Localisation par immunocytologie de sécrétions apparentées aux hormones thyréotropes,gonadotropes et corticotropes dans l'hypophyse distale de l'Axolotl (Ambystoma mexicanum,Shaw)

Résumé Les anticorps anti-TSH (hormone thyréotrope) d'origine bovine, anti-LH (hormone lutéinisante) d'origine ovine et anti (1–24) corticotropine de synthèse ont été appliqués à l'hypophyse de l'Axolotl (Ambystoma mexicanum, Shaw) selon une technique d'immunofluorescence histologique indirecte. Les anticorps anti-TSH se fixent sur une population de cellules fusiformes localisées dans les régions caudale et ventrale de l'hypophyse distale. Les éléments révélés par les anticorps anti-LH possèdent un noyau ovalaire entouré d'un cytoplasme intensément fluorescent et sont répartis dans l'ensemble du lobe hypophysaire distal. Les cellules fixant l'anticorps anti (1–24) corticotropine sont disposées en bordure de l'éminence médiane et présentent un noyau ovalaire et un cytoplasme abondant. Il s'agirait respectivement des cellules à fonction thyréotrope, gonadotrope et corticotrope. L'examen des caractéristiques cytologiques et histochimiques de ces trois types cellulaires complète ces données immunologiques.

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Localisation by immunofluorescence of secretions related to thyreotropins, gonadotropins and corticotropins in axolotl pituitary (Ambystoma mexicanum, Shaw)

Summary Antisera prepared respectively against TSH (thyreotropin) of bovine origin, against LH (luteotropin) of ovine origin and against synthetic (1–24) corticotropin were applied to histological sections of Axolotl pituitary (Ambystoma mexicanum, Shaw) with the indirect immunofluorescence procedure. The anti-TSH antibodies from the first antiserum attach themselves to a population of fusiform cells situated in the caudal and ventral areas of the pars distalis. The cells revealed by the second antiserum show an oval nucleus surrounded by a strongly fluorescent cytoplasm. They are scattered throughout the whole pars distalis. The cells revealed by the third antiserum are arranged around the median eminence. Their nucleus is oval and their cytoplasm abundant. Presumably, these three cell types correspond respectively to the thyreotropic, gonadotropic and corticotropic cells as indicated by a parallel examination of their cytological and histochemical characteristics.

     

Recherche du cytochrome b-563 et du p700 chez trois mutants non photosynthétiques de Chlamydomonas reinhardti

Studies of cytochrome b-563 and P 700 in three non-photosynthetic mutants of Chlamydomonas reinhardAn investigation into the presence of cytochrome b-563 and of P700 in three non-photosynthetic mutants (Fl 5, Fl 9, Fl 15) of Chlamydomonas reinhardti was carried out. These three mutants exhibit several functional anomalies (described elsewhere), which indicate that the electron transport chain between the two photoreactions is blocked. In addition, Fl 5 is unable to carry out any reaction related to System I.Mutants Fl 9 and Fl 15 had less than 19% of the cytochrome b-563 content found in the wild type (which was about 0.27 mole per 100 moles chlorophyll); mutant Fl 5 had more than 80% of this content. The deficiencies (only traces) in bound cytochrome c-553, previously observed with mutants Fl 9 and Fl 15, but not Fl 5, were confirmed (in the wild type, there is about 0.20 mole bound cytochrome per 100 moles chlorophyll).Photosystem I particles, prepared from wild type and mutants Fl 9 and Fl 15 chloroplast fragments, had about 2 (Fl 9, Fl 15) and 3 (wild type) moles P700 per 100 moles chlorophyll. Mutant Fl 5 particles showed neither P700 spectroscopic characteristics nor photooxidation activity; their chlorophyll a/b ratio was lower by a factor of 2 and protein/chlorophyll ratio about 8 times higher than in the wild type particles. This mutant appears to lack P700.     

Détection par immunofluorescence des cellules corticotropes et mélanotropes dans l'hypophyse de la grenouille,Rana temporaria L., au cours du développément

Summary During the embryonic and larval developmental stages of the frog, Rana temporaria L, anti- 1–24, 17–39 corticotropine, and MSH antibodies were used to define, with immunofluorescence technique, the appearence of corticotropic and melanotropic cells.A very small number of fluorescent corticotropic cells appears for the first time during the embryonic stage (10 mm), just before the differentiation of the pars intermedia. The cells are small, their large nucleus is surrounded by a fine rim of fluorescent cytoplasm.During premetamorphic stage, the anti-ACTH antibodies (anti- 1–24 and anti- 17–39 corticotropine) reveal more fluorescent cells in the whole pars distalis. The pars intermedia cells can also be visualized by both antisera.At the end of prometamorphosis and during climax the corticotropic cells show a more precise localization. As in adult frog pars distalis, they are concentrated in the rostral half of the lobe. With the same technique anti- and MSH antibodies reveal only the cells of the pars intermedia. No other cell type of the pars distalis reacts with these antibodies. This technique has the advantage to show that the ACTH and the MSH cells appear very early during the embryonic life.

     

The Biochemical Properties of the Arabidopsis Ecto-Nucleoside Triphosphate Diphosphohydrolase AtAPY1 Contradict a Direct Role in Purinergic Signaling

The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three – GDP, IDP and UDP – were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca²⁺, Mg²⁺ and Mn²⁺ were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent K_m values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in purinergic signaling.     

Role of pyrimidine salvage pathway in the maintenance of organellar and nuclear genome integrity

Nucleotide biosynthesis proceeds through a de novo pathway and a salvage route. In the salvage route, free bases and/or nucleosides are recycled to generate the corresponding nucleotides. Thymidine kinase (TK) is the first enzyme in the salvage pathway to recycle thymidine nucleosides as it phosphorylates thymidine to yield thymidine monophosphate. The Arabidopsis genome contains two TK genes ?TK1a and TK1b? that show similar expression patterns during development. In this work, we studied the respective roles of the two genes during early development and in response to genotoxic agents targeting the organellar or the nuclear genome. We found that the pyrimidine salvage pathway is crucial for chloroplast development and genome replication, as well as for the maintenance of its integrity, and is thus likely to play a crucial role during the transition from heterotrophy to autotrophy after germination. Interestingly, defects in TK activity could be partially compensated by supplementation of the medium with sugar, and this effect resulted from both the availability of a carbon source and the activation of the nucleotide de novo synthesis pathway, providing evidence for a compensation mechanism between two routes of nucleotide biosynthesis that depend on nutrient availability. Finally, we found differential roles of the TK1a and TK1b genes during the plant response to genotoxic stress, suggesting that different pools of nucleotides exist within the cells and are required to respond to different types of DNA damage. Altogether, our results highlight the importance of the pyrimidine salvage pathway, both during plant development and in response to genotoxic stress.     

Semi-automatic classification of skeletal morphology in genetically altered mice using flat-panel volume computed tomography

Rapid progress in exploring the human and mouse genome has resulted in the generation of a multitude of mouse models to study gene functions in their biological context. However, effective screening methods that allow rapid noninvasive phenotyping of transgenic and knockout mice are still lacking. To identify murine models with bone alterations in vivo, we used flat-panel volume computed tomography (fpVCT) for high-resolution 3-D imaging and developed an algorithm with a computational intelligence system. First, we tested the accuracy and reliability of this approach by imaging discoidin domain receptor 2- (DDR2-) deficient mice, which display distinct skull abnormalities as shown by comparative landmark-based analysis. High-contrast fpVCT data of the skull with 200 microm isotropic resolution and 8-s scan time allowed segmentation and computation of significant shape features as well as visualization of morphological differences. The application of a trained artificial neuronal network to these datasets permitted a semi-automatic and highly accurate phenotype classification of DDR2-deficient compared to C57BL/6 wild-type mice. Even heterozygous DDR2 mice with only subtle phenotypic alterations were correctly determined by fpVCT imaging and identified as a new class. In addition, we successfully applied the algorithm to classify knockout mice lacking the DDR1 gene with no apparent skull deformities. Thus, this new method seems to be a potential tool to identify novel mouse phenotypes with skull changes from transgenic and knockout mice on the basis of random mutagenesis as well as from genetic models. However for this purpose, new neuronal networks have to be created and trained. In summary, the combination of fpVCT images with artificial neuronal networks provides a reliable, novel method for rapid, cost-effective, and noninvasive primary screening tool to detect skeletal phenotypes in mice.     

Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library

4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca(2+) -dependent chaperone and a Ca(2+) -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K(d) = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein.