Purification of androgen-binding protein from rat testis using high-performance liquid chromatography and physicochemical properties of the iodinated molecule

AbstractThe androgen-binding protein (ABP) has been purified 87 500-fold from rat testis using 4 steps of HPLC, with a yield of 14%. The molecule was 99% pure with a specific activity estimated to 16 600 pmol/mg protein. The iodinated molecule was eluted in 2 peaks in Sephacryl S300 gel filtration with a molecular mass estimated to be 92 600 ± 3300 and 50 300 ± 4000 Da. The column isoelectrofocusing of 125I-ABP demonstrated 3 isoproteins isoelectric at pH 4.7, 4.9 and 5.3 and the sedimentation coefficient was estimated to be 4.7 S in sucrose gradient ultracentrifugation. The 125I-ABP had similar physicochemical properties to the non-labelled ABP of epididymis

Anchoring Secreted Proteins in Endoplasmic Reticulum by Plant Oleosin: The Example of Vitamin B12 Cellular Sequestration by Transcobalamin

BACKGROUND: Oleosin is a plant protein localized to lipid droplets and endoplasmic reticulum of plant cells. Our idea was to use it to target functional secretory proteins of interest to the cytosolic side of the endoplasmic reticulum of mammalian cells, through expressing oleosin-containing chimeras. We have designed this approach to create cellular models deficient in vitamin B12 (cobalamin) because of the known problematics associated to the obtainment of effective vitamin B12 deficient cell models. This was achieved by the overexpression of transcobalamin inside cells through anchoring to oleosin. METHODOLOGY: chimera gene constructs including transcobalamin-oleosin (TC-O), green fluorescent protein-transcobalamin-oleosin (GFP-TC-O) and oleosin-transcobalamin (O-TC) were inserted into pAcSG2 and pCDNA3 vectors for expression in sf9 insect cells, Caco2 (colon carcinoma), NIE-115 (mouse neuroblastoma), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster ovary cells). The subcellular localization, the changes in vitamin B12 binding activity and the metabolic consequences were investigated in both Caco2 and NIE-115 cells. PRINCIPAL FINDINGS: vitamin B12 binding was dramatically higher in TC-O than that in O-TC and wild type (WT). The expression of GFP-TC-O was observed in all cell lines and found to be co-localized with an ER-targeted red fluorescent protein and calreticulin of the endoplasmic reticulum in Caco2 and COS-7 cells. The overexpression of TC-O led to B12 deficiency, evidenced by impaired conversion of cyano-cobalamin to ado-cobalamin and methyl-cobalamin, decreased methionine synthase activity and reduced S-adenosyl methionine to S-adenosyl homocysteine ratio, as well as increases in homocysteine and methylmalonic acid concentration. CONCLUSIONS/SIGNIFICANCE: the heterologous expression of TC-O in mammalian cells can be used as an effective strategy for investigating the cellular consequences of vitamin B12 deficiency. More generally, expression of oleosin-anchored proteins could be an interesting tool in cell engineering for studying proteins of pharmacological interest

Vitamin B12-Impaired Metabolism Produces Apoptosis and Parkinson Phenotype in Rats Expressing the Transcobalamin-Oleosin Chimera in Substantia Nigra

International audienceThe development of fearfulness and the capacity of animals to cope with stressful events are particularly sensitive to early experience with mothers in a wide range of species. However, intrinsic characteristics of young animals can modulate maternal influence. This study evaluated the effect of intrinsic fearfulness on non-genetic maternal influence. Quail chicks, divergently selected for either higher (LTI) or lower fearfulness (STI) and from a control line (C), were cross-fostered by LTI or STI mothers. Behavioural tests estimated the chicks' emotional profiles after separation from the mother. Whatever their genotype, the fearfulness of chicks adopted by LTI mothers was higher than that of chicks adopted by STI mothers. However, genetic background affected the strength of maternal effects: the least emotional chicks (STI) were the least affected by early experience with mothers. We demonstrated that young animal's intrinsic fearfulness affects strongly their sensitivity to non-genetic maternal influences. A young animal's behavioural characteristics play a fundamental role in its own behavioural development processes

Early incidence of occupational asthma among young bakers, pastry-makers and hairdressers: design of a retrospective cohort study

<p>Abstract</p> <p>Background</p> <p>Occupational exposures are thought to be responsible for 10-15% of new-onset asthma cases in adults, with disparities across sectors. Because most of the data are derived from registries and cross-sectional studies, little is known about incidence of occupational asthma (OA) during the first years after inception of exposure. This paper describes the design of a study that focuses on this early asthma onset period among young workers in the bakery, pastry making and hairdressing sectors in order to assess early incidence of OA in these "at risk" occupations according to exposure duration, and to identify risk factors of OA incidence.</p> <p>Methods/Design</p> <p>The study population is composed of subjects who graduated between 2001 and 2006 in these sectors where they experience exposure to organic or inorganic allergenic or irritant compounds (with an objective of 150 subjects by year) and 250 young workers with no specific occupational exposure. A phone interview focusing on respiratory and 'Ear-Nose-Throat' (ENT) work-related symptoms screen subjects considered as "possibly OA cases". Subjects are invited to participate in a medical visit to complete clinical and lung function investigations, including fractional exhaled nitric oxide (FE_NO) and carbon monoxide (CO) measurements, and to collect blood samples for IgE (Immunoglobulin E) measurements (total IgE and IgE for work-related and common allergens). Markers of oxidative stress and genetic polymorphisms exploration are also assessed. A random sample of 200 "non-cases" (controls) is also visited, following a nested case-control design.</p> <p>Discussion</p> <p>This study may allow to describ a latent period between inception of exposure and the rise of the prevalence of asthma symptoms, an information that would be useful for the prevention of OA. Such a time frame would be suited for conducting screening campaigns of this emergent asthma at a stage when occupational hygiene measures and adapted therapeutic interventions might be effective.</p> <p>Trial registration</p> <p>Clinical trial registration number is NCT01096537.</p

Implication de la cubiline et du récepteur du facteur intrinsèque dans la physiopathologie moléculaire de la malabsorption sélective congénitale de la vitamine B12 (syndrome de Gräsbeck-Imerslund)

NANCY1-SCD Medecine (545472101) / SudocSudocFranceF

Déterminants nutritionnels et métaboliques de l'homocystéine au niveau hépatique (évaluation de 3 modèles expérimentaux)

La physiopathologie et les conséquences cellulaires de l'hyperhomocystéinemie observée après gastrectomie ou thyroïdectomie restent mal élucidées. Nous avons étudié 3 modèles expérimentaux. Le rat gastrectomisé présente une carence en vitamine B12, permettant d'obtenir une hyperhomocystéinémie en rapport avec une diminution de l'activité MTR. Cependant, la méthylation de l'ADN était corrélée à l'activité MTR et à la B12, mais pas directement à l'homocystéinémie. La thyroïdectomie chez le rat entraîne un défaut de synthèse des coenzymes flaviniques par la riboflavine kinase qui se traduit par une baisse des activités MTHFR et MTR. Cependant, les rats thyroïdectomisés présentent une hypohomocystéinémie en rapport avec une stimulation de la voie de la transulfuration via l'augmentation de l'activité CBS. Les ratons nés de rates soumise à une carence présentent une hyperhomocystéinémie liée à un effondrement de l'activité MTR, due à une véritable carence hépatique en B12.NANCY1-SCD Medecine (545472101) / SudocSudocFranceF

Influence des déterminants nutritionnels de l'homocystéïne sur la stéatose hépatique chez le rongeur

[Résumé en français] L'homocystéine (Hcy) est un acide aminé porteur de groupement thiol réducteur qui joue un rôle central dans les processus de méthylation cellulaire. L'hyperhomocystéinémie (HHcy) est associée à diverses anomalies au niveau du cœur, du cerveau ou du foie. Les maladies qui en résultent vont de la stéatose hépatique aux maladies cardiovasculaires, défauts de fermeture du tube neural ou encore à la maladie d'Alzheimer. Des perturbations des activités de transméthylations, SAM dépendantes, et/ou une augmentation du stress oxydant, sont impliquées dans la pathogenèse de ces maladies. L'objectif de ce travail est de déterminer les facteurs conduisant d'une HHcy, sévère ou modérée, à l'apparition de stéatose hépatique. La stéatose est caractérisée par une accumulation de triglycérides dans les hépatocytes qui, à terme, peut conduire à une fibrose, voire une cirrhose. Dans un deuxième temps, nous avons évalué le statut redox hépatique, généralement associé à la stéatose. La carence maternelle (folates, vitamine B12 et choline) induit chez le raton âgé de 21 jours une HHcy modérée, en même temps qu'apparaît une stéatose hépatique. L'HHcy est dûe à une baisse du ratio SAM/SAH. Cela provoque une chute de la synthèse de camitine et aboutit à une stéatose hépatique. L'HHcy induit une augmentation des marqueurs du stress oxydant. Chez les souris transgéniques cbs -/-, il y a une accumulation de SAH, inhibiteur des méthyltransférases, qui perturbe le cycle de reméthylation de l'Hcy. Notre modèle nutritionnel d'HHcy modérée, dûe à un methyldeficient, a permis d'établir une relation entre reméthylation de l'Hcy, synthèse hépatique de la camitine et stéatose hépatique.[Résumé en anglais] Homocysteine (Hcy) is a sulphur-containing amino acid that plays a central role in cellular methylation processes. hyperhomocysteinernia (HHcy) has been linked to various abnormalities occurring in liver, heart and brain. The relevant pathologies can range from hepatic steatosis to cardiovascular disease, neural tube defect and Alzheimer disease. Although the mechanisms for these are not clear, the perturbation of the SAM dependant transméthylation and the elevation of the oxidant stress could be involved in the pathogenesis of these diseases. The aim of this work was to deterrnine the factors leading from moderate or severe HHcy, to hepatic steatosis, a condition characterized by triglycerides accumulation that can lead to fibrosis and cirrhosis. ln addition, we have studied the changes of hepatic redox status as a consequence of HHcy. ln our study, we used 21 days-old young rats born to mothers that had undergone a feeding of methyl-donor deficient diet (in folate, vitamin B12 and choline) prior to the pregnancy. The maternal deficiency in methyl donor, induced in these pups moderate HHcy. Additionally, hepatic steatosis appeared in these pups. HHcy induced the drop of the SAM/SAH ratio and 100 to a decrease in camitine synthesis, causing the hepatic steatosis. We found that Hcy accumulation also 100 to an increase of the oxidant stress. ln transgenic mice cbs -/-, there is SAH accumulation, potent inhibitor of methyltransferases, that disturbs Hcy remethylation cycle. So, we have established a relationship between Hcy remethylation, camitine synthesis and hepatic steatosis using the moderate HHcy nutritional model through using the methyl deficient diet.NANCY1-SCD Medecine (545472101) / SudocSudocFranceF

Relations gène-environnement des déterminants du métabolisme des monocarbones et associations avec la pathologie en Afrique de l'Ouest

Nous avons évalué les déterminants nutritionnels et génétiques du métabolisme de l homocystéine en fonction des associations avec des pathologies prévalentes, en Afrique de l Ouest. Nous avons montré qu il existe une traduction phénotypique du polymorphisme MTHFR 677C>T sur l homocystéinémie en rapport avec la prévalence de la carence en folates. Le statut en folates est associé avec la fréquence allélique MTHFR 677T, en comparant des populations de différents continents. Nous avons trouvé une association favorable de cet allèle avec l hépatite virale B en Afrique. Nous avons remarqué également que le polymorphisme TCN 776C>G n a pas d influence sur l homocystéinémie. Par contre la comparaison des populations migrantes et autochtones pour le polymorphisme TCN 776C>G fait ressortir une pression sélective par rapport au paludisme. De plus TCN776C>G est le seul déterminant génétique du métabolisme des monocarbones associé à la gravité de la maladie, en période d accès palustre. L association des déterminants de l homocystéine avec les marqueurs clinico-biologiques du syndrome métabolique est surtout observée chez les femmes en Afrique et porte essentiellement sur les marqueurs du transport lipidique, dont ApoA et HDL.In this work we had analysed nutritional and genetics determinants involved in homocysteine metabolism according to geographical areas, environment-gene interactions and their association with the most prevalent diseases in West Africa, viral B hepatitis, malaria and metabolic syndrome linked to obesity. We had shown that MTHFR 677C>T polymorphism had a dramatic influence on homocysteinemia, in relation with folates deficit. Folates status was associated with MTHFR 677T allelic frecuency, when comparing populations worldwide. There was not migrational influence, when African populations were compared with those from North America. We had found an association between T allele and favourable evolution of viral B hepatitis in Africa. In the other hand, we observed no influence of TCN776C>G on homocysteinemia. When comparing TCN 776C>G polymorphism in migrant and autochthonous populations, our results suggested a selection pressure related to malaria. This polymorphism was associated with the severe form of malaria fever. Influence of homocysteine determinants on clinical and biological markers of metabolic syndrome in Africa was preferably observed in women, with a significant association with ApoA and HDL.NANCY1-Bib. numérique (543959902) / SudocSudocFranceF