Expression of an Artificial Cl<Superscript>−</Superscript> Channel in Microperfused Renal Proximal Tubules

To better understand the process of fluid movement driven by Cl^– conductance, a Cl^– channel-forming peptide was delivered to the luminal membrane of microperfused rabbit renal proximal tubules. When the peptide (NK₄-M2GlyR) was perfused, a significant new conductance was observed within 3 min and stabilized at 10 min. Alteration of the ion composition revealed it to be a Cl^–-specific conductance. Reabsorption of Cl^– (J _Cl) was increased by NK₄-M2GlyR, but not by a scramble NK₄-M2GlyR sequence, suggesting that the active peptide formed de novo Cl^– channels in the luminal membrane of the perfused tubules. In the presence of the peptide, reabsorption of fluid (J _v) was dramatically increased and J _Na and J _Ca were concomitantly increased. We propose that introduction of the new Cl^– conductance in the luminal membrane leads to a coordinated efflux of water across the membrane and an increase in cation translocation via the paracellular pathway, resulting in an increase in J _v. This novel method could prove useful in characterizing mechanisms of fluid transport driven by Cl^– gradients.     

Enhanced Th2 cell-mediated allergic inflammation in Tyk2-deficient mice

Allergic inflammation is mediated by Th2 cell-derived cytokines, including IL-4, IL-5, and IL-13, and down-regulated by IFN-gamma and IL-12. Tyk2 is a member of the Janus family of protein tyrosine kinases and is activated by a variety of cytokines: IFN-alphabeta, IL-6, IL-10, IL-12, and IL-13. In this study, we investigated the role of Tyk2 in the regulation of Ag-induced Th cell differentiation and Ag-induced allergic inflammation in the airways using Tyk2-deficient (Tyk2(-/-)) mice. When splenocytes were stimulated with antigenic peptide, IL-12-mediated Th1 cell differentiation was decreased, but IL-4-mediated Th2 cell differentiation was increased in Tyk2(-/-) mice. In vivo, Ag-specific IgE and IgG1 production was increased, but Ag-specific IgG2a production was decreased in Tyk2(-/-) mice as compared with those in control mice. In addition, Ag-induced eosinophil and CD4(+) T cell recruitment, as well as the production of Th2 cytokines in the airways, was increased in Tyk2(-/-) mice. Adoptive transfer experiments revealed that CD4(+) T cells were responsible for the enhanced Ag-induced eosinophil recruitment in Tyk2(-/-) mice. In contrast, although the level of IL-13 was increased in the airways of Tyk2(-/-) mice after Ag inhalation, the number of goblet cells, as well as Muc5ac mRNA expression, was decreased in Tyk2(-/-) mice. Together, these results indicate that Tyk2 plays a bilateral role in the regulation of allergic inflammation in the airways: Tyk2 plays a role in the down-regulation of Th2 cell-mediated Ab production and eosinophil recruitment in the airways by regulating Th1/Th2 balance toward Th1-type, while Tyk2 is necessary for the induction of IL-13-mediated goblet cell hyperplasia in the airways.     

Targeting tuberculosis and malaria through inhibition of Enoyl reductase: compound activity and structural data

Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD+ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.     

Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells

Pancreatic beta-cells exposed to hyperglycemia produce reactive oxygen species (ROS). Because beta-cells are sensitive to oxidative stress, excessive ROS may cause dysfunction of beta-cells. Here we demonstrate that mitochondrial ROS suppress glucose-induced insulin secretion (GIIS) from beta-cells. Intracellular ROS increased 15min after exposure to high glucose and this effect was blunted by inhibitors of the mitochondrial function. GIIS was also suppressed by H(2)O(2), a chemical substitute for ROS. Interestingly, the first-phase of GIIS could be suppressed by 50 microM H(2)O(2). H(2)O(2) or high glucose suppressed the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and inhibitors of the mitochondrial function abolished the latter effects. Our data suggested that high glucose induced mitochondrial ROS, which suppressed first-phase of GIIS, at least in part, through the suppression of GAPDH activity. We propose that mitochondrial overwork is a potential mechanism causing impaired first-phase of GIIS in the early stages of diabetes mellitus.     

Regulation by nectin of the velocity of the formation of adherens junctions and tight junctions

Cadherins are key Ca(2+)-dependent cell-cell adhesion molecules at adherens junctions (AJs) in fibroblasts and epithelial cells, whereas claudins are key Ca(2+)-independent cell-cell adhesion molecules at tight junctions (TJs) in epithelial cells. The formation and maintenance of TJs are dependent on the formation and maintenance of AJs. Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules which comprise a family of four members, nectin-1, -2, -3, and -4, and are involved in the formation of AJs in cooperation with cadherins, and the subsequent formation of TJs. We show here that the velocity of the formation of the E-cadherin-based AJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in L cells stably expressing E-cadherin and Madin-Darby canine kidney cells. Moreover, the velocity of the formation of the claudin-based TJs is increased by overexpression of nectin-1 and is reduced by addition of the nectin-1 inhibitors to the medium in Madin-Darby canine kidney cells. These results indicate that nectins regulate the velocity of the formation of the E-cadherin-based AJs and the subsequent formation of the claudin-based TJs.     

Crystal structure of chloroplastic ascorbate peroxidase from tobacco plants and structural insights into its instability

Ascorbate peroxidase (APX) is a heme-containing protein that plays a central role in scavenging H(2)O(2) in higher plants. The structure of stromal APX (sAPX) was determined at 1.6 A to an R-factor of 19.1% and an R-free-factor of 22.3%. The electrostatic potential of the gamma-channel that connects the molecular surface of sAPX to the gamma-edge of heme was more positive than that of cytosolic APX (cAPX) from pea, so sAPX might bind more easily with ascorbate than cAPX. The overall structure of sAPX was similar to those of cAPX from pea and cytochrome c peroxidase (CCP) from yeast, with a substantial difference in a loop structure located in the vicinity of the heme. The side chain of Arg169 in sAPX corresponding to His169 in cAPX and His181 in CCP extended in the opposite direction from the heme, forming two hydrogen bonds with carbonyl groups in the loop structure. The rapid inactivation of sAPX might be due to the characteristic conformation of Arg169 owing to the loop structure of sAPX.     

Effect of circular motion exercise on bone modeling and bone mass in young rats: an animal model of isometric exercise

The aims of the study are to develop a non-invasive animal model of circular motion exercise and to evaluate the effect of this type of exercise on bone turnover in young rats. The circular motion exercise simulates isometric exercise using an orbital shaker that oscillates at a frequency of 50 Hz and is capable of speeds from 0-400 rpm. A cage is fixed on top of the shaker and the animals are placed inside. When the shaker is turned on, the oscillatory movement should encourage the animals to hold on to the cage and use various muscle forces to stabilize themselves. Rats at 8 weeks of age were trained on the shaker for 6 weeks and static and dynamic histomorphometric analyses were performed for the proximal tibial metaphysis and the tibial shaft. The exercise resulted in no significant effect on animal body weight, gastrocnemius muscle weight and femoral weight. Although the bone formation rate of cancellous and cortical periosteum was increased by the exercise, trabecular bone volume was decreased. The exercise increased periosteal and marrow perimeters and the cross-sectional diameter of cortical bone from medial to lateral without a significant increase in the cortical bone area. These results suggest that circular motion exercise under force without movement or additional weight loading will cause bone-modeling drift with an increase in bone turnover to reconstruct bone shape in adaptation to the demand in strength. Since there is no additional weight loading during circular motion exercise, the net mass of bone is not increased. The bone mass lost in trabecular bone could possibly be due to a re-distribution of mineral to the cortical bone.     

Wine has activity against entero-pathogenic bacteria in vitro but not in vivo

We studied the activity of wine against entero-pathogenic bacteria both in vitro and in vivo. The food-borne bacteria were killed in both red and white wine within 30 min. However, the results of a Salmonella infection experiment using mice suggested that wine was not effective in preventing food-borne diseases in vivo.     

Entire sequence of a mouse chromosomal segment containing the gene Rhced and a comparative analysis of the homologous human sequence

The mouse genomic sequence of the region containing the gene Rhced, the orthologue to the human gene RH30, was determined to elucidate the structure of Rhced and its flanking regions and to compare these with the corresponding human genomic region. Two genes, Smp1 and AK003528 (an orthologue of FLJ10747), flank Rhced. Neither sequences homologous to the characteristic nucleotide elements flanking the RHD gene in humans (rhesus boxes) nor an additional Rh gene were found within the mouse region sequenced. This result and that of a previous report demonstrate that this chromosomal region of the mouse comprises five genes (FLJ10747-RHCE-SMP1-NPD014-P29) that exhibit syntenic homology with the corresponding human region, which suggests that the RHD gene and rhesus boxes were inserted later. Evaluations of tissue distribution and subcellular localization of these genes indicate that the SMP1 orthologue has a ubiquitous tissue distribution and cytoplasmic localization, whereas AK003528 is expressed slightly higher in testis with a strong subcellular localization in the nucleus. Despite the steady improvements in the draft sequence of the human genome, this study demonstrates the continuing benefits of comparative genetic analyses in increasing our understanding of human genomic structure.