferation at the site of the non-polarizing anode, probably due to electrolysis and electro-osmotic processes affecting the chromatin of the cell nucleus. It resulted in cessation of the growth of the root-germs of the onion. Plant germs lost their ability to germinate 18–20 h after the application of the weak direct current.     

C-peptide fragments stimulate glucose utilization in diabetic rats

Studies of C-peptide cellular effects show that not only the full-length native peptide but also specific C-terminal fragments are biologically active in in vitro systems. In the present study, the effect of five C-peptide fragments and the native peptide on whole-body glucose turnover was studied in streptozotocin diabetic rats using the insulin clamp technique. Insulin was infused intravenously at 18 pmol kg^–1 min^–1 for 90 min and blood glucose concentration was clamped at 8 and 4 mM in diabetic and non-diabetic animals. A steady state was reached during the last 30 min of the study period. Rat C-peptide II and fragments comprising residues 27–31 and 28–31 were effective in augmenting glucose turnover in diabetic rats (+100% to 150%), while no significant effects were seen for segments 1–26, 11–19 and 11–15. The metabolic clearance rate for glucose during infusion of C-peptide or fragments 27–31 and 28–31 in diabetic rats was similar to that seen in non-diabetic animals. We conclude that C-terminal tetra- and pentapeptides, but not fragments from the middle segment of C-peptide, are as effective as the full-length peptide in stimulating whole-body glucose turnover in diabetic rats.Received 18 December 2003; received after revision 19 January 2004; accepted 21 January 2004     

Complement evasion by the Lyme disease spirocheteBorrelia burgdorferi grown in host-derived tissue co-cultures: role of fibronectin in complement-resistance

The effectiveness of complement-mediated killing ofBorrelia burgdorferi, the causative agent of Lyme disease, in the presence of host-derived tissues was studied. Second and high passage forms ofB. burgdorferi 297 isolate were grown in a LEW/N rat joint tissue co-culture system and in artificial BSK medium. Guinea pig complement and third week immune serum from hamsters with experimental Lyme disease were added to the cultures. Both high and low passage borrelia grown in BSK medium died and did not revive after 3 weeks incubation in BSK medium. However, 5–12% of tissue co-cultured borrelia survived the first complement-mediated lysis. Repeated re-growth and lysis cycles in tissue co-culture resulted in isolation of an 85% complement-resistant population ofB. burgdorferi. Joint tissue culture supernatant collected on the third day of tissue culture, and fibronectin (25 g/ml), also protected spirochetes from complement-mediated lysis in contrast to BSK or fresh co-culture medium. Complement-mediated lysis may not be an effective mechanism in eradication of borrelia, and the chronicity of Lyme disease may be due to resistance ofB. burgdorferi variants to host immune defense mechanisms in the presence of host-derived tissues.     

In vitro biosynthesis of juvenile hormone III by the corpora allata ofCalliphora vomitoria and its role in ovarian maturation and sexual receptivity

Summary JH III is the only JH detected by GLC-MS in medium from in vitro incubations of corpora allata of adult females ofCalliphora vomitoria. When corpora allata were removed from females at various times during the reproductive cycle and the JH III produced by the glands in vitro measured by a JH III radioimmunoassay, an increase in the level of synthesis was found to occur before previtellogenesis (0–24 h). A second increase appeared at the onset of vitellogenesis (72–83 h) and continued until the end of vitellogenesis (96 h) and the occurrence of chorionation (120 h). Since sexual receptivity develops with vitellogenesis, the significantly higher levels of JH III biosynthesis in vitro at this time supports a possible role for JH in the acquisitive of receptivity.     

Rat pancreatic islet cells in primary culture: occurrence of giant cells amenable to patch clamping

Summary Neonatal and adult rat pancreatic islet cells were maintained in dissociated cell culture for up to three weeks. The unexpected occurrence of giant (40–50 m) cells was noted, some of which reacted positively to an insulin antiserum, indicating the presence of insulin. The giant cells were amenable to study using the extracellular patch clamp technique, which was used to demonstrate a population of membrane channels gating outwardly directed current in these cells.     

Nucleotide metabolism in the rat liver following whole-body X-irradiation

Summary Evidence has been presented to show that, like synthesis of RNA, syntheses of ribonucleotide precursors of RNA in the rat liver were stimulated for 6–18 h following whole-body X-irradiation (1000 R).     

Fibronectin peptide DRVPHSRNSIT and fibronectin receptor peptide DLYYLMDL arrest gastrulation ofRana pipiens

Gastrulation is characterized by dramatic cell migration which is thought to require the interaction of cell adhesion molecules with extracellular molecules. We have tested two novel peptides, a fibronectin peptide and a fibronectin receptor peptide, for their effects on gastrulation of the leopard frogRana pipiens. The fibronectin peptide DRVPHSRNSIT corresponds to residues 1373–1383 of the cell-binding domain of fibronectin; the receptor peptide DLYYLMDL corresponds to residues 124–131 of 1 subunit of a variety of integrins including 51. Either of these peptides significantly inhibited gastrulation after being microinjected into mid-blastulae. These results indicate that these sequences may correspond to the ligand/receptor interaction sites of fibronectin and its receptor(s).     

DNA synthesis in the nuclei of differentiating muscle fibers of the silkworm,Bombyx mori L.

Summary Incorporation of³H-thymidine (³HTdr) into the nuclei of myofibril-containing myofibers of larvae of the silkworm,Bombyx mori, was shown by means of light microscope (LM) and electron-microscope (EM) autoradiography. The number of DNA-synthesizing myonuclei attains 42% 12–18 h after each molt. Thus in the developing silkworm DNA replication and myofibrillogenesis are coexisting and not mutually exclusive processes as is the rule in vertebrate somatic myogenesis.     

The inhibitory effect of sodium on the contraction of frog's heart perfused with sucrose solution

Résumé Le cur de grenouille continue à battre de lui-même et répond à une stimulation électrique durant 5–6 h s'il reçoit une injection contenant une demi solution isotonique de saccharose, de pyrophosphate de sodium et d'adénosinetriphosphate ou de citrate de sodium. Le chloride de sodium annule ces deux réactions.