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.     

Effect of GnRH injection timing in the production of pronuclear-stage zygotes used for DNA microinjection

This study was aimed at developing a hormonal treatment protocol in order to optimize the proportion of pronuclear-stage embryos to be used for DNA microinjection in a goat transgenic founder production programme. A total of 46 adult BELE and 47 adult standard goats (1-5 years old) were used as donors and recipients, respectively. They were heat-synchronized using intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days with an injection of 125 microg cloprostenol on the morning of the eighth day. Recipients were injected with 400 IU eCG at the time of sponge removal while donors received a total of 133 mg NIH-FSH-P1 (Folltropin-V) given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced in donors by injecting 100 microg of GnRH at 24 h (GnRH24) or 36 h (GnRH36) after sponge removal. Embryo recovery was performed by oviduct flushing following a standard mid-ventral laparotomy procedure. The proportion of embryos in the pronuclear stage of development was higher in the GnRH36 group (90% vs 34%, p < 0.01). Embryos were microinjected with a DNA expression cassette followed by transfer to the oviduct of synchronized recipients. A higher, yet not statistically significant, pregnancy rate was found in the recipients transferred with pronuclear-stage embryos compared with those transferred with 2-cell-stage embryos (64% vs 37%, chi-square p = 0.06). One transgenic female founder was produced from the group of recipients transferred with pronuclear-stage microinjected embryos.     

Bioluminescent imaging: applications to cancerology and endocrinology

Our laboratory has been using various bioluminescent imaging systems for more than 20 years to visualize and quantify bioluminescent and chemiluminescent reactions. This equipment allowed us to establish numerous cell lines expressing bioluminescent reporter genes to study the mechanism of action of nuclear receptors. Cells expressing the luciferase gene under the control of a constitutive promoter were used to follow in vivo proliferation of cancer cells. Intensities of in vitro and in vivo bioluminescent signals were compared and the conditions of bioluminescent reaction measurements were determined. These bioluminescent models are new tools for evaluating cancer treatment efficiencies and the role of hormone receptors in invasion. Cells expressing the luciferase gene under the control of hormones are used as in vivo biosensors for studying analog bioavailabilities and in vivo response kinetics. They are complementary models to in vitro models that have been developed in our laboratory for several years. In the future, targeting reporter gene (luciferase and GFP) expression to specific tissues should allow the detailed localisation of the action of nuclear receptor ligands.