Compartmentalized expression of three novel sarco/endoplasmic reticulum Ca2+ATPase 3 isoforms including the switch to ER stress, SERCA3f, in non-failing and failing human heart

The human sarco/endoplasmic reticulum (ER) Ca(2+)ATPase 3 (SERCA3) gene gives rise to SERCA3a-3f isoforms, the latter inducing ER stress in vitro. Here, we first demonstrated the co-expression of SERCA3a, -3d and -3f proteins in the heart. Evidence for endogenous proteins was obtained by using isoform-specific antibodies including a new SERCA3d-specific antibody, and either Western blotting of protein lysates or immunoprecipitation of membrane proteins. An immunolocalization study of both left ventricle tissue and isolated cardiomyocytes showed a distinct compartmentalization of the SERCA3 isoforms, as a uniform distribution of SERCA3a was detected while -3d and -3f isoforms were observed around the nucleus and in close vicinity of plasma membrane, respectively. Second, we studied their expressions in failing hearts including mixed (MCM) (n=1) and idiopathic dilated (IDCM) cardiomyopathies (n=4). Compared with controls (n=5), similar expressions of SERCA3a and -3d mRNAs were observed in all patients. In contrast, SERCA3f mRNA was found to be up-regulated in failing hearts (125+/-7%). Remarkably, overexpression of SERCA3f paralleled an increase in ER stress markers including processing of X-box-binding protein-1 (XBP-1) mRNA (176+/-24%), and expression of XBP-1 protein and glucose-regulated protein (GRP)78 (232+/-21%). These findings revisit the human heart's Ca(2+)ATPase system and indicate that SERCA3f may account for the mechanism of ER stress in vivo in heart failure.     

Mitochondrial Carbonic Anhydrase VA Deficiency Resulting from CA5A Alterations Presents with Hyperammonemia in Early Childhood

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.     

Mass Spectrometry Detection of G3m and IGHG3 Alleles and Follow-Up of Differential Mother and Neonate IgG3

Mass spectrometry (MS) analysis for detection of immunoglobulins (IG) of the human IgG3 subclass is described that relies on polymorphic amino acids of the heavy gamma3 chains. IgG3 is the most polymorphic human IgG subclass with thirteen G3m allotypes located on the constant CH2 and CH3 domains of the gamma3 chain, the combination of which leads to six major G3m alleles. Amino acid changes resulting of extensive sequencing previously led to the definition of 19 IGHG3 alleles that have been correlated to the G3m alleles. As a proof of concept, MS proteotypic peptides were defined which encompass discriminatory amino acids for the identification of the G3m and IGHG3 alleles. Plasma samples originating from ten individuals either homozygous or heterozygous for different G3m alleles, and including one mother and her baby (drawn sequentially from birth to 9 months of age), were analyzed. Total IgG3 were purified using affinity chromatography and then digested by a combination of AspN and trypsin proteases, and peptides of interest were detected by mass spectrometry. The sensitivity of the method was assessed by mixing variable amounts of two plasma samples bearing distinct G3m allotypes. A label-free approach using the high-performance liquid chromatography (HPLC) retention time of peptides and their MS mass analyzer peak intensity gave semi-quantitative information. Quantification was realized by selected reaction monitoring (SRM) using synthetic peptides as internal standards. The possibility offered by this new methodology to detect and quantify neo-synthesized IgG in newborns will improve knowledge on the first acquisition of antibodies in infants and constitutes a promising diagnostic tool for vertically-transmitted diseases.     

Individual dispersal,landscape connectivity and ecological networks

Connectivity is classically considered an emergent property of landscapes encapsulating individuals' flows across space. However, its operational use requires a precise understanding of why and how organisms disperse. Such movements, and hence landscape connectivity, will obviously vary according to both organism properties and landscape features. We review whether landscape connectivity estimates could gain in both precision and generality by incorporating three fundamental outcomes of dispersal theory. Firstly, dispersal is a multi‐causal process; its restriction to an ‘escape reaction’ to environmental unsuitability is an oversimplification, as dispersing individuals can leave excellent quality habitat patches or stay in poor‐quality habitats according to the relative costs and benefits of dispersal and philopatry. Secondly, species, populations and individuals do not always react similarly to those cues that trigger dispersal, which sometimes results in contrasting dispersal strategies. Finally, dispersal is a major component of fitness and is thus under strong selective pressures, which could generate rapid adaptations of dispersal strategies. Such evolutionary responses will entail spatiotemporal variation in landscape connectivity. We thus strongly recommend the use of genetic tools to: (i) assess gene flow intensity and direction among populations in a given landscape; and (ii) accurately estimate landscape features impacting gene flow, and hence landscape connectivity. Such approaches will provide the basic data for planning corridors or stepping stones aiming at (re)connecting local populations of a given species in a given landscape. This strategy is clearly species‐ and landscape‐specific. But we suggest that the ecological network in a given landscape could be designed by stacking up such linkages designed for several species living in different ecosystems. This procedure relies on the use of umbrella species that are representative of other species living in the same ecosystem.     

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.