Synthèse d'ARN et de protéines dans des disques de patte de drosophile cultivés in vitro

Résumé Des disques imaginaux de patte, prélevés dans des larves de fin de 3e stade de drosophile, ont été cultivés dans le milieu M de Mandaron, en l'absence d'hormone ou en présence soit d'- soit de -ecdysone. L'incorporation de précurseurs marqués (³H-uridine,³H-leucine ou³H-proline) a été étudiée en fonction du stade de développement des disques.En l'absence d'hormone de mue, l'incorporation d'uridine décroît dès que les disques ont été explantés; l'incorporation de leucine et de proline ne décroît que 6 à 12 heures après l'explantation.- et -ecdysone stimulent l'incorporation des trois précurseurs; toutefois celle-ci varie en fonction du développement morphologique du disque.Les maxima et les minima d'incorporation d'uridine précèdent dans le temps ceux de la leucine et de la proline.Les maxima d'incorporation peuvent être mis en rapport avec des évènements morphologiques marquants du développement: évagination, sécrétion des cuticules nymphale et maginale.Il n'y a pas de différences significatives d'incorporation d'uridine en présence d'-ecdysone ou de -ecdysone; en revanche les maxima d'incorporation de leucine et de proline sont plus élevés en présence d'-ecdysone que de -ecdysone.Ces résultats montrent que l'-ecdysone—et à un degré moindre la -ecdysone—peuvent induire les synthèses de macromolécules nécessaires au développement des appendices in vitro.

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RNA and protein synthesis inDrosophila leg discs cultured in vitro

Summary Imaginal leg discs from late third instarDrosophila larvae were cultured in Mandaron's medium without hormone or with -ecdysone or -ecdysone. Incorporation of labelled precursors (tritiated uridine, tritiated leucine or tritiated proline) was studied as a function of the stage of in vitro disc development.In the absence of moulting hormone, uridine incorporation decreased as soon as the discs were explanted; leucine and proline incorporation however began to decrease only after 6 to 12 h.- and -ecdysone stimulated the incorporation of all three precursors; however the rate of the incorporation varied as a function of the morphological disc development.The maxima and minima of uridine incorporation preceeded in time those of proline and leucine incorporation.The peaks of incorporation were coincident with salient morphological events of development: evagination, secretion of pupal and imaginal cuticles.There were no significant differences in uridine incorporation in the presence of -ecdysone or -ecdysone. Leucine and proline incorporation maxima however were significantly higher in the presence of -ecdysone than of -ecdysone.The results show that -ecdysone—and to a lesser extent also -ecdysone—can induce the macromolecular syntheses required for the development of the appendage in vitro.

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Ce travail a été réalisé avec l'aide du CNRS (Action thématique programmée «Différenciation cellulaire», contrat n^o A 6554324)

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Ce mémoire représente une partie de la thèse qui sera soutenue par l'auteur devant l'Université Scientifique et Médicale de Grenoble     

Ultrastructure des disques de patte de drosophile cultivésin vitro. Evagination,sécrétion de la cuticule nymphale et apolysis

Summary Ultrastructural observations were made on imaginal leg discs ofDrosophila cultured in vitro in Mandaron's medium M in the presence of - or -ecdysone.During evagination of discs in vitro the tight epidermal folds of the disc flatten out progressively and the cells change their shape.As the discs evaginate, the pupal cuticle is secreted at the apex of the cells: it comprises a three-layered cuticulin, an epicuticle and a thick endocuticle. Secretion of pupal cuticle always starts by the appearance of dense deposits at the apex of microvilli. Only the latter apparently participate in the construction of the three successive layers of the pupal cuticle.During disc development an abundant rough endoplasmic reticulum is formed. Nuclei and nucleoli become progressively enlarged. At the same time, the nucleus, initially located near the base of the cell, moves closer to the apical cell surface. Microtubules become fragmented as soon as evagination begins and are reorientated parallel to the cell surface (i.e. parallel to the new long axis of the cell) at the end of evagination.

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Ce travail a été réalisé avec l'aide du CNRS (Action thématique programmée) «Différenciation cellulaire», contrat n^o A 6554324.

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Ce mémoire représente une partie de la thèse qui sera soutenue par l'auteur devant l'Université Scientifique et Médicale de Grenoble.     

A specific radioimmunoassay for PGE2 using an antibody with high specificity and a sephadex LH-20 microcolumn for the separation of prostaglandins

Antiserum against PGE₂ was raised in rabbits following immunization with prostaglandin-hen-γ-globulin conjugate. The antiserum exhibited 14% cross reactivity with PGE₁ and far less cross-reaction with heterologous prostaglandins. A microcolumn of Sephadex LH-20 was used for a partial, but sufficient separation of PGE₂ from PGE₁ and a complete separation from heterologous prostaglandins to ensure a specific RIA for PGE₂. The precision of the method in the range 10–500 picograms showed a coefficient of variation varying between 4 and 13%. The detection limit was 10 picograms corresponding to 15 pg/ml of PGE₂ in serum.In order to demonstrate the validity of the method values obtained for non-diuretic rat renal venous serum were compared with those obtained using the isotope derivative method of Bojesen & Buckhave (1972) on the same samples. The concentrations of PGE₂ obtained were 239 ± 25 pg/ml and 250 ± 58 pg/ml, respectively.     

A comparison of the structures of apo dogfish M4 lactate dehydrogenase and its ternary complexes

Details are recorded of the X-ray diffraction data collection, heavy atom refinement and preliminary structure refinement for two different dogfish M₄ lactate dehydrogenase structures. One of these is the 2.0 Å resolution apoenzyme structure; the other is a 3.0 Å resolution abortive ternary complex. Two other ternary substrate inhibitory complexes (LDHase²: NAD: oxalate and LDHase: NADH: oxamate), isomorphous with the abortive ternary complex (LDHase: NAD-pyruvate), have also been examined. The apo-LDHase and LDHase: NAD-pyruvate structures are systematically compared to determine significant differences in their conformation. These are related to differences in structure amongst the three studied ternary complexes. These differences all occur in regions of the protein around the active site, particularly the flexible loop covering the active center pocket and the C-terminal helix αH. The changes are suggestive of a domino effect whereby the closing of the loop on binding coenzyme and substrate triggers the critical reactive residues into assuming their catalytically active positions.     

Characterization of a monoclinic crystal form of an enzyme of steroid metabolism, Δ5-3-ketosteroid isomerase

A monoclinic crystal form ($\text{P2}{}_1\text{, a = 140.4}\text{A}\text{?}\text{, b = 85.0}\text{A}\text{?}\text{, c = 94.5}\text{A}\text{?}$, β= 130.1 °) of Δ⁵-3-ketosteroid isomerase from Pseudomonas testosteroni (EC 5.3.3.1), grown at pH 7.0, has been characterized. Crystal-density measurements show that the asymmetric unit contains 12 protomers (M_r = 13,394).     

The pineal gland of the mole-rat (Spalax ehrenbergi,Nehring)

Summary A comparative investigation of the distribution of monoaminergic neurons in non-malacostracan crustaceans was performed with the histochemical fluorescence method of Falck-Hillarp.Two fluorophores were found: the more widespread of the two emits a green fluorescence; and the more sparsely distributed emits a yellow to brown-yellow fluorescence.Specific green fluorescent areas were shown to exist in the protocerebrum. The central body and the optic ganglia of the compound eye (where present) are always fluorescent. Moreover, the centre of the nauplius eye may have a green fluorophore, as in ostracods, and a neuropile area, here called the frontal area. These neuropile centres are known from ordinary histological studies of the nervous system. In addition, there are specific monoaminergic centres, such as the so-called dorsal area of phyllopods and anostracans as well as the copepod specific areas. Specific monoaminergic areas appear in the deutocerebrum and the suboesophageal ganglion where they are particularly well developed.Presumed sensory neurons in the cavity receptor organ of Artemia salina are shown to be monoaminergic. Monoaminergic sensory neurons have not been described previously in Arthropods.Presumed motor innervation of hind-gut and trunk muscles is also found, and it is concluded that in crustaceans neurons of every type (sensory, internuncial, motor) may be monoaminergic.We have enjoyed unrestricted laboratory facilities at the Department of Histology, Faculty of Medicine, and with great pleasure express our sincere thanks to Prof. Bengt Falck. — Grants from the Swedish Natural Science Research Council (2760-007), the Swedish Medical Research Council (04X-712), the Royal Swedish Academy of Science (Hierta-Retzius), the Royal Physiographic Society of Lund, and the University of Lund supported the work.