Pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists

A series of 1-substituted 3-(t-butyl/trifluoromethyl)pyrazole C-region analogues of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1 antagonism. The structure activity relationship indicated that the 3-chlorophenyl group at the 1-position of pyrazole was the optimized hydrophobic group for antagonistic potency and the activity was stereospecific to the S-configuration, providing exceptionally potent antagonists 13S and 16S with K_i(CAP) = 0.1 nM. Particularly significant, 13S exhibited antagonism selective for capsaicin and NADA and not for low pH or elevated temperature. Both compounds also proved to be very potent antagonists for rTRPV1, blocking in vivo the hypothermic action of capsaicin, consistent with their in vitro mechanism. The docking study of compounds 13S and 16S in our hTRPV1 homology model indicated that the binding modes differed somewhat, with that of 13S more closely resembling that of GRT12360.     

The Mitochondrial Genome of Arctica islandica; Phylogeny and Variation

Arctica islandica is known as the longest-lived non-colonial metazoan species on earth and is therefore increasingly being investigated as a new model in aging research. As the mitochondrial genome is associated with the process of aging in many species and bivalves are known to possess a peculiar mechanism of mitochondrial genome inheritance including doubly uniparental inheritance (DUI), we aimed to assess the genomic variability of the A. islandica mitochondrial DNA (mtDNA). We sequenced the complete mitochondrial genomes of A. islandica specimens from three different sites in the Western Palaearctic (Iceland, North Sea, Baltic Sea). We found the A. islandica mtDNA to fall within the normal size range (18 kb) and exhibit similar coding capacity as other animal mtDNAs. The concatenated protein sequences of all currently known Veneroidea mtDNAs were used to robustly place A. islandica in a phylogenetic framework. Analysis of the observed single nucleotide polymorphism (SNP) patterns on further specimen revealed two prevailing haplotypes. Populations in the Baltic and the North Sea are very homogenous, whereas the Icelandic population, from which exceptionally old individuals have been collected, is the most diverse one. Homogeneity in Baltic and North Sea populations point to either stronger environmental constraints or more recent colonization of the habitat. Our analysis lays the foundation for further studies on A. islandica population structures, age research with this organism, and for phylogenetic studies. Accessions for the mitochondrial genome sequences: KC197241 Iceland; KF363951 Baltic Sea; KF363952 North Sea; KF465708 to KF465758 individual amplified regions from different speciemen     

Inhibition of the myosin light chain kinase prevents hypoxia-induced blood-brain barrier disruption

Increased mortality after stroke is associated with development of brain edema. The aim of the present study was to examine the contribution of endothelial myosin light chain (MLC) phosphorylation to hypoxia-induced blood-brain barrier (BBB) opening. Measurements of trans-endothelial electrical resistance (TEER) were performed to analyse BBB integrity in an in vitro co-culture model (bovine brain microvascular endothelial cells (BEC) and rat astrocytes). Brain fluid content was analysed in rats after stroke induction using a two-vein occlusion model. Dihydroethidium was used to monitor intracellular generation of reactive oxygen species (ROS) in BEC. MLC phosphorylation was detected using immunohistochemistry and immunoblot analysis. Hypoxia caused a decrease of TEER values by more than 40%, which was prevented by inhibition of the MLC-kinase (ML-7, 10 micromol/L). In addition, ML-7 significantly reduced the brain fluid content in vivo after stroke. The NAD(P)H-oxidase inhibitor apocynin (500 micromol/L) prevented the hypoxia-induced TEER decrease. Hypoxia-dependent ROS generation was completely abolished by apocynin. Furthermore, ML-7 and apocynin blocked hypoxia-dependent phosphorylation of MLC. Our data demonstrate that hypoxia causes a breakdown of the BBB in vitro and in vivo involving ROS and the contractile machinery.     

Differential processing of autoantigens in lysosomes from human monocyte-derived and peripheral blood dendritic cells

Dendritic cells (DC) initiate immunity and maintain tolerance. Although in vitro-generated DC, usually derived from peripheral blood monocytes (MO-DC), serve as prototype DC to analyze the biology and biochemistry of DC, phenotypically distinct primary types of DC, including CD1c-DC, are present in peripheral blood (PB-DC). The composition of lysosomal proteases in PB-DC and the way their MHC class II-associated Ag-processing machinery handles a clinically relevant Ag are unknown. We show that CD1c-DC lack significant amounts of active cathepsins (Cat) S, L, and B as well as the asparagine-specific endopeptidase, the major enzymes believed to mediate MHC class II-associated Ag processing. However, at a functional level, lysosomal extracts from CD1c-DC processed the multiple sclerosis-associated autoantigens myelin basic protein and myelin oligodendrocyte glycoprotein in vitro more effectively than MO-DC. Although processing was dominated by CatS, CatD, and asparagine-specific endopeptidase in MO-DC, it was dominated by CatG in CD1c-DC. Thus, human MO-DC and PB-DC significantly differ with respect to their repertoire of active endocytic proteases, so that both proteolytic machineries process a given autoantigen via different proteolytic pathways.     

A J-like protein influences fatty acid composition of chloroplast lipids in Arabidopsis

A comprehensive understanding of the lipid and fatty acid metabolic machinery is needed for optimizing production of oils and fatty acids for fuel, industrial feedstocks and nutritional improvement in plants. T-DNA mutants in the poorly annotated Arabidopsis thaliana gene At1g08640 were identified as containing moderately high levels (50-100%) of 16∶1Δ7 and 18∶1Δ9 leaf fatty acids and subtle decreases (5-30%) of 16∶3 and 18∶3 (http://www.plastid.msu.edu/). TLC separation of fatty acids in the leaf polar lipids revealed that the chloroplastic galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were the main lipid types affected by this mutation. Analysis of the inferred amino acid sequence of At1g08640 predicted the presence of a transit peptide, three transmembrane domains and an N-terminal J-like domain, and the gene was named CJD1 for Chloroplast J-like Domain 1. GFP reporter experiments and in vitro chloroplast import assays demonstrated CJD1 is a chloroplast membrane protein. Screening of an Arabidopsis cDNA library by yeast-2-hybrid (Y2H) using the J-like domain of CJD1 as bait identified a plastidial inner envelope protein (Accumulation and Replication of Chloroplasts 6, ARC6) as the primary interacting partner in the Y2H assay. ARC6 plays a central role in chloroplast division and binds CJD1 via its own J-like domain along with an adjacent conserved region whose function is not fully known. These results provide a starting point for future investigations of how mutations in CJD1 affect lipid composition.     

Association between the plasma proteome and serum ascorbic acid concentrations in humans

Vitamin C has been associated with a reduced risk of chronic diseases, but the biological pathways regulated by vitamin C are not all known. The objective was to use a proteomics approach to identify plasma proteins associated with circulating levels of ascorbic acid. Men and women (n= 1022) 20–29 years of age from the Toronto Nutrigenomics and Health Study completed a general health and lifestyle questionnaire and a 196-item food frequency questionnaire and provided a fasting blood sample. Circulating ascorbic acid was analyzed by high-performance liquid chromatography, and a mass-spectrometry-based multiple reaction monitoring method was used to measure 54 proteins abundant in plasma that are involved in numerous physiologic pathways. Mean protein concentrations were compared across tertiles of serum ascorbic acid using analysis of covariance adjusted for sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference and tertiles of plasma α-tocopherol. A Bonferroni significance level of P<.0009 was applied, and analyses were adjusted for multiple comparisons using the Tukey–Kramer procedure. Levels of complement C9, ceruloplasmin, alpha-1-anti-trypsin, angiotensinogen, complement C3, vitamin D binding protein and plasminogen were inversely associated with levels of ascorbic acid. The inverse association between ascorbic acid and vitamin D binding protein was highest in those with higher levels of serum 25-hydroxyvitamin D. In conclusion, several plasma proteins from various physiologic pathways are significantly associated with circulating levels of ascorbic acid. These findings suggest that vitamin C may have novel physiological effects.     

The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells

Bartonella henselae is an arthropod-borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans. Remarkably, B. henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis. Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B. henselae with the vascular endothelium. However, little is known about the bacterial virulence factors required for this pathogenic process. Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required to establish intraerythrocytic infection in the mammalian reservoir. Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated with the interaction of B. henselae during the interaction with human endothelial cells. These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor kappaB-dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival. Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB-independent mitogenic activity. We conclude that the VirB T4SS is a major virulence determinant of B. henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen.     

Colloidal structure and stability of DNA/polycations polyplexes investigated by small angle scattering

Polyplexes of short DNA-fragments (300 b.p., 100 nm) with tailor-made amine-based polycations of different architectures (linear and hyperbranched) were investigated in buffer solution as a function of the mixing ratio with DNA. The resulting dispersed polyplexes were characterized using small-angle neutron and X-ray scattering (SANS, SAXS) as well as cryo-TEM with respect to their mesoscopic structure and their colloidal stability. The linear polyimines form rather compact structures that have a high tendency for precipitation. In contrast, the hyperbranched polycation with enzymatic-labile pentaethylenehexamine arms (PEHA) yields polyplexes colloidally stable for months. Here the polycation coating of DNA results in a homogeneous dispersion based on a fractal network with low structural organization at low polycation amount. With increasing polycation, bundles of tens of aligned DNA rods appear that are interconnected in a fractal network with a typical correlation distance on the order of 100 nm, the average length of the DNA used. With higher organization comes a decrease in stability. The 3D network built by these beams can still exhibit some stability as long as the material concentration is large enough, but the structure collapses upon dilution. SAXS shows that the complexation does not affect the local DNA structure. Interestingly, the structural findings on the DNA polyplexes apparently correlate with the transfection efficiency of corresponding siRNA complexes. In general, these finding not only show systematic trends for the colloid stability, but may allow for rational approaches to design effective transfection carriers.