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Five human clinical isolates of an unknown, strictly anaerobic, slow-growing, Gram-stain-negative, rod-shaped micro-organism were subjected to a polyphasic taxonomic study. Comparative 16S rRNA gene sequence-based phylogeny showed that the isolates grouped in a clade that included members of the generaPyramidobacter,Jonquetella, andDethiosulfovibrio; the type strain ofPyramidobacter piscolenswas the closest relative with 91.5–91.7 % 16S rRNA gene sequence similarity. The novel strains were mainly asaccharolytic and unreactive in most conventional biochemical tests. Major metabolic end products in trypticase/glucose/yeast extract broth were acetic acid and propionic acid and the major cellular fatty acids were C13 : 0and C16 : 0, each of which could be used to differentiate the strains fromP. piscolens. The DNA G+C content based on whole genome sequencing for the reference strain 22-5-S 12D6FAA was 57 mol%. Based on these data, a new genus,Rarimicrobiumgen. nov., is proposed with one novel species,Rarimicrobium hominissp. nov., named after the exclusive and rare finding of the taxon in human samples.Rarimicrobiumis the fifth genus of the 14 currently characterized in the phylumSynergistetesand the third one in subdivision B that includes human isolates. The type strain ofRarimicrobium hominisis ADV70T( = LMG 28163T = CCUG 65426T).

La première partie de ce travail a été consacrée à l’étude de l’expression du système IGF dans les gonades de deux espèces de poisson : la truite arc-en-ciel (gonochorique) et la daurade royale (hermaphrodite successif protandre). Une des approches pour montrer que les IGFs sont produits localement dans les gonades de poisson, a été de rechercher leurs ARN messagers, d’identifier les types cellulaires exprimant ces facteurs de croissance et leur cellules cibles. La régulation hormonale de l’expression des ARNm des IGFs a également été abordée in vivo et in vitro. Les ARNm des IGFs sont préférentiellement exprimés dans les gonades immatures. L’expression des transcrits de l’IGF-I chute brutalement au cours du développement gonadique, tandis que les niveaux des ARNm de l’IGF-II se maintiennent. D’autre part, l’expression préférentielle de l’IGF-II et la distribution ubiquitaire de son transcrit dans les gonades, suggère un rôle important de ce facteur de croissance dans la physiologie gonadique. L’effet stimulateur de l’hormone de croissance (x 8 versus témoins) et des gonadotropines (x 2-3 versus témoins) sur l’expression des ARNm des IGFs dans le testicule de truite a été mis en évidence. Par immunohistochimie, nous avons montré l’expression de l’IGF-I dans les spermatogonies A, mais aussi dans les spermatogonies engagées dans le processus de spermatogenèse, les cellules de sertoli et les cellules interstitielles du testicule. Dans l’ovaire, l’IGF-I a été localisé dans le cytoplasme des follicules prévitellogéniques, ainsi que dans les cellules folliculaires des ovocytes prévitellogéniques, vitellogéniques et matures. Par Northern blot, 3 types d’ARNm des IGF-IR, de 11, 7 et 2.4 kb ont été détectés dans les gonades de daurade, quel que soit leur stade. Par immunohistochimie, les deux types de récepteurs aux IGFs, récemment mis en évidence chez les poissons, ont été détectés dans les spermatogonies A, les cellules de sertoli et les cellules interstitielles du testicule. Dans l’ovaire, les cellules folliculaires des ovocytes prévitellogéniques, vitellogéniques et matures expriment ces deux récepteurs. En conclusion, nos travaux ont permis de montrer l’existence d’un système IGF intragonadique chez le poisson. Le rôle biologique de ce sytème dera être étudié. La seconde partie de ce travail a été consacrée à l’étude de l’ontogenèse des IGFs et de leurs récepteurs au cours du développement embryonnaire et larvaire chez la daurade. L’expression de ces différents transcrits a été détectée dans les œufs non-fécondés, les embryons et les larves. Les ARNm de l’IGF-II sont plus abondants que ceux de l’IGF-I dans les larves. Par immunohistochimie, nous avons mis en évidence la présence de deux IGF-IR dans la plupart des tissus larvaires. Dans la limite temporelle de notre étude, l’IGF-I est préférentiellement exprimé dans le muscle squelettique et le pancréas. La localisation des IGF-IR est similaire, excepté dans le corps du cervelet et le muscle, où seul SpIR6 a été détecté. L’ensemble de ces résultats suggère l’apparition d’un système IGF très tôt au cours de développement chez la daurade.

International audience; The deadenylation of maternal mRNAs in the Xenopus embryo is a sequence-specific process. One cis element that targets maternal mRNAs for deadenylation after fertilization is the embryo deadenylation element (EDEN). This element, composed of U/R repeats, is specifically bound by a protein, EDEN-BP. In the present study we show that the rate at which an RNA containing an EDEN is deadenylated can be increased by the presence of an additional cis element composed of three AUU repeats. This effect was observed for a natural EDEN (c-mos) and two synthetic EDENs. Hence, the enhancement of EDEN-dependent deadenylation conferred by the (AUU)3 motif is not due to an interaction with a particular EDEN sequence. Mutation of the (AUU)3 motif abrogated the enhancement of EDEN-dependent deadenylation. These data indicate that the rate at which a specific maternal mRNA is deadenylated in Xenopus embryos is probably defined by a cross talk between multiple cis elements.

The structure-activity relationship study of one of recently described aromatase inhibitors, compound 1 (MR20814), allowed us to design some related derivatives as potential new inhibitors. Among those we synthesized, chlorophenylpyridylmethylenetetrahydroindolizinone 5 (MR20492) exhibited in vitro a ten-fold higher inhibition of the enzyme (IC50 = 0.2 +/- 0.0 microM and Ki = 10.3 +/- 3.3 nM).

International audience; The poly(A) tail of mRNAs plays an important role in translational control. In Xenopus laevis matured oocytes, maternal mRNAs that contain a cytoplasmic polyadenylation element (CPE) are polyadenylated, whereas CPE deficient mRNAs are deadenylated by a default process. Eg mRNAs are maternal transcripts that are poly(A)+ in matured oocytes and rapidly deadenylated after fertilization. This post-fertilization deadenylation of Eg mRNAs requires specific sequence information. Such a deadenylation element has been identified previously in the 3'UTR of Eg2 mRNA. In this study, we show that cell-free extracts made from embryos or activated eggs contain two kinetically distinct deadenylation activities, with different substrate specificities. One, responsible for the slow deadenylation of RNAs that are devoid of a functional CPE, has the characteristics of a default PAN activity. The other effectuates the rapid deadenylation of RNAs containing a deadenylation element. The in vitro system described here will allow the characterization of factors controlling the deadenylation of Eg mRNAs in embryos.

International audience; Rainbow trout hepatocytes were isolated by a two step perfusion technique and maintained either in monolayer culture for 5 days or in aggregate culture for 30 days. Cytochrome P450 content decreased from day 0 to day 5 in both culture systems, and then was preserved at the same level after one month in aggregated cells. 7-Ethoxyresorufin O-deethylase (EROD) and UDP-glucuronosyl transferase were not significantly different in freshly isolated cells and in 30-day aggregated hepatocytes, whereas a substantial increase in glutathione S-transferase was observed. Two-day exposure of cells to beta-naphthoflavone led to a significant increase in EROD activity in both culture systems, especially after one month of aggregation (10-fold increase). According to these results, aggregate culture of rainbow trout hepatocytes seems to be a promising in vitro model to investigate the biotransformation pathways in fish and their regulation by endogenous and exogenous compounds.

The decapeptide gonadotropin-releasing hormone (GnRH) is a key hormone for the central regulation of reproduction. The distribution of salmon GnRH (sGnRH), which is the major form in salmonids, has been studied in different fish species by immunocytochemistry. Discrepancies in data concerning the distribution of sGnRH perikarya led us to investigate this problem in two species, the Atlantic salmon and the rainbow trout, with in situ hybridizaiton of sGnRH messenger, a highly specific molecular tool. By Northern blot analysis, the rainbow trout sGnRH messenger appears to be about 500 bases in length, which is close to those isolated from Atlantic salmon or masu salmon and characterized previously. In situ hybridization with riboprobes generated with Atlantic salmon sGnRH cDNA demonstrated that sGnRH perikarya are restricted to the ventral part of olfactory bulbs, telencephalon, and preoptic area. They are distributed on a nearly continuous line extending from the olfactory bulbs to the preoptic area in both salmonid species studied. Despite the presence of GnRH-like immunoreactivity in the preoptic magnocellular nucleus (NPOm) and in the tegmentum of the midbrain (MT), the sGnRH mRNA is not present in these two structures. Stained cells in NPOm could be target cells for GnRH and immunoreactive neurons in MT are likely to be chicken GnRH-II containing cells. Our study not only gives a precise distribution of the sGnRH system in two salmonids, Atlantic salmon and rainbow trout, but also clarifies the ambiguous data published up to now in rainbow trout.

International audience; Elongation factor 1 mediates the elongation step of mRNA translation. The transfer of aminoacyl-tRNA to ribosomes under hydrolysis of GTP is catalyzed by a GTP binding protein, EF1Ia. A guanine-nucleotide exchange complex now referred as EF1beta gamma delta replaces GDP by GTP on EF1alpha (1). A complex, purified from Xenopus oocytes as a substrate for the meiotic and mitotic p34Cdc2 kinase, was shown to contain the guanine-nucleotide exchange activity (2). The Xenopus complex is composed of three main proteins, p30, p36 and p47. Surprisingly, microsequencing of two of its components, p36 and p30 suggested the presence of two related proteins (2). We have previously cloned and sequenced the cDNA encoding for p47 or EF1gamma (3) and p36 or EF1delta (4), we present here the molecular cloning of p30 or EF1beta. This result allows for the first time, sequence analysis of EF1beta and delta proteins, both present in the same complex.

International audience; In the amphibian Xenopus laevis, the elongation factor 1 alpha proteins (EF-1 alpha) synthesised in oocytes and somatic cells correspond to distinct gene products. Furthermore, the somatic EF-1 alpha gene (EF-1 alpha S) produces one of the most highly expressed early zygotic transcripts in the embryo. The functional recycling of EF-1 alpha (conversion of EF-1 alpha-GDP to EF-1 alpha-GTP) is assured by the EF-1 beta gamma complex. We show here that in Xenopus laevis embryos, contrary to the situation for EF-1 alpha, EF-1 beta, and EF-1 gamma mRNAs are transcribed from the same genes in oocytes and somatic cells. In addition, the onset of transcription of the EF-1 beta and EF-1 gamma genes from the zygotic genome occurs several hours after that of the somatic EF-1 alpha S gene. Therefore, during early Xenopus development the expression of these three elongation factors is not co-ordinated at the transcriptional level. The consequences of this uncoupling on the efficiency of translational elongation in the early Xenopus embryo are discussed.

Maternal Xenopus Eg mRNAs have been previously identified as transcripts that are specifically deadenylated after fertilization and degraded after the mid blastula transition. Destabilizing cis sequences were previously localised in the 3 untranslated region of Eg2 mRNA. In order to characterize possible trans-acting factors which are involved in the post-transcriptional regulation of Eg mRNAs, gel-shift and u.v. cross-linking experiments were performed, which allowed the identification of a p53-p55 RNA-binding protein doublet specific for the 3 untranslated regions of Eg mRNAs. These p53-p55 proteins do not bind to the 3 untranslated regions of either ornithine decarboxylase or phosphatase 2Ac mRNAs, which remain polyadenylated in embryos. These novel RNA-binding proteins are distinct from the cytoplasmic polyadenylation element-binding protein that controls the polyadenylation of maternal mRNAs in maturing Xenopus oocytes, and from previously identified thermoresistant RNA-binding proteins present in oocyte mRNP storage particles. The p53-p55 bind a portion of the Eg2 mRNA 3 untranslated region, distinct from the previously identified destabilizing region, that is able to confer the postfertilization dead-enylation of CAT-coding chimeric mRNAs. This suggests that the p53-p55 RNA-binding proteins are good candidates for trans-acting factors involved in the deadenylation of Eg mRNAs in Xenopus embryos.

International audience

International audience; The highly specialized membranes of the filter chamber found in the digestive tract of some homopteran insects could represent a favorable material for characterizing water channels. In order to demonstrate that membrane proteins of this epithelial complex serve as water channels, we have investigated the membrane permeability for water in Xenopus oocytes injected with RNA isolated from the filter chamber. Volumes of oocytes injected with filter chamber RNA were increased by 15% following a 16-min osmotic shock, while volumes of oocytes injected with RNA from midgut not of filter chamber or with water were increased only by 8.5 and 10%, respectively. This significant difference in oocyte swelling leads us to conclude that RNA isolated from the filter chamber contains mRNA coding for water channel proteins.

WOS:A1992HP40200015; International audience; Seasonal changes of several parameters related to sexual activity were studied in the gerbil (Rodentia: Gerbillidae). The weight of the testes, seminal vesicles, and adrenals fluctuate throughout the year. Plasma androgen levels and histological aspect of the testes also vary throughout the year. Spermatogonial and steroid activities are synchronous and are maximal in winter and spring. The relationship between these activities and environmental climatic parameters is discussed: the beginning of sexual activity seems correlated with the first rains.

National audience; To study the control of prolactin (PRL) secretion in rainbow trout, an in vivo technique using a monolayer cell culture system of pituitary glands was developped.

International audience; The effects of estradiol-17beta (E2) implants on plasma prolactin (PRL) concentrations, pituitary PRL content and pituitary PRL mRNA levels were examined in rainbow trout (Oncorhynchus mykiss). Intact immature fish treated with 1 mg estradiol-17beta did not show significant changes in both PRL mRNA levels and pituitary PRL content after 3 days of treatment. In a similar experiment, no changes were observed in plasma PRL levels followed during 7 days. Similarly, lack of estradiol-17beta effect on plasma PRL levels and on final PRL pituitary content was observed in ovariectomized female rainbow trout treated during 48 days with 25 mg estradiol-17beta and in mature male fish over a 3-week treatment period. Localization of estradiol receptor (ER) mRNAs in the pituitary was carried out by Northern blot analysis using a full-length rainbow trout estrogen receptor (rtER) cDNA as a probe. The rostral pars distalis of the pituitary which contained mostly PRL cells showed the lower amount of rtER mRNA when compared to other parts of the pituitary. Moreover, two mRNAs of different size (3.5 and 1.4 kb) were detected in different parts of the pituitary. Further hybridization experiments using probes containing part of the rtER cDNA (E domain or C and D domains) indicated that the small-sized mRNA (1.4 kb) probably encodes a truncated ER protein lacking hormone binding domain or an ER-related protein. Thus, only the 3.56 kb mRNA appeared to be involved in the regulation of pituitary function by estradiol. In situ hybridization analysis allowed a more precise localization of this rtER mRNA in the pituitary. No labeling was discernible over PRL cells whereas other parts of the pituitary were labeled. Grain counting corroborated this localization. These results indicate that (1) in vivo estradiol-17beta treatment did not modify PRL cell activity in rainbow trout, (2) PRL secretion, in this fish species, is not directly regulated by estradiol.

National audience

International audience; A polyclonal antibody was raised against bacterially produced catalytic alpha subunit of protein phosphatase 2A (PP2AC) cloned from Xenopus ovarian library. The amount of PP2AC in Xenopus oocytes determined by Western blot analysis was 1 ng/microgram of cytosolic protein. The antibody depleted PP2AC from oocyte extracts in association with 6 components (40, 62, 65, 80, 85 and 90 kDa). Prophase- and metaphase-arrested oocytes contained identical amounts of PP2AC. Metaphase oocytes showed one specific change in the 62 kDa protein associated with PP2AC.

International audience; The respective roles of sex steroids and hormones related to growth and metabolism, on SBP regulation have been studied in rainbow trout. In vivo, oestradiol (E2) supplementation induces a slow but significant increase of plasma SBP concentration. Testosterone or cortisol injections have no effect. In vitro, the steroid binding protein that accumulates in incubation medium of hepatic cell primary cultures has been characterized and found to be similar to blood SBP. Its production is increased by addition of E2 (maximum: +300%). This effect develops slowly over several days of culture and is dose dependent; as little as 1-10 nM E2 is effective. Recombinant rainbow trout GH (rtGH)--0.01 to 1 microgram/ml--also increases SBP accumulation as compared to control cells and seems to maintain SBP production over culture duration. In preliminary experiments, (1) insulin-like growth factor (IGF) and SBP concentrations were found to change inversely after a 4 days stimulation with increasing concentrations of GH; (2) recombinant human IGF1 (250 ng/ml) tended to be inhibitory when SBP production was expressed per mg of total cellular protein, and a micromolar concentration of bovine insulin was clearly inhibitory. Other hormones tested in vitro: triiodothyronine (10-1000 nM), thyroxine (100 nM), 17 alpha, 20 beta-dihydroprogesterone (10-2000 nM), and testosterone (1-1000 nM) did not influence SBP concentration in hepatic cells culture media.

International audience

International audience; We have previously described the cloning, sequencing and in vitro expression of a full-length rainbow trout estrogen receptor cDNA (rtER cDNA). This full cDNA randomly labelled was used to study the estrogen induction of hepatic rtER mRNA in correlation with vitellogenin (Vg) mRNA in different physiological situations. In this paper, we show that in the liver two mRNA species are under hormonal control and their level increases about 8-fold after estrogen stimulation. These two mRNAs are expressed and induced in the liver as early as the hatching stage in correlation with the expression of Vg mRNA. A long-term analysis of rtER mRNA after estradiol (E2) injection shows a transient induction of the nuclear ER and its mRNA which recover to the basal level after 2 weeks. Nevertheless, a memory effect was observed on the expression of the Vg gene which does not appear to be directly related to the estrogen receptor level.

International audience; The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

International audience; We previously reported the isolation of a partial cDNA clone encoding the rainbow trout estrogen receptor (rtER). A 0.4 kb 5'-end insert of this cDNA was used to screen the trout liver lambda gt10 cDNA library, and a full-length cDNA was isolated and sequenced. The principal structural characteristics of the complete coding sequence of the rtER are: first a remarkable homology of the DNA binding (C) and hormone binding (E) domains with those of other species, and second the lack of an A region, the function of which is not yet known but which is well conserved in other species. In vitro expression of the full-length rtER cDNA was carried out after transcription by T7 RNA polymerase and translation in rabbit reticulocyte lysate. Translation product analysis shows three major proteins, the largest one of which probably corresponds to the translation of the complete open reading frame of mRNA. The rtER in vitro translation products specifically bind estrogens (estradiol and diethylstilbestrol), without competition from testosterone or cortisol. The equilibrium dissociation constant (Kd), deduced from the Scatchard plot, is in the same order of magnitude as those determined heretofore in salmon livers during classical experiments. The tissue distribution of rtER mRNA shows that the same mRNA size (3.5 kb) is also present in the pituitary and hypothalamus. However, in the pituitary, a smaller sized mRNA (1.4 kb) is also detected.

International audience

WOS:A1987H760000010; International audience; Maternal responses and variations in plasma levels of prolactin and testosterone have been studied in incubating and in non-incubating, non-laying hens during forced adoption experiments. The results demonstrate the ability of incubating hens to display complete maternal behavior as early as the 10th day of incubation after being exposed to stimulation by chicks during one night. Maternal responses also emerged in non-laying hens but more gradually. In both groups, a decline in plasma testosterone occurred after the introduction of the chicks and, in the incubating hens, prolactin levels fell as they abandoned their nests.

WOS:A1983QV73800005; International audience; Variations in the plasma concentrations of testosterone (T) and delta 4-androstenedione (delta 4) levels were measured by radioimmunoassay in individual young Hubbard Chickens from the age of 16 days until 100 days. Plasma testosterone was low and remained relatively stable between days 16 and 63 (0.10 to 0.30 ng/ml), but increased then rapidly (0.32 +/- 0.07 ng/ml on day 65 vs. 1.49 +/- 0.27 ng/ml on day 100). During the same period delta 4 levels increased too (0.23 +/- 0.06 ng/ml on day 65 vs. 0.75 +/- 0.18 ng/ml on day 100). During the first weeks of life, delta 4-androstenedione prevailed over testosterone. The [delta 4]/[T] ratio reached a maximum between days 30 and 40 and was then reversed to negative values. Results are discussed in relation to those obtained for Chicken from other stocks.

International audience; The results presented here show that a decrease in the concentration of total polyamines, due to a decrease in putrescine and spermine, occurs during oogenesis in Xenopus laevis. The microinjection of spermine or spermidine decreases the hormonal responsiveness (maturation) of the fully-grown oocytes. This effect is synergistic with that already described for the microinjection of casein kinase II (Mulner-Lorillon, O. et al. (1987) Eur. J. Biochem. 171, 107-117), a polyamine dependent enzyme. Therefore a decrease in polyamine concentration, via its effect on endogeneous casein kinase II, could constitute one of the molecular changes required for the acquisition of competence to mature.

WOS:A1987J061900008; International audience; 1. An experiment was conducted with 4 groups of hens: a control group of laying hens, a group subjected to food deprivation for 7 d, a group subjected to food deprivation for 7 d, then re‐fed for 10 d and a group subjected to food deprivation for 7 d then re‐fed for 10 d with two chicks per hen introduced during the last 7 d of re‐feeding.2. Food deprivation provoked the involution of the oviduct and ovarian regression, as well as a decrease in the plasma concentrations of luteinising hormone (LH) and sex steroids (progesterone, testosterone and oestradiol).3. After 3 d of re‐feeding, there was a significant increase in the plasma concentrations of LH and steroid hormones. This phenomenon was even more marked after 10 d of re‐feeding; most of the hens of the third group which were not given chicks, were at the point of lay.4. The presence of chicks resulted in the expression of maternal behaviour and suppressed a rapid return to laying. This was especially marked in hens showing typical maternal behaviour traits, for which the morphological and endocrine measurements indicated a decrease in the activity of the hypothalamo‐hypophyseal‐gonadal axis.

International audience; The distribution of four specific RNA species between the poly(A)+ and poly(A)- fractions has been studied during the first hours of Xenopus laevis development, before the mid-blastula transition (MBT). Two of these specific RNA species correspond to clones selected by differential hybridization from a Xenopus egg cDNA library. Another corresponds to Xenopus c-raf mRNA and the last one to RNA revealed by a mouse ornithine decarboxylase probe. We show that two of these RNAs are adenylated after fertilization and remain in the poly(A)+ population. During the same period, the other two RNAs are deadenylated and these new poly(A)- RNAs remain stable at least until the MBT. These results show (i) that polyadenylation of specific RNA species occurs after fertilization in Xenopus and (ii) that, in the absence of transcription, adenylation and deadenylation can occur simultaneously in the fertilized egg.

WOS:A1983RW07500001; International audience; The manifestation of maternal behavior and the variations of plasmatic levels of gonadic hormones (testosterone, Δ4-androstenedione, and 17β-estradiol) have been studied in two groups of domestic hens (layers and nonlayers) during a forced adoption experiment. Maternal behavior appears later in actively laying hens than in nonlayers. The former hens show a higher level of androgens (T and Δ4). The appearance of typical maternal behavior coincides with a pause in laying and a decrease in all the plasmatic steroid levels measured.

International audience; Investigated maternal behavior and endocrine concomitants in 10 adult hens without prior maternal experience. Throughout continuous exposure to newly hatched chicks, Ss' aggressive behaviors disappeared and maternal responses emerged. After 13 days' exposure, gonadic steroids were dramatically depressed, and egg laying was almost stopped.