VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.     

Reproductive strategies and population structure in Leishmania: substantial amount of sex in Leishmania Viannia guyanensis

Leishmania species of the subgenus Viannia and especially Leishmania Viannia guyanensis are responsible for a large proportion of New World leishmaniasis cases. Since a recent publication on Leishmania Viannia braziliensis, the debate on the mode of reproduction of Leishmania parasites has been reopened. A predominant endogamic reproductive mode (mating with relatives), together with strong Wahlund effects (sampling of strains from heterogeneous subpopulations), was indeed evidenced. To determine whether this hypothesis can be generalized to other Leishmania Viannia species, we performed a population genetic study on 153 human strains of L. (V.) guyanensis from French Guiana based on 12 microsatellite loci. The results revealed important homozygosity and very modest linkage disequilibrium, which is in agreement with a high level of sexual recombination and substantial endogamy. These results also revealed a significant isolation by distance with relatively small neighbourhoods and hence substantial viscosity of Leishmania populations in French Guiana. These results are of epidemiological relevance and suggest a major role for natural hosts and/or vectors in parasite strain diffusion across the country as compared to human hosts.     

Metabolic channeling of carbamoyl phosphate, a thermolabile intermediate: evidence for physical interaction between carbamate kinase-like carbamoyl-phosphate synthetase and ornithine carbamoyltransferase from the hyperthermophile Pyrococcus furiosus

Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate synthetase (CKase) and ornithine carbamoyltransferase (OTCase) from the hyperthermophilic archaeon Pyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment.     

Proteomic Analysis of the Reproductive Organs of the Hermaphroditic Gastropod Lymnaea stagnalis Exposed to Different Endocrine Disrupting Chemicals

Many studies have reported perturbations of mollusc reproduction following exposure to low concentrations (ng/L range) of endocrine disrupting chemicals (EDCs). However, the mechanisms of action of these molecules on molluscs are still poorly understood. Investigation of the modifications of protein expression in organisms exposed to chemicals using proteomic methods can provide a broader and more comprehensive understanding of adverse impacts of pollution on organisms than conventional biochemical biomarkers (e.g., heat-shock proteins, metallothioneins, GST, EROD). In this study we have investigated the impacts of four chemicals, which exhibit different endocrine disrupting properties in vertebrates, on the proteome of the hermaphroditic freshwater pulmonate gastropod Lymnaea stagnalis after 21 days of exposure. Testosterone, tributyltin, chlordecone and cyproterone acetate were chosen as tested compounds as they can induce adverse effects on the reproduction of this snail. The 2D-DIGE method was used to identify proteins whose expression was affected by these compounds. In addition to modifying the expression of proteins involved in the structure and function of the cytoskeleton, chemicals had impacts on the expression of proteins involved in the reproduction of L. stagnalis. Exposure to 19.2 µg/L of chlordecone increased the abundance of ovipostatin, a peptide transmitted during mating through seminal fluid, which reduces oviposition in this species. The expression of yolk ferritin, the vitellogenin equivalent in L. stagnalis, was reduced after exposure to 94.2 ng Sn/L of tributyltin. The identification of yolk ferritin and the modification of its expression in snails exposed to chemicals were refined using western blot analysis. Our results showed that the tested compounds influenced the abundance of yolk ferritin in the reproductive organs. Alteration in proteins involved in reproductive pathways (e.g., ovipostatin and yolk ferritin) could constitute relevant evidence of interaction of EDCs with reproductive pathways that are under the control of the endocrine system of L. stagnalis.     

PIK3R1 Mutations Cause Syndromic Insulin Resistance with Lipoatrophy

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.     

Influence of Nutrient Stress on the Relationships between PAM Measurements and Carbon Incorporation in Four Phytoplankton Species

Two methods of measuring primary production, modulated fluorimetry (PAM) and the traditional carbon incorporation method (¹³C), were compared in four phytoplankton species, two diatoms (Pseudo-nitzschia pungens and Asterionellopsis glacialis), and two dinoflagellates (Heterocapsa sp and Karenia mikimotoï), under N (nitrogen), P (phosphorus) and Si (silicon) limited semi-continuous culture. N and Si-limited cultures showed relatively high quantum efficiency of the PSII (F_v/F_m) values, confirming that F_v/F_m is not a good proxy for nutrient stress in balanced systems, whereas P limitation had a drastic effect on many physiological parameters. In all species, the physiological capacity of phytoplankton cells to acclimate to nutrient limitations led to changes in the cellular biochemical composition and the structure of the photosynthetic apparatus. The observed physiological responses were species and nutrient specific. The values of the chlorophyll-specific absorption cross section (a*) increased with nutrient limitation due to package effect, while the carbon/Chl a ratio was higher under N and P limitations. In diatoms, Si limitation did not affect photosynthesis confirming the uncoupling between Si and carbon metabolisms. In all four species and under all treatments, significant relationships were found between photosynthetic activities, ETR^Chl (electron transport rate) and P^Chl (carbon fixation rate) estimated using PAM measurements and ¹³C incorporation, showing that the fluorescence technique can reliably be used to estimate carbon fixation by phytoplankton. The relationship between ETR^Chl and P^Chl can be described by the shape and the slope of the curve (Φ_C.e). Linear relationships were found for dinoflagellates and P. pungens under all treatments. A decrease in Φ_C.e was observed under N and P limitation probably due to structural damage to the photosynthetic apparatus. A. glacialis showed a logarithmic relationship in N and P limited conditions, due to the alternative electron flow which takes place to optimise photosynthetic performances under high light and/or nutrient stress.     

Haemosporidian Parasites of Antelopes and Other Vertebrates from Gabon,Central Africa

Re-examination, using molecular tools, of the diversity of haemosporidian parasites (among which the agents of human malaria are the best known) has generally led to rearrangements of traditional classifications. In this study, we explored the diversity of haemosporidian parasites infecting vertebrate species (particularly mammals, birds and reptiles) living in the forests of Gabon (Central Africa), by analyzing a collection of 492 bushmeat samples. We found that samples from five mammalian species (four duiker and one pangolin species), one bird and one turtle species were infected by haemosporidian parasites. In duikers (from which most of the infected specimens were obtained), we demonstrated the existence of at least two distinct parasite lineages related to Polychromophilus species (i.e., bat haemosporidian parasites) and to sauropsid Plasmodium (from birds and lizards). Molecular screening of sylvatic mosquitoes captured during a longitudinal survey revealed the presence of these haemosporidian parasite lineages also in several Anopheles species, suggesting a potential role in their transmission. Our results show that, differently from what was previously thought, several independent clades of haemosporidian parasites (family Plasmodiidae) infect mammals and are transmitted by anopheline mosquitoes.