Les protéines des corps d'inclusion des baculovirus

La comparaison des protéines des polyèdres des Baculovirus deGalleria mellonella L.,Lymantria dispar L.,Autographa californica Speyer,Mamestra brassicae L.,Arctia caja L.,Bombyx mori L. etAntheraea pernyi Guer. en immunodiffusion montre qu'il existe entre elles une étroite parenté antigénique. Des différences entre ces protéines peuvent être mises en évidence à l'aide de sérums spécifiques obtenus dans des conditions particulières d'immunisation.     

Hydrolyse enzymatique des protéines par les bactéries du rumen

The hydrolysis of proteins in the rumen is a process brought about mainly by bacteria, of which many species produce proteases. The majority of endopeptidases are cysteine proteases, whereas exopeptidases are mainly aminopeptidases. Prevotella ruminicola is distinguished from other bacterial species by its capacity to produce dipeptidases such as type I dipeptidyl aminopeptidase. The mechanisms controlling the synthesis of endo- and exopeptidases have been little studied. Enzyme production seems to depend on the concentrations of peptides, amino acids and carbohydrates. Proteolytic activity varies in relation to pH, and the concentrations of ions and phenolic compounds. Various works have shown that hydrolysis of a protein by enzymes depends on its three-dimensional structure and possible bonding to non-protein structures. These properties determine the peptide and amino acid concentrations that occur in the rumen. The molecular weight, hydrophobic property and primary structure of the peptides are the main factors that affect the hydrolysis and/or uptake of these compounds by rumen bacteria. The methodological problems inherent to assaying these compounds do however lead to current divergences of opinion concerning the physico-chemical characteristics of the peptides that escape rumen fermentation.     

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     

Le rôle des protéines liant les odeurs (OBP) dans la transduction olfactive

This paper reviews biochemical and functional properties of a family of proteins involved in the transduction process of chemical signals. Odorant-binding proteins (OBPs) are small soluble proteins highly concentrated in the chemosensory organs of Insects and Vertebrates. They are preferentially expressed in the nasal mucus of Vertebrates and in the sensillar lymph of Insects. They have been found to bind reversibly small hydrophobic molecules detected via the olfactory system. The vertebrate OBPs bind non-specific odorants with low affinities. They belong to the lipocalin family as well as other proteins involved in chemical communication and associated to different organs and functions. Nevertheless, no specific ligand for OBPs has been yet identified in Vertebrates. However, the large microdiversity of OBPs in the same animal suggests that OBPs could be involved in the discrimination of odors. Chemical communication in Noctuid moths was used as a model to study the molecular mechanisms of odor recognition. The pheromonal system is extremely sensitive and specific since the male is able to detect only few molecules of the pheromone and to recognize specific blends of the same molecules. The chemical signals were identified for a large number of Lepidopteran species and the associated behaviours they elicit were fully characterized. The Lepidopteran OBPs are divided into two sub-classes according to their ligands: pheromone-binding proteins (PBP) are expressed in sensilla trichodea responding to pheromonal compounds while general odorant binding proteins (GOBP) are associated with sensilla basiconica tuned to the detection of general odors, such as plant volatiles. The PBPs selectively bind components of the female sex-pheromone with measurable affinities. The ligand binding site was localized in the 40–60 aminoacids region. Substitutions in the binding site of different proteins are correlated with the fixation of different ligands, leading to the hypothesis that the primary structure encodes the ligand specificity. Other proteins expressed in chemosensory organs of other orders of Insects were cloned or purified. In absence of functional data, they were called OBP-like. Some of them were localized in the gustatory organs and could be common carriers of odors in both olfactory and gustatory systems. Many arguments are in accordance with an active role of the OBPs in the early steps of odor discrimination. The heterogeneity of OBPs inside species and between species, the spatial segregation in their expression and the different binding affinities of PBPs towards pheromonal compounds support the hypothesis that the coding of odors is realized as soon as the level of OBPs. More, the complex OBP/odor could be the stimulus for olfactory receptors cloned in Vertebrates and still putative in Insects. This hypothesis suggests that OBPs take part as an essential element of the chemosensory transduction.     

Polymorphisme électrophorétique des protéines et enzymes sériques et érythrocytaires chez le chimpanzé (Pan troglodytes)

Summary The electrophoretic mobilities of serum enzymes and other proteins were investigated at 35 loci in 25 unrelated individuals of the chimpanzee Pan troglodytes. In this population the mean average of individual polymorphism is =0181, and the average level of heterozygoty =0.022. The same pattern of individual variability is found in Pan troglodytes and Homo sapiens.     

Distances éntre l'homme et les singes anthropoïdes basées sur la mobilité électrophorétique des protéines et enzymes

Electrophoretic mobilities of ten homologous serum proteins and enzymes in Man and anthropoid apes led to estimations of the genetic distances between five species (Homo, Pan, Gorilla, Pongo and Symphalangus). The separation of the Symphalangus (Siamang) lineage from that leading to the great apes and Man is obvious. Less evident is the cluster containing only humans and chimpanzees, and also the fact that orang-utans are placed closer to Man than gorillas.     

Côntrole neuroendocrine des protéines sanguines vitellogenes d'Anacridium aegyptium sain et parasite

The changing patterns of haemolymph proteins were followed in male and female adults of normal and parasitized Anacridium aegyptium during diapause (autumn, winter) or during activity (spring) of their endocrine system without or with electrostimulations of the pars intercerebralis (PI).The haemolymph protein concentration is high in winter and decreases in spring. It is comparatively depleted in locusts infected by the fly Metacemyia calloti. However, the depletion is significant only in ‘castrated’ females.Fifteen protein fractions were resolved by polyacrylamide disk gel electrophoresis in haemolymph of normal and infected locusts during diapause and activity. Some fractions decrease in quantity during activity in males, normal females, and parasitized females with complete ovarian development. One fraction disappears in females with mature eggs and seems correlated with formation of the eggshell. Eight others protein fractions exhibit electrophoretic mobility identical to the 7 protein fractions of homogenates of eggs. There is little doubt that these haemolymph protein fractions are involved in yolk synthesis and are thus ‘vitellogenic’. One of these ‘vitellogenic’ fractions (band 6) is larger in yolk than in blood.Five protein fractions were demonstrated by electrophoresis of homogenates of parasites. Their electrophoretic mobilities are similar to those of 5 of the 8 haemolymph ‘vitellogenic’ fractions of the host. There is little doubt that these 5 haemolymph protein fractions (one of them is the band 6) are involved in the nutritional requirements of the parasite.Electrostimulation of the PI, during diapause and activity, increase the haemolymph protein concentration and chiefly the protein concentration of the blood band 6. Thus, the median neurosecretory cells of the brain (M-NSC) regulate protein synthesis and chiefly the synthesis of ‘vitellogenic’ proteins.In parasitized females, the increase of the haemolymph protein concentration after electrostimulations of the PI is associated with an enhancement of ovarian development. The depletion of the haemolymph protein concentration in ‘castrated’ females is thus involved in the inability of the oöcytes to sequester available proteins from the haemolymph. The haemolymph protein deficiency may be attributed to (1) an impairment of protein synthesis, attendant upon the hypoactivity of the M-NSC, and (2) the nutritional requirements of the parasite.     

Les protéines des corps d'inclusion desBaculovirus III. Étude comparée de la granulose dePieris brassicae et de la polyédrose deBombyx mori

Résumé L'étude des protéines des granules dePieris brassicae L. et des polyèdres nucléaires deBombyx mori L. par les techniques de filtration sur gel, d'électrophorèse et d'immunodiffusion révèle de nombreuses ressemblances. La comparaison des protéines des corps d'inclusion engendrés par lesBaculovirus des Lépidoptères contribue à montrer l'homogénéité de ce groupe.

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Summary It is generally admitted that the viruses of nuclear polyhedroses and granuloses of Lepidoptera form two distinct groups serologically. A comparative study of proteins of nuclear polyhedral bodies ofBombyx mori and of granules ofPieris brassicae was carried by using techniques of gel filtration, electrophoresis and immunodiffusion. The characteristics of proteins of granules studied by gel chromatography and electrophoresis are closely related to those of proteins of polyhedral bodies. In immunodiffusion, the extraction of proteins of polyhedral inclusion bodies by the carbonate reveals a common antigen (C antigen) which is recognized by homologue serums and anti-protein heterologue serums of inclusion bodies of seven otherBaculovirus. The extraction by the thioglycolate reveals, in addition to C antigens, specific antigens: T antigen for the granulosis ofPieris brassicae and B antigens for the nuclear polyhedrosis ofBombyx mori. The comparison of proteins of inclusion bodies generated by granulosis and nuclear polyhedrosis viruses contributes to show the homogeneity of the LepidopterBaculovirus group.

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Avec la collaboration technique de LilianeCroizier.     

Sur des aspects particuliers des protéines élaborées dans la glande mammaire. Etude au microscope électronique chez la lapine en lactation

Résumé L'étude au microscope électronique de la glande mammaire de la lapine en lactation a montré 1) des remaniements dans le cytoplasme des protéines élaborées et 2) une localisation inhabituelle des grains de protéine dans les espaces inter-cellulaires et le tissu conjonctif péri-acineux. Ces phénomènes répondent vraisemblablement à une stase lactée avec réabsorption du produit sécrétoire.

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Summary The electron microscopic study of the mammary gland of the lactating rabbit has shown 1) a fusion and probably rearrangement within the cytoplasm of the secreted proteins, and 2) an unusual localization of protein granules in the intercellular spaces and the periacinar connective tissue. These phenomena probably indicate a stasis of the secretory product accompanied by its reabsorption.

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En hommage au Professeur W. Bargmann.     

Nature et évolution des protéines basiques au cours de la spermiogenèse chez Pleurodeles waltlii Michah., Amphibien urodèle

Résumé La spermatogenèse du Triton Pleurodeles waltlii a été étudiée au moyen de techniques cytochimiques et autoradiographiques. Les phénomènes spermiogénétiques ont été divisés en six stades caractéristiques en fonction de la forme du noyau et de l'évolution des structures extranucléaires: cou et filament de soutien de la queue.Les protéines basiques du noyau du spermatozoïde sont du type protamine, tandis que certaines structures extranucléaires se montrent riches en protéines basiques libres. Les transitions depuis les histones de type somatique présentes dans les jeunes spermatides I et II jusqu'aux protamines du spermatozoïde mûr par l'intermédiaire d'histones enrichies en arginine, montrent des phénomènes intéressants.La première transition depuis les histones de type somatique riches en lysine vers les histones enrichies en arginine est progressive. Elle se produit au cours des stades III et IV. Un gradient apparaît dans le noyau depuis la base vers le sommet. Au même moment, les protéines basiques libres apparaissent dans le cou.La perte des histones enrichies en arginine puis leur substitution par des protamines au niveau des noyaux des spermatides V est beaucoup plus brutale, elle intéresse tout le noyau. Ce dernier montre de même une disparition complète des acides aminés soufrés. Au même moment, les protéines basiques apparaissent dans la queue, de même que des acides aminés soufrés.Toutes ces observations plaident en faveur d'une origine nucléaire possible de certains composants de ces structures extranucléaires originales et riches en protéines basiques libres.

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Nature and development of the basic proteins during the spermiogenesis in Pleurodeles waltlii michah., urodele amphibian

Summary The spermiogenesis of the newt Pleurodeles waltlii has been studied with cytochemical and autoradiographical methods. The process of spermiogenesis has been divided into six characteristic steps in relation with the nuclear shape and the evolution of the extranuclear structures: the neck and the supporting filament of the tail.The nuclear basic proteins of the spermatozoon are of protamine type while the well developed extra-nuclear structures show a free basic proteins rich content. The transition from early spermatids somatic type histone to the mature sperm protamine via arginine-rich histone and the appearance of the free basic proteins in the tail show different interesting features. The first transition from the somatic rich lysine histone to the arginine rich histone which occurs at the spermatid III and IV steps, is gradual. A gradient appears in the nuclei from the base to the apex. Concurrently the free basic proteins appear in the neck. The loss of the arginine rich histone and its substitution by protamine in the spermatid V is more sudden and affects all the nuclei without a gradient, the sulfhydryl group content of the nuclei also disappear. At the same time, the basic protein of the tail appear as well as the sulfhydryl group.All these results are discussed in view of the hypothetical nuclear origin of these very unusual extranuclear basic proteins.

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Equipe de Recherche associée au C.N.R.S. Cytologie Ultrastructurale No. 129.

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Avec la collaboration technique de Melle F. de Sallier Dupin et de M. B. Morille.     

Caractérisation d'une hémopexine sérique de truite par utilisation d'une protéine recombinante

Rainbow trout hemopexin was produced by Sf9 cells through the Baculovirus system; the protein is released into the culture medium. It has been purified through two successive chromatographic steps: ion exchange (MonoQ) and chelation (Zn⁺ affinity). Treatment of the protein by N-glycosidase F reduced its relative mass by 3 000, demonstrating N-glycosylation. Spectrophotometric analysis in the Soret band of protein + hemin mixture revealed characteristics of a hemoprotein (oxidized: λ = 413 nm, dithionite-reduced: λ = 422 nm); the titration curve suggested a 1:1 molar ratio. An anti tHx antiserum was produced; it detected an intense band in the trout serum, but also in the serum of two perciforms: gilt-head sea bream and sea bass; it did not cross-react with the serum of the phylogenetically distant eel.     

Inhibiteurs anti-trypsine et activités protéolytiques des Albumines de graine de Vigna unguiculata

Summary The extracellular space of tentacles of Drosera capensis L. is divided into two compartments by cuticular material between cells of an endodermoid layer and by the nonporous cuticle of the stalk and neck. The distal compartment includes the mucilaginous secretion as well as the free space of the secretory cap, since the cuticle covering the cap is perforated by numerous 0.05–0.3 m pores. The proximal compartment includes xylem and the intercellular space of the stalk. The existence of the endodermoid partition is consistent with the observation that action potentials recorded extracellularly from the head may be positive-going while those recorded extracellularly from the stalk are negative-going. The partitioning is also consistent with the hypothesis previously proposed to explain why the amplitude of action potentials recorded from the mucilage varies as a function of the amplitude of the receptor potential.The living cells are united by plasmodesmata. Unusually abundant plasmodesmata were observed in the walls between endodermoid cells and neck cells, between neck cells and the next row of outer stalk cells, in the end walls connecting the outer stalk cells, and the end walls connecting the inner stalk cells: these strategically located plasmodesmata presumably permit the electrotonic spread of receptor potentials and action potentials between cells.     

Sur les ferments protéolytiques duVibrio cholerae et duVibrio El Tor

Conclusions On peut conclure, en résumé, que l'hémodigestion et la liquéfaction de la gélatine sont des manifestations de 2 ferments.Au surplus, les comportements du pouvoir de scission de la gélatine et du pouvoir hémodigestif furent comparés chez 4 souches deV. El Tor cultivées sur gélose et conservées sur un milieu renfermant du blanc d'oeuf. A cette occasion on a eu recours au viscosimètre de Ostwald.Il fut observé que, chez les souches deV. El Tor cultivées sur gélose, la scission de la gélatine et l'hémodigestion furent plus intenses que chez des souches conservées sur milieu à l'oeuf. En cutre, chez ces dernières, le pouvoir hémodigestif avait entièrement disparu.     

Détection des différentes formes du prostate-specific antigen et autres kallicréines dans le cancer de la prostate

Prostate cancer is the most common cancer in men over 50 years old and the second leading cause of cancer death. Prostate-specific antigen (PSA) is widely used for the diagnosis and follow-up of prostate cancer. PSA, also called kallikrein 3, is a member of the human kallikrein-type serine protease family and circulates in the blood stream in the form of complexes with serum protease inhibitors and in free form. However, free PSA is also a heterogeneous mixture of different molecular PSA forms: proPSA, intact and clived mature forms. The clinical significance of these different forms is still unclear but their specific measurement in serum could improve the specificity of PSA for detecting cancer or predicting treatment outcome. Others kallikreins including kallikrein 2, 4, 11, 14 and 15 are also emerging as complementary markers to PSA for prostate cancer. Multiple detection of the different molecular forms of PSA, as well as of these kallikreins, in addition to total PSA, could significantly increase the diagnostic utility of PSA and may add prognostic value by bringing clinical information on the cancer progression.