Ultrastructure of scales in a teleost (Carassius auratus L.) after use of rapid freeze-fixation and freeze-substitution

Summary New data on the ultrastructural features of the elasmoid scales ofCarassius auratus have been obtained by use of rapid freezing with subsequent freeze-substitution in anhydrous solvents. These are compared with the results obtained using conventional aqueous fixatives.The external layer of the scales is composed of randomly oriented collagen fibres. In the first stages of mineralization, mineral deposits are located in the interfibrillary substance where dense granules appear to be active sites of mineralization. Spheritic mineralization occurs in this layer.The fibrillary plate is composed of two kinds of collagen fibres. Most of them are organized in lamellae forming the plywood-like structure. They are thicker than the so-called TC fibres, which are oriented from the basal part towards the superficial layer. These TC fibres are involved in the first stages of mineral deposition in the fibrillary plate where inotropic mineralization occurs.The mineral phase is almost always located in the interfibrillary matrix in both layers of the elasmoid scale. In this respect, teleost scales differ from those described so far in other lower vertebrates.

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Resumé Des précisions concernant les aspects ultrastructuraux des dépôts minéraux dans les écailles deCarassius auratus ont été obtenues grâce à l'utilisation de la congélation ultra-rapide suivie d'une cryosubstitution en milieu anhydre. Ces données sont comparées à celles fournies par les méthodes usuelles utilisant des fixateurs aqueux.La couche externe des écailles comprend des fibres collagènes disposées sans ordre apparent. Les dépôts minéraux se produisent surtout dans la substance interfibrillaire où des granules denses semblent représenter des sites actifs au cours de la minéralisation apparentée au type sphéritique.La plaque basale comporte deux catégories de fibres collagènes. Les unes, les plus nombreuses, de plus fort diamètre, sont organisées en lamelles formant une structure en contre-plaqué; les autres appelées fibres TC, orientées de la base de l'écaille vers la zone superficielle, jouent un rôle important dans les premières phases de la minéralisation de type inotropique dans cette partie de l'écaille.Dans les deux couches de l'écaille, la phase minérale est surtout trouvée dans la substance interfibrillaire. De ce fait, les écailles élasmoides des Téleostéens peuvent être distinguées des autres écailles dermiques connues de Vertébrés inférieurs.

     

Dark modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast

The properties of the system which reverses light modulation of NADP-dependent malate dehydrogenase and glucose-6-phosphate dehydrogenase activity in pea chloroplasts were examined. A factor catalyzing dark modulation of these enzymes was found. This factor cochromatographed with thioredoxin in all systems used (Sephacryl S-200, Sephadex G-75, DEAE-cellulose). Inhibition of dithiothreitol-dependent modulation and of dark reversal by antibody against Escherichia coli thioredoxin further suggest that the dark factor is in fact thioredoxin. It appears that the reaction is the reverse of the previously described dithiothreitol-dependent thioredoxin-catalyzed modulation of enzymes. The limiting step in vitro seems to be the oxidation of thioredoxin during the dark period.     

Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations

Summary Postreplication repair of nuclear DNA was examined in an excision defective haploid strain of yeast lacking mitochondrial DNA (ral ⁰). The size of the DNA synthesized in cells exposed to various fluences of ultraviolet light (UV) corresponds approximately to the average interdimer distance in the parental DNA. Upon further incubation of cells following exposure to 2.5 J/m², the DNA increases in size; by 4 h, it corresponds to DNA from uniformly labeled cells. The alkaline sucrose sedimentation pattern of DNA pulse labeled at various times after UV irradiation, for up to 4 h, does not change substantially, indicating that dimers continue to block DNA replication. A significant amount of postreplication repair requires de novo protein synthesis, as determined by its inhibition by cycloheximide. The rad6 mutant does not carry out postreplication repair, the rad18 and rad52 mutants show great inhibition while the rev3 mutation does not affect postreplication repair. Both recombinational and nonrecombinational repair mechanisms may function in postreplication repair and most of postreplication repair is error free.     

Precocious recovery from allotype suppression in transiently chimeric rabbits

Rabbits suppressed for paternal immunoglobulin allotypes specified by the b locus were injected with spleen cells from non-inbred donors at the age of 1 month. This resulted in transient chimerism as shown by the appearance of 10 to >200 μg of donor type Ig per milliliter recipient's serum 1 to 2 weeks after cell transfer. Antibody production by donor cells could not be demonstrated during their survival in the recipients. If, and only if, the donor cells produced Ig of the suppressed allotype, the release from suppression was expedited as judged by the time of appearance and increase of lymphoid cells with membrane-bound Ig of the suppressed type, and also by the onset and rise of secreted Ig of this type in the recipients' sera.     

A restriction cleavage map of ?X 174 DNA by pulse-chase labelling using E.coli DNA polymerase

A pulse chase labelling technique using E. coli DNA polymerase I has been used to determine a number of restriction endonuclease cleavage maps of ØX 174 DNA. In addition to verifying the accuracy of the method by confirming some previously published maps, the Hha I and Alu I maps have also been constructed.     

Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36–54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351–727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.     

Triploidy—Observations in 154 Diandric Cases

Hydatidiform moles (HMs) are abnormal human pregnancies with vesicular chorionic villi, imposing two clinical challenges; miscarriage and a risk of gestational trophoblastic neoplasia (GTN). The parental type of most HMs are either diandric diploid (PP) or diandric triploid (PPM). We consecutively collected 154 triploid or near-triploid samples from conceptuses with vesicular chorionic villi. We used analysis of DNA markers and/or methylation sensitive-MLPA and collected data from registries and patients records. We performed whole genome SNP analysis of one case of twinning (PP+PM).In all 154 triploids or near-triploids we found two different paternal contributions to the genome (P1P2M). The ratios between the sex chromosomal constitutions XXX, XXY, and XYY were 5.7: 6.9: 1.0. No cases of GTN were observed. Our results corroborate that all triploid human conceptuses with vesicular chorionic villi have the parental type P1P2M. The sex chromosomal ratios suggest approximately equal frequencies of meiosis I and meiosis II errors with selection against the XYY conceptuses or a combination of dispermy, non-disjunction in meiosis I and meiosis II and selection against XYY conceptuses. Although single cases of GTN after a triploid HM have been reported, the results of this study combined with data from previous prospective studies estimate the risk of GTN after a triploid mole to 0% (95% CI: 0–1,4%).