Étude du phénomène de l’induction de facteurs bactériolytiques chez les lépidoptères: Inhibition de la production du lysozyme chez les larves deGalleria mellonella [Lep.: Pyralidae] par l’Actinomycine D et la Cycloheximide

Résumé L’introduction de corps étrangers dans les larves de lépidoptères entra?ne une augmentation parfois considérable du taux de lysozyme dans l’hémolymphe. L’efficacité inductrice de diverses substances varie dans de larges mesures. L’action de l’Actinomycine D et de la Cycloheximide sur la production du lysozyme induit a été recherchée chez des larves deGalleria mellonella L. recevant un stimulus peu spécifique (chlorure de sodium) ou plus spécifique (Micrococcus luteus vivants). L’action de ces inhibiteurs sur la quantité de lysozyme présente dans l’hémolymphe 24 h après induction, indique que les variations du taux de l’enzyme sont sous le contr?le d’un mécanisme unique indépendant de la spécificité des stimuli inducteurs. L’augmentation du taux de lysozyme chez les larves deG. mellonella induites, fait suite à une synthèse protéique et non à la libération d’enzyme préformé.

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Summary Foreign bodies introduced into lepidopteran larvae induce an increase, often considerable, in amount of lysozyme in the hemolymph. The efficiency of various substances to elicit the release of lysozyme in the larvae varies to a great extent. The effect of Actinomycin D and Cycloheximide introduced in theGalleria mellonella L. larvae stimulated to produce lysozyme by injection of saline or liveMicrococcus luteus was studied. The action of these metabolic inhibitors on the quantity of lysozyme in the hemolymph, 24 h after injection of the inducers, showed that the variations of the amount of the enzyme are under the control of only one mechanism which is not affected by the nature of the inducers. The increase in lysozyme amount of the hemolymph of the inducedG. mellonella larvae is due to the protein synthesis; it does not result from the release of a preformed enzyme as it was demonstrated in other animal species.

     

Discovery and structure-guided drug design of inhibitors of 11β-hydroxysteroid-dehydrogenase type I based on a spiro-carboxamide scaffold

Spiro-carboxamides were identified as inhibitors of 11β-hydroxysteroid-dehydrogenase type 1 by high-throughput screening. Structure-based drug design was used to optimise the initial hit yielding a sub-nanomolar IC₅₀ inhibitor (0.5 nM) on human 11β-HSD1 with a high binding efficiency index (BEI of 32.7) which was selective against human 11β-HSD2 (selectivity ratio > 200000).     

“I Don’t See That as a Medical Problem”: Clinicians’ Attitudes and Responses to Requests for Cosmetic Genital Surgery by Adolescents

Labiaplasty is a form of genital surgery to reduce large or protruding labia minora. Internationally, the rates of this surgery among women and girls is increasing and is viewed as a worrying trend. Currently, the main clinical strategy is to reassure adolescents that they are normal by talking about the variation of labia size and appearance and showing pictures demonstrating the wide range of normal female genital appearance. For the most part, policy documents recommend against labiaplasty in adolescents, claiming that it is medically non-essential surgery. In this paper, we contrast findings from our interviews with clinicians with the existing literature and policy documents and we point out areas needing more thought. This is qualitative research using semi-structured interviews. We set out to find out on what basis clinicians decide how to treat or manage adolescent patients seeking labiaplasty. We interviewed clinicians who are likely to be approached by under-eighteens requesting labiaplasty. We use interpretive content analysis and thematic analysis to analyse the data. Our findings support the emphasis on education and reassurance as the first step for all patients, but other issues that have not figured previously in the literature that would alter clinical strategies for managing patients emerge as well. Key findings are that reassurance does not always work and that the distinction between functional and appearance concerns is not a solid foundation in itself for deciding whether surgery is ethically appropriate. We conclude that the distinction between functional and appearance concerns is not ethically relevant. It is open to different interpretations and is not regarded by all clinicians as the definitive factor in relation to surgery. The focus of clinicians should be on relieving distress whatever the cause. Appearance reasons may sometimes justify surgery but, also, functional reasons may sometimes not be sufficient justification for surgery.     

UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity.

The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 units.mg-1 of protein. In the absence of GTP, the activity of B. subtilis enzyme is less than 10% of its maximum activity. Only dGTP and 3'-anthraniloyl-2'-deoxyguanosine-5'-triphosphate (Ant-dGTP) can increase catalysis significantly. Binding of Ant-dGTP to B. subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three. UTP and GTP completely displaced Ant-dGTP, whereas GMP and UMP were ineffective. UTP inhibits UMP kinase of B. subtilis with a lower affinity than that shown towards the Escherichia coli enzyme. Among nucleoside monophosphates, 5-fluoro-UMP (5F-UMP) and 6-aza-UMP were actively phosphorylated by B. subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium. A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N-acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B. subtilis and E. coli enzymes.     

Peptides as tools and drugs for immunotherapies.

Peptides are essential tools for discovery and pre-clinical and pharmaceutical development of viral and cancer vaccines ('active immunotherapies') as well as for therapeutic antibodies ('passive immunotherapies'). They help to trigger and analyze immune responses at a molecular level (B-cell, T-helper and CTL epitopes). They contribute largely to the design of new vaccine candidates and to the generation of monoclonal antibodies. They are also valuable analytical reference compounds for the structural characterisation by liquid chromatography and mass spectrometry of recombinant proteins used as biopharmaceuticals. As for other therapeutic applications, formulation, solubilisation, batch consistency and stability, issues have to be addressed to allow the pre-clinical and clinical development of this class of compounds as immunotherapeutic drugs. In the present review, three case studies dealing with (i) the design and the characterisation of Respiratory Syntycial Virus subunit vaccines, (ii) peptide-based melanoma vaccines, and (iii) therapeutic monoclonal antibodies, all investigated in clinical trials, are reported and discussed.     

Inhibitory effects of anions and active site amino acid sequence of chicken liver L-2-hydroxyacid oxidase A, a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family

Monocarboxylic acids with aliphatic chains were found to be mixed inhibitors of chicken liver L-2-hydroxyacid oxidase A when L-2-hydroxy-4-methylthiobutanoic acid was used as the substrate. The finding that the binding affinity of the enzyme for monocarboxylic acids was directly proportional to the number of carbon atoms in the chain strongly suggests that in addition to the electrostatic interaction due to the carboxyl moiety, hydrophobic forces may also be involved in the binding affinity of monocarboxylic acids to the enzyme's active site. Oxalate, a dicarboxylic acid, also resulted in a mixed-type inhibition of chicken liver L-2-hydroxyacid oxidase A, and, surprisingly, its binding affinity to the enzyme was found to be quite high as compared with monocarboxylic acids. This is probably due to the fact that the two carboxyl groups of oxalate give rise to electrostatic interactions with the positively charged side chains of two adjacent residues in the polypeptide chain. The inhibitory effects of other dicarboxylic acids was found to decrease as the number of carbon atoms in the chain increased. Oxamate was found however to be a novel type of potent inhibitor of the enzyme. All in all, these kinetic studies and the amino acid sequence determination in the active site region after limited proteolysis of the polypeptide chain definitely establish that chicken liver NADH/FMN containing L-2-hydroxyacid oxidase A is a member of the FMN-dependent α-hydroxyacid oxidizing enzyme family.     

Decline and recovery of olfactory receptor expression following unilateral bulbectomy

The effects of unilateral olfactory bulb ablation upon the odorant receptor expression were studied during the degeneration/regeneration process in the olfactory epithelium of adult rats. Using the in situ hybridization approach, we compared the time course of decay and recovery of expression for three different receptor subtypes (OR14, OR5, OR124). The number of neurons expressing receptor subtypes dramatically decreased in the olfactory epithelium on the lesioned side and reached a minimum at day 5 postsurgery. A progressive recovery was then observed from day 5 to day 15 postlesion, when a plateau was reached. Noticeable differences in the recovery level of receptor expression were observed according to the zonal patterning: the recovery level for neurons located in the lateral zone reached 70% of the control side value while the recovery levels in the dorsal and medial zones represented 35% and 53% of this value, respectively. Axotomy experiments suggest that zone-specific differences in receptor reexpression reported after bulbectomy might be related to the trophic influence of the olfactory bulb.