Transgenic goats produced by DNA pronuclear microinjection of in vitro derived zygotes

This study was undertaken to investigate various factors affecting the outcomes of in vitro fertilization (IVF) of oocytes retrieved by laparoscopic ovum pick-up (LOPU) technique from prepubertal and adult goats, as well as to evaluate the developmental competence of in vitro produced embryos. Oocyte-cumulus complexes recovered by LOPU from donors stimulated with gonadotrophins were matured in vitro. Fresh semen was used for IVF following various capacitation treatments. In vitro produced zygotes were either cultured to assess in vitro development or were transferred into recipients for full term development. The results indicated that successful IVF of the goat oocytes was affected by factors such as sperm capacitation treatment, oocyte quality, and abundance of cumulus cells on zona pellucida. Oocytes from both prepubertal and adult goats demonstrated similar full term developmental competence despite the fact that in vitro developmental rates were lower for prepubertal goats. The births of transgenic offspring demonstrated that the established LOPU-IVF technology combined with pronuclear microinjection can be successfully used to produce transgenic goats.     

Effect of previous culture conditions and the presence of the rpoS gene on the survival of Salmonella typhimurium in sea water exposed to sunlight

The effect of sunlight exposure on Salmonella typhimurium isogenic strains harboring an rpoS gene functional (rpoS+) or not functional (rpoS-) was investigated in microcosms of sterile sea water at 20 degrees C. The two strains rapidly lost their ability to produce colonies on solid culture media. The detrimental action of sunlight was more important when the salinity of sea water increased. The survival of stationary phase cells was influenced by RpoS. Bacteria grown in media with high salinity or osmolarity and transferred to sea water in stationary phase were more resistant to irradiation than those grown in media with low salinity. Prior growth under oxidative (0.2 mmol/L of H2O2) or amino acid starved (minimal medium) conditions did not modify the survival of either strain when they were exposed to sunlight. Bacteria were more resistant when cells were incubated in sea water in the dark prior to being exposed to sunlight. The resistance to sunlight irradiation was also greater in clones of both strains isolated from microcosms exposed to sunlight for 90 min, then further inoculated into sea water and reexposed to sunlight.     

Community ecology of the terrestrial small mammals of Zakouma National Park,Chad

The terrestrial small mammal community of the Zakouma National Park (Chad) was assessed by live trapping in various habitats during the dry season. Nearly 3000 trap-nights yielded 505 captures of nine rodent and two shrew species, making up a representative small mammal community for the Sudanian savanna biotic zone. Murine rodents of the genusMastomys dominated, withM. erythroleucus andM. cf.kollmannspergeri occuring at similar abundances. The former was widespread and especially abundant in habitats with high tree cover. The latter was more localized, predominantly in annually flooded habitats characterized by a clay-rich soil, often withAcacia seyal as the main vegetation. Population structure differed between the two species, suggesting distinct reproductive strategies possibly linked with habitat preferences: theM. erythroleucus population comprised mainly young, immature individuals, whereasM. cf.kollmannspergeri was represented by older individuals, a significant fraction of which had already reproduced.Taterillus congicus andTatera kempi (gerbilline rodents), together withLemniscomys zebra, were mainly found in more open habitats with sand-rich soils.Arvicanthis niloticus, Acomys cf.johannis, Mus mattheyi andXerus erythropus were the other rodents captured, whereas shrews were represented byCrocidura fulvastra andSuncus sp.     

CD14 is an acute-phase protein

The origin of soluble CD14 (sCD14) in the circulation is uncertain. To examine whether CD14 could be an acute-phase protein (APP), the levels of sCD14, IL-6, and C-reactive protein were determined by ELISA in serum and synovial fluid (SF) of patients with various arthropathies, and the regulation of CD14 synthesis was examined in liver cells. In patients with crystal-mediated or immunologically mediated arthritis (rheumatoid arthritis), serum levels of sCD14 were higher than or similar to those found in infection-mediated arthritis (reactive arthritis), precluding a relation with bacteria exposure. Levels of sCD14 were similar in SF and serum, and did not correlate with the number of SF leukocytes, excluding an important source from leukocyte membrane-bound CD14, by protease-mediated shedding. In contrast, serum levels of sCD14 in patients correlated with those of C-reactive protein, a classical APP, and IL-6, a cytokine known to regulate the synthesis of APP in the liver. Serum levels of sCD14 also correlated with disease activity in rheumatoid arthritis and reactive arthritis patients. IL-6 stimulated the production of CD14 by HepG2 hepatoma cells. By real-time PCR, the inducibility of CD14 by IL-6 was also observed at the mRNA level both in HepG2 cells and human primary hepatocytes. These in vitro results were confirmed by in vivo studies in IL-6(-/-) mice injected with turpentine, an experimental model of acute-phase response. Liver levels of CD14 mRNA increased in IL-6(+/+), but not in IL-6(-/-) mice. These results indicate that sCD14 can be considered as a type 2 APP.     

The Acrolepiopsis assectella silk cocoon: kairomonal function and chemical characterisation

Two soluble sericin-like polypeptides, B1 and B2, from leek moth (Acrolepiopsis assectella) cocoons trigger host-acceptance behaviour in the parasitoid, Diadromus pulchellus (Proc. Roy. Soc. London B 269 (2002) 1879). We found that these polypeptides were particularly cysteine-rich and lost their ability to trigger host-acceptance behaviour after being denatured and purified. This suggests that inter-disulphide bonds and the secondary structure of B1 and B2 are important for their biological activity. We also isolated six insoluble polypeptides (or polypeptides of low solubility) from A. assectella cocoons. At least four of these polypeptides triggered host-acceptance behaviour. The strongest responses were observed with P22, a light-chain fibroin or a seroin-peptide, and P100, a sericin-like polypeptide that is probably more strongly associated with the silk core than are B1 and B2. In conclusion, several polypeptides from different parts of the A. assectella silk-cocoon (the insoluble core and coating of the silk thread) are able to elicit host-acceptance behaviour in D. pulchellus females. These polypeptides belong to different silk protein families and are used as kairomones by this specialist parasitoid.     

Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules

Polyglutamine expansion (polyQ) in the protein huntingtin is pathogenic and responsible for the neuronal toxicity associated with Huntington's disease (HD). Although wild-type huntingtin possesses antiapoptotic properties, the relationship between the neuroprotective functions of huntingtin and pathogenesis of HD remains unclear. Here, we show that huntingtin specifically enhances vesicular transport of brain-derived neurotrophic factor (BDNF) along microtubules. Huntingtin-mediated transport involves huntingtin-associated protein-1 (HAP1) and the p150(Glued) subunit of dynactin, an essential component of molecular motors. BDNF transport is attenuated both in the disease context and by reducing the levels of wild-type huntingtin. The alteration of the huntingtin/HAP1/p150(Glued) complex correlates with reduced association of motor proteins with microtubules. Finally, we find that the polyQ-huntingtin-induced transport deficit results in the loss of neurotrophic support and neuronal toxicity. Our findings indicate that a key role of huntingtin is to promote BDNF transport and suggest that loss of this function might contribute to pathogenesis.     

Microrobots for in vitro fertilization applications.

The Micromanipulation and Micro-actuation Research Group at the LAB has activities related to biological and surgical applications. Concerning cells micromanipulation, our laboratory works in collaboration with the research team "Genetic and Reproduction" of the Besan?on's hospital (France). The global final objective is the development of an automatic intra cytoplasmic sperm injection (ICSI) device in order to improve performances and ergonomics of current devices. In the future this new device will contain various modules: module for removal of cumulus cells, modules for characterization of oocytes, microinjection module, cells transport system. The first subsystem developed is a new single cell transport system. It consists in a so-called micropusher which pushes single cells without having contact with the external environment. This micropusher is a ferromagnetic particle (from 400 x 400 x 20 microm3 to 100 x 100 x 5 microm3) which follows the movement of a permanent magnet located under the biological medium. A 2D micro-positioning table moves this magnet under the glass slide. The pusher and cells positions are measured through an optical microscope with a CCD camera located above the biological medium. The second subsystem is developed to measure oocytes mechanical stiffness in order to sort them. We have then developed a micro/nano-force sensor based on the diamagnetic levitation principle: a glass tip end-effector (with 20 microm in diameter) is fixed on the equipment which is in levitation (0.5 mm in diameter, 100 mm in length). When a force is applied to the levitated glass tip, it moves to a new equilibrium position. Thanks to themeasurement of this displacement, the applied force can be measured. Since there is no contact and friction between the levitated tip and the fixed part, the resolution of this sensor is very high (10 nN).     

Ultrastructure of Mycobacterium marinum granuloma in striped bass Morone saxatilis

An emerging epizootic of mycobacteriosis currently threatens striped bass Morone saxatilis populations in Chesapeake Bay, USA. Several species of mycobacteria, including Mycobacterium marinum, species resembling M. avium, M. gordonae, M. peregrinum, M. scrofulaceum and M. terrae, and the new species M. shottsii have been isolated from diseased and healthy bass. In this study, we describe the ultrastructure of developing M. marinum granulomas in experimentally infected bass over a period of 45 wk. The primary host response to injected mycobacteria was formation of large macrophage aggregations containing phagocytosed bacilli. M. marinum were always contained within phagosomes. Close association of lysosomes with mycobacterial phagosomes, as well as the presence of electron-opaque material within phagosomes, suggested phagolysosomal fusion. Development of granulomas involved epithelioid transformation of macrophages, followed by appearance of central necrosis. Desmosomes were present between mature epithelioid cells. The necrotic core region of M. marinum granulomas was separated from overlying epithelioid cells by several layers of flattened, electron-opaque spindle-shaped cells. These cells appeared to be formed by compression of epithelioid cells and, aside from a flattened nucleus, did not possess recognizable organelles. Following the development of well-defined, paucibacillary granulomas, secondary disease was observed. Recrudescence was marked by bacterial replication followed by disruption of granuloma architecture, including loss of epithelioid and spindle cell layers. In advanced recrudescent lesions, normal tissue was replaced by macrophages, fibroblasts, and other inflammatory leukocytes. Large numbers of mycobacteria were observed, both intracellular and suspended in cellular debris.     

Attempts at correlation of the radiolytic species of irradiated solid-state captopril studied by multi-frequency EPR and HPLC

The purpose of this study was to provide insight into the processes that occur after the irradiation of solid-state drugs. Electron paramagnetic resonance (EPR) experiments were performed at two different frequencies, X-band (about 9.5 GHz) and Q-band (about 34 GHz), to identify the radicals present in irradiated captopril. The results confirmed that an irradiated drug can trap several main radicals. Moreover, the radical composition varied as a function of the treatment. In addition, non-volatile final products were studied by liquid chromatography coupled to UV and to mass spectrometry (LC-MS). The variation of the radical composition did not influence the profile of the final products; this appears to indicate that, in the case of captopril, the trapped radicals observed by EPR are not the main precursors of the final products. Finally, high-performance liquid chromatography data appear to indicate that radiosterilization of captopril is feasible.     

In vitro response of the striped bass natural resistance-associated macrophage protein, Nramp, to LPS and Mycobacterium marinum exposure

Mycobacteriosis in Chesapeake Bay (USA) striped bass Morone saxatilis is an ongoing disease problem with important economic implications for a large commercial and recreational fishery. Additionally, striped bass serve as a reservoir of potential mycobacterial zoonoses. Recently, we described a striped bass gene homolog of the natural resistance-associated macrophage protein family (MsNramp), which is responsible for resistance to mycobacterial infections in mice. Striped bass MsNramp is strongly induced in peritoneal exudate cells (PE) in vivo after intraperitoneal injection with Mycobacterium spp. The purpose of the present study was to investigate short-term in vitro MsNramp expression and reactive oxygen intermediate (ROI) production in primary cultures of adherent PE after exposure to bacterial lipopolysaccharide (LPS), or live- or heat-killed (HK) Mycobacterium marinum. PE expressed significantly higher levels of MsNramp at 4 and 24 h post-treatment with live and HK M. marinum. MsNramp response to LPS was dose-dependent in these cells, with maximum expression at 4 h and 20 microg/ml LPS. Treatment of PE with LPS resulted in increased intracellular superoxide anion levels, whereas treatment with live M. marinum caused a significant depression. This study is the first report of induction of a teleost Nramp in vitro by mycobacteria, and supports findings of teleost Nramp induction by LPS.