A DNA Damage Checkpoint Pathway Coordinates the Division of Dikaryotic Cells in the Ink Cap Mushroom Coprinopsis cinerea

The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus.     

Fate of biomedical research protocols and publication bias in France: retrospective cohort study

Objectives To describe the fate of protocols approved by the French research ethics committees, a national system created by the French 1988 Huriet-Sérusclat Act; to assess publication bias at a national level.Design Retrospective cohort study.Setting Representative sample of 25/48 French research ethics committees in 1994.Protocols 649 research protocols approved by committees, with follow-up information.Main outcome measures Protocols'' initial characteristics (design, study size, investigator) abstracted from committees'' archives; follow-up information (rates of initiation, completion, and publication) obtained from mailed questionnaire to principal investigators.Results Completed questionnaires were available for 649/976 (69%) protocols. Of these, 581 (90%) studies were initiated, 501/581 (86%) were completed, and 190/501 (38%) were published. Studies with confirmatory results were more likely to be published as scientific papers than were studies with inconclusive results (adjusted odds ratio 4.59, 95% confidence interval 2.21 to 9.54). Moreover, studies with confirmatory results were published more quickly than studies with inconclusive results (hazard ratio 2.48, 1.36 to 4.55).Conclusion At a national level, too many research studies are not completed, and among those completed too many are not published. We suggest capitalising on research ethics committees to register and follow all authorised research on human participants on a systematic and prospective basis.     

Effects of cytochalasin B on Na+ content and cell volume of Entamoeba histolytica

Cells of Entamoeba histolytica accumulated K+ and extruded Na+ compared to the concentrations of those ions present in the growth medium. Pinocytic activity, measured by the uptake of horseradish peroxidase of 125I-polyvinylpyrrolidone, was high (up to 0.3 ml/ml cells per h). Upon addition of cytochalasin B, at a concentration (20 microM) that completely blocked pinocytosis, cells lost up to 40% of their Na+ content within 90 min; K+ content was not affected or increased slightly compared to control cells without the inhibitor. Cation loss was associated with cell shrinkage. The dose-response curves for the effects of cytochalasin B on pinocytosis and Na+ content were identical. These data provide direct evidence that pinocytosis is an important component of the homeostatic system for Na+.     

The role of histones H3 and H1 in the formation of thymine-lysine cross-links during UV-irradiation of deoxyribonucleoprotein

The effect of UV light (lambda = 254 nm) on calf thymus DNP at low ionic strengths was studied. It was found that at the irradiation doses used the protein in the DNA-protein complex increases as the irradiation dose rises. Thermal treatment and acid hydrolysis resulted in a predominant release of histones H3 and H1 from the complex. Data from liquid high performance chromatography, amino acid analysis, thin-layer chromatography point to the induction by UV-light of a thymine-lysine bond, whose formation involves DNA thymines and histone lysine residues, predominantly H3 and H1 fractions.     

Effect of serotonin on the yield of UV- and x ray-induced DNA damage

Using two-dimensional thin-layer chromatography, the effect of serotonin on the yield of thymine dimers and on cleavage of the N-glycosidic bond in the DNA irradiated with ultraviolet (UV) light and X-ray was studied. Bound serotonin was shown to reduce the synthesis of UV-induced thymine dimers but had no effect on the number of X-ray-induced breaks in the N-glycoside bonds in thymidine residues. The data obtained are discussed in terms of the mechanisms of serotonin involvement in the photoprotection of yeast cells from the lethal action of UV and X-ray irradiations.     

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ascorbic acid on the binding of 3H-GABA and 3H-glutamic acid to synaptosomes of the rat cerebral cortex

The effects of different concentrations of L-ascorbic acid (Asc) on Na+-dependent binding of 3H-GABA and 3H-DL-glutamic acid to rat brain cortical synaptosomes were studied. Asc, at a concentration nearly equal to brain extracellular one (3 X 10(-4) M), had no effect on specific and nonspecific 5H-GABA binding. At higher concentrations (10(-3) M) Asc strongly inhibited, and at lower concentrations (10(-6) M) considerably stimulated 3H-GABA binding. At a concentration of 10(-5)-10(-3) M Asc tended to decrease 3H-DL-glutamic acid binding.