Insulin-mediated down-regulation of apolipoprotein A5 gene expression through the phosphatidylinositol 3-kinase pathway: role of upstream stimulatory factor

The apolipoprotein A5 gene (APOA5) has been repeatedly implicated in lowering plasma triglyceride levels. Since several studies have demonstrated that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 is regulated by insulin. Here, we show that cell lines and mice treated with insulin down-regulate APOA5 expression in a dose-dependent manner. Furthermore, we found that insulin decreases human APOA5 promoter activity, and subsequent deletion and mutation analyses uncovered a functional E box in the promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that this APOA5 E box binds upstream stimulatory factors (USFs). Moreover, in transfection studies, USF1 stimulates APOA5 promoter activity, and the treatment with insulin reduced the binding of USF1/USF2 to the APOA5 promoter. The inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway abolished insulin's effect on APOA5 gene expression, while the inhibition of the P70 S6 kinase pathway with rapamycin reversed its effect and increased APOA5 gene expression. Using an oligonucleotide precipitation assay for USF from nuclear extracts, we demonstrate that phosphorylated USF1 fails to bind to the APOA5 promoter. Taken together, these data indicate that insulin-mediated APOA5 gene transrepression could involve a phosphorylation of USFs through the PI3K and P70 S6 kinase pathways that modulate their binding to the APOA5 E box and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in men showed a decrease in the plasma ApoAV level. These results suggest a potential contribution of the APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.     

Molecular evolution of rickettsia surface antigens: evidence of positive selection

The Rickettsia genus is a group of obligate intracellular parasitic alpha-proteobacteria that includes human pathogens responsible for the typhus disease and various types of spotted fevers. rOmpA and rOmpB are two members of the "surface cell antigen" (Sca) autotransporter (AT) protein family that may play key roles in the adhesion of the Rickettsia cells to the host tissue. These molecules are likely determinants for the pathogenicity of the Rickettsia and represent good candidates for vaccine development. We identified the 17 members of this family of outer-membrane proteins in nine fully sequenced Rickettsia genomes. The typical architecture of the Sca proteins is composed of an N-terminal signal peptide and a C-terminal AT domain that promote the export of the central passenger domain to the outside of the bacteria. A characteristic of this family is the frequent degradation of the genes, which results in different subsets of the sca genes being expressed among Rickettsia species. Here, we present a detailed analysis of their phylogenetic relationships and evolution. We provide strong evidence that rOmpA and rOmpB as well as three other members of the Sca protein family--Sca1, Sca2, and Sca4--have evolved under positive selection. The exclusive distribution of the predicted positively selected sites within the passenger domains of these proteins argues that these regions are involved in the interaction with the host and may be locked in "arms race" coevolutionary conflicts.     

New insights into plant transaldolase

The oxidative pentose phosphate pathway (OPPP) provides plants with important substrates for both primary and secondary metabolism via the oxidation of glucose-6-phosphate. The OPPP is also thought to generate large amounts of reducing power to drive various anabolic processes. In animals this major pathway is located within the cytoplasm of cells, but in plants its subcellular compartmentation is far from clear. Although several enzymes of the OPPP were demonstrated to have both cytosolic and plastidic counterparts, there is yet no evidence for a full set of functional enzymes in each compartment. We report here the isolation of two coding sequences from tomato (Lycopersicon esculentum L.) which encode phylogenetically distant sequences (ToTal1 and ToTal2) that putatively encode distinct plastidic TA isoforms. The kinetic characterization of ToTal1 revealed that, unlike other enzymes of the non-oxidative branch of the OPPP, ToTal1 does not follow a Michaelis-Menten mode of catalysis which has implications for its role in regulating carbon flux between primary and secondary metabolism. TA genes appear to be differentially regulated at the level of gene expression in plant tissues and in response to environmental factors which suggests that TA isoforms have a non-overlapping role for plant metabolism.     

Multiple origin of metallicolous populations of the pseudometallophyte Arabidopsis halleri (Brassicaceae) in central Europe: the cpDNA testimony

The population structure of the pseudo-metallophyte herb, Arabidopsis halleri, was studied using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) on chloroplast DNA (cpDNA). The history of metallicolous (M) populations showing increased zinc tolerance was investigated. Eight primer-enzyme combinations out of 72 tested were applied to a total of 625 individuals from 28 widespread populations, 14 of them being M. Eleven distinct chlorotypes were found: five were common to nonmetallicolous (NM) and M populations, whereas six were only observed in one edaphic type (five in NM and one in M). No difference in chlorotype diversity between edaphic types was detected. Computed on the basis of chlorotype frequencies, the level of population differentiation was high but remained the same when taking into account levels of molecular divergence between chlorotypes. Isolation by distance was largely responsible for population differentiation. Geographically isolated groups of M populations were more genetically related to their closest NM populations than to each other. Our results suggest that M populations have been founded separately from distinct NM populations without suffering founding events and that the evolution towards increased tolerance observed in the distinct M population groups occurred independently.     

Oligomerization of the alpha and beta isoforms of the thromboxane A2 receptor: relevance to receptor signaling and endocytosis

Thromboxane A(2) (TXA(2)) is a potent mediator of inflammation, vasoconstriction and oxidative stress. The TXA(2) receptor (TP) is a G protein-coupled receptor (GPCR) that is expressed as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues) that share the first 328 residues. For many years GPCRs were assumed to exist and function as monomeric species, but increasing evidence suggests that a dimer is the minimal functional unit of GPCRs. In the present report, using co-immunoprecipitation of differentially tagged TP expressed in HEK293 cells, we demonstrate that TPalpha and TPbeta form homo- and hetero-oligomers. Immunoblotting of lysates from human platelets with an anti-TP specific antibody revealed the presence of endogenously expressed TP oligomers. We show that TP oligomerization is an agonist-independent process highly affected by the reducing agent dithiothreitol suggesting the involvement of disulfide bonds in TP oligomerization. Over-expression of G protein-coupled receptor kinases and arrestins did not modulate the extent of receptor dimerization/oligomerization. Co-expression of two TP signaling-deficient mutants, R60L and E2402R, resulted in rescuing of receptor signal transduction suggesting that dimers/oligomers constitute the functional units of this receptor. Interestingly, TPalpha which does not undergo constitutive or agonist-induced endocytosis on its own was subjected to both types of endocytosis when co-expressed with TPbeta, indicating that TPalpha can display intracellular trafficking when complexed through hetero-oligomerization with TPbeta.     

Detection of Mycobacterium leprae in nasal mucosa biopsies by the polymerase chain reaction

Several discoveries about leprosy indicate that Mycobacterium leprae transmission mainly occurs by inhalation, and the nose is major port of entry and exit. The present study evaluated the clinical application of PCR for detection of M. leprae DNA in nasal mucosa biopsies in untreated leprosy patients (52) and their contacts (99) from the State Reference Center in Sanitary Dermatology and Leprosy, Uberlandia, MG, Brazil. PCR detection of a 372-base pair DNA fragment from M. leprae was accomplished in 36 (69.2%) patients, from which 34 (91.9%) of them were multibacillaries. Furthermore, PCR was positive in 3 (16.7%) of 18 slit-skin smear negative, 4 (25.0%) of 16 skin lesion BI negative, 8 (33.3%) of 24 nasal mucosa BI negative patients, and 10 of 99 contacts (10.1%). The presence of bacilli in 10.1% of the contacts may potentially reflect an occult leprosy, and these patients must be accompanied, followed by a chemoprophylaxy treatment. Considering all PCR results against clinical and BI classification of patients and controls, we have found a sensitivity of 69.2%, a specificity of 89.9%, and an accuracy of 82.8%. It has been demonstrated here through PCR of nasal biopsies that the bacillus invades the mucosa, passing through the nasal inferior turbinate to reach peripheral blood. Therefore, the molecular investigation of invasive nasal biopsies by PCR tests has proven to be useful in defining patients of higher risk of transmission and risk-group contacts, which is an important step to reach the World Health Organization objective towards the elimination of leprosy as a public health problem.     

Activation of the prostaglandin D2 receptor DP2/CRTH2 increases allergic inflammation in mouse

Allergic pathologies are often associated with IgE production, mast cell activation, and eosinophilia. PGD2 is the major eicosanoid, among several inflammatory mediators, released by mast cells. PGD2 binds to two membrane receptors, D prostanoid receptor (DP)1 and DP2, endowed with antagonistic properties. In humans, DP2 is preferentially expressed on type 2 lymphocytes, eosinophils, and basophils and mediates chemotaxis in vitro. Although not yet supported by in vivo studies, DP2 is thought to be important in the promotion of Th2-related inflammation. Herein, we demonstrate that mouse eosinophils express both DP1 and DP2 and that PGD2 exerts in vitro chemotactic effects on eosinophils through DP2 activation. Furthermore, 13,14-dihydro-15-keto-PGD2, a specific DP2 agonist not only increases eosinophil recruitment at inflammatory sites but also the pathology in two in vivo models of allergic inflammation: atopic dermatitis and allergic asthma. By contrast, DP1 activation tends to ameliorate the pathology in asthma. Taken together, these results support the hypothesis that DP2 might play a critical role in allergic diseases and underline the interest of DP2 antagonists in human therapy.     

Expression of the delta-protocadherin gene Pcdh19 in the developing mouse embryo

Protocadherins constitute a large family of transmembrane proteins primarily involved in weak homophilic adhesion in the brain and several other tissues. In a screen for potential regulators of kidney development, we have identified Pcdh19, a poorly characterized member of the delta-protocadherin subfamily. Here, we report the spatio-temporal expression pattern of Pcdh19 during mouse embryonic development. In midgestation embryos, Pcdh19 mRNA was detected in the mesonephros and in the neuroepithelium of the forebrain and midbrain. At later stages, Pcdh19 was expressed in other neural tissues such as the neural retina, nasal epithelium and spinal cord, as well as in the collecting duct and differentiating nephrons of the metanephros, in the glandular stomach, the exocrine pancreas and the hair follicles. Hence, the Pcdh19 gene is developmentally regulated during mouse organogenesis and shows a unique expression profile among protocadherins.     

ANKRD13C acts as a molecular chaperone for G protein-coupled receptors

Although the mechanisms that regulate folding and maturation of newly synthesized G protein-coupled receptors are crucial for their function, they remain poorly characterized. By yeast two-hybrid screening, we have isolated ANKRD13C, a protein of unknown function, as an interacting partner for the DP receptor for prostaglandin D(2). In the present study we report the characterization of this novel protein as a regulator of DP biogenesis and trafficking in the biosynthetic pathway. Co-localization by confocal microscopy with an endoplasmic reticulum (ER) marker, subcellular fractionation experiments, and demonstration of the interaction between ANKRD13C and the cytoplasmic C terminus of DP suggest that ANKRD13C is a protein associated with the cytosolic side of ER membranes. Co-expression of ANKRD13C with DP initially increased receptor protein levels, whereas siRNA-mediated knockdown of endogenous ANKRD13C decreased them. Pulse-chase experiments indicated that ANKRD13C can promote the biogenesis of DP by inhibiting the degradation of newly synthesized receptors. However, a prolonged interaction between ANKRD13C and DP resulted in ER retention of misfolded/unassembled forms of the receptor and to their proteasome-mediated degradation. ANKRD13C also regulated the expression of other GPCRs tested (CRTH2, thromboxane A(2) (TPα), and β2-adrenergic receptor), whereas it did not affect the expression of green fluorescent protein, GRK2 (G protein-coupled receptor kinase 2), and VSVG (vesicular stomatitis virus glycoprotein), showing specificity toward G protein-coupled receptors. Altogether, these results suggest that ANKRD13C acts as a molecular chaperone for G protein-coupled receptors, regulating their biogenesis and exit from the ER.