Posttranslational regulation of I-Ed by affinity for CLIP

Several MHC class II alleles linked with autoimmune diseases form unusually low stability complexes with CLIP, leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. To investigate cellular consequences of altering class II/CLIP affinity, we evaluated invariant chain (Ii) mutants with varying CLIP affinity for a mouse class II allele, I-E(d), which has low affinity for wild-type CLIP and is associated with a mouse model of spontaneous, autoimmune joint inflammation. Increasing CLIP affinity for I-E(d) resulted in increased cell surface and total cellular abundance and half-life of I-E(d). This reveals a post-endoplasmic reticulum chaperoning capacity of Ii via its CLIP peptides. Quantitative effects on I-E(d) were less pronounced in DM-expressing cells, suggesting complementary chaperoning effects mediated by Ii and DM, and implying that the impact of allelic variation in CLIP affinity on immune responses will be highest in cells with limited DM activity. Differences in the ability of cell lines expressing wild-type or high-CLIP-affinity mutant Ii to present Ag to T cells suggest a model in which increased CLIP affinity for class II serves to restrict peptide loading to DM-containing compartments, ensuring proper editing of antigenic peptides.     

E(z), a depth-dependent potential for assessing the energies of insertion of amino acid side-chains into membranes: derivation and applications to determining the orientation of transmembrane and interfacial helices

We have developed an empirical residue-based potential (E(z) potential) for protein insertion in lipid membranes. Propensities for occurrence as a function of depth in the bilayer were calculated for the individual amino acid types from their distribution in known structures of helical membrane proteins. The propensities were then fit to continuous curves and converted to a potential using a reverse-Boltzman relationship. The E(z) potential demonstrated a good correlation with experimental data such as amino acid transfer free energy scales (water to membrane center and water to interface), and it incorporates transmembrane helices of varying composition in the membrane with trends similar to those obtained with translocon-mediated insertion experiments. The potential has a variety of applications in the analysis of natural membrane proteins as well as in the design of new ones. It can help in calculating the propensity of single helices to insert in the bilayer and estimate their tilt angle with respect to the bilayer normal. It can be utilized to discriminate amphiphilic helices that assume a parallel orientation at the membrane interface, such as those of membrane-active peptides. In membrane protein design applications, the potential allows an environment-dependent selection of amino acid identities.     

The Himalayas as a directional barrier to gene flow

High-resolution Y-chromosome haplogroup analyses coupled with Y-short tandem repeat (STR) haplotypes were used to (1) investigate the genetic affinities of three populations from Nepal--including Newar, Tamang, and people from cosmopolitan Kathmandu (referred to as "Kathmandu" subsequently)--as well as a collection from Tibet and (2) evaluate whether the Himalayan mountain range represents a geographic barrier for gene flow between the Tibetan plateau and the South Asian subcontinent. The results suggest that the Tibetans and Nepalese are in part descendants of Tibeto-Burman-speaking groups originating from Northeast Asia. All four populations are represented predominantly by haplogroup O3a5-M134-derived chromosomes, whose Y-STR-based age (+/-SE) was estimated at 8.1+/-2.9 thousand years ago (KYA), more recent than its Southeast Asian counterpart. The most pronounced difference between the two regions is reflected in the opposing high-frequency distributions of haplogroups D in Tibet and R in Nepal. With the exception of Tamang, both Newar and Kathmandu exhibit considerable similarities to the Indian Y-haplogroup distribution, particularly in their haplogroup R and H composition. These results indicate gene flow from the Indian subcontinent and, in the case of haplogroup R, from Eurasia as well, a conclusion that is also supported by the admixture analysis. In contrast, whereas haplogroup D is completely absent in Nepal, it accounts for 50.6% of the Tibetan Y-chromosome gene pool. Coalescent analyses suggest that the expansion of haplogroup D derivatives--namely, D1-M15 and D3-P47 in Tibet--involved two different demographic events (5.1+/-1.8 and 11.3+/-3.7 KYA, respectively) that are more recent than those of D2-M55 representatives common in Japan. Low frequencies, relative to Nepal, of haplogroup J and R lineages in Tibet are also consistent with restricted gene flow from the subcontinent. Yet the presence of haplogroup O3a5-M134 representatives in Nepal indicates that the Himalayas have been permeable to dispersals from the east. These genetic patterns suggest that this cordillera has been a biased bidirectional barrier.     

Differential regulation of cholera toxin-inhibited Na-H exchange isoforms by butyrate in rat ileum

Electroneutral Na absorption occurs in the intestine via sodium-hydrogen exchanger (NHE) isoforms NHE2 and NHE3. Bicarbonate and butyrate both stimulate electroneutral Na absorption through NHE. Bicarbonate- but not butyrate-dependent Na absorption is inhibited by cholera toxin (CT). Long-term exposure to butyrate also influences expression of apical membrane proteins in epithelial cells. These studies investigated the effects of short- and long-term in vivo exposure to butyrate on apical membrane NHE and mRNA, protein expression, and activity in rat ileal epithelium that had been exposed to CT. Ileal loops were exposed to CT in vivo for 5 h and apical membrane vesicles were isolated. 22Na uptake was measured by using the inhibitor HOE694 to identify NHE2 and NHE3 activity, and Western blot analyses were performed. CT reduced total NHE activity by 70% in apical membrane vesicles with inhibition of both NHE2 and NHE3. Reduced NHE3 activity and protein expression remained low following removal of CT but increased to control values following incubation of the ileal loop with butyrate for 2 h. In parallel there was a 40% decrease in CT-induced increase in cAMP content. In contrast, NHE2 activity partially increased following removal of CT and was further increased to control levels by butyrate. NHE2 protein expression did not parallel its activity. Neither NHE2 nor NHE3 mRNA content were affected by CT or butyrate. These results indicate that CT has varying effects on the two apical NHE isoforms, inhibiting NHE2 activity without altering its protein expression and reducing both NHE3 activity and protein expression. Butyrate restores both CT-inhibited NHE2 and NHE3 activities to normal levels but via different mechanisms.     

Noradrenergic afferents and receptors in the medial preoptic area: neuroanatomical and neurochemical links between the regulation of sleep and body temperature

Several studies have shown the importance of the medial preoptic area in the regulation of sleep-wakefulness and of body temperature. The medial preoptic area has a rich noradrenergic innervation, coming mostly from the lateral tegmental noradrenergic system. The accumulating evidences show that the noradrenergic afferents to the medial preoptic area are involved in the induction of sleep. This hypnogenic mechanism operates through the postsynaptic alpha1 and alpha2-adrenergic receptors. Noradrenergic afferents are also involved in the thermoregulatory mechanisms, and the activation of these fibers brings about a fall in body temperature. Though the body temperature changes are brought about by the same receptor subtypes as those involved in hypnogenesis, observations suggest the possibility of separate sets of noradrenergic afferents in the medial preoptic area for sleep regulation and thermoregulation. In this review, we present the compelling evidences, which showed that the noradrenergic afferents of the medial preoptic area bring about a fall in body temperature and other thermoregulatory behavioral alterations associated with sleep.     

A computational docking study for prediction of binding mode of diospyrin and derivatives: Inhibitors of human and leishmanial DNA topoisomerase-I

A computational approach was utilized to study the relative binding modes of diospyrin (bisnaphthoquinonoid) with the crystal structure of human DNA-TopoI and the recently reported Leishmania donavani DNA-TopoI. Additionally, the binding site interactions of amino derivatives of diospyrin with human TopoI were studied extensively. Based on the docking results, binding modes of diospyrin with the human and leishmanial TopoI catalytic core were predicted. The parallel use of two efficient and predictive docking programs, GOLD and Ligandfit, allowed mutual validation of the predicted binding poses. A reasonably good correlation coefficient between the calculated docking scores and the experimentally determined cytotoxicity helped in validating the docking method. Furthermore, a structure-based pharmacophore model was developed for L. donavani DNA-TopoI inhibition which helped in elucidating the topological and spatial requirements of the ligand-receptor interactions. This study provides an understanding of the structural basis of ligand binding to the topoisomerase receptor, which may be used for the structure-based design of potent and novel ligands for anticancer and antileishmanial therapy. To our knowledge, this is the first report of a binding mode exploration study for diospyrin and its derivatives as inhibitors of the leishmanial and human TopoI enzymes.     

Commensal Bacteria-induced Interleukin 1β (IL-1β) Secreted by Macrophages Up-regulates Hepcidin Expression in Hepatocytes by Activating the Bone Morphogenetic Protein Signaling Pathway

The liver hormone hepcidin is the central regulator of systemic iron metabolism. Its increased expression in inflammatory states leads to hypoferremia and anemia. Elucidation of the mechanisms that up-regulate hepcidin during inflammation is essential for developing rational therapies for this anemia. Using mouse models of inflammatory bowel disease, we have shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition. Here we investigate how two commensal bacteria, Bifidobacterium longum and Bacteroides fragilis, representative members of the gut microbiota, affect hepcidin expression. We found that supernatants of a human macrophage cell line infected with either of the bacteria up-regulated hepcidin when added to a human hepatocyte cell line. This activity was abrogated by neutralization of IL-1β. Moreover, purified IL-1β increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes and when injected into mice. IL-1β activated the bone morphogenetic protein (BMP) signaling pathway in hepatocytes and in mouse liver, as indicated by increased phosphorylation of small mothers against decapentaplegic proteins. Activation of BMP signaling correlated with IL-1β-induced expression of BMP2 in human hepatocytes and activin B in mouse liver. Treatment of hepatocytes with two different chemical inhibitors of BMP signaling or with a neutralizing antibody to BMP2 prevented IL-1β-induced up-regulation of hepcidin. Our results clarify how commensal bacteria affect hepcidin expression and reveal a novel connection between IL-1β and activation of BMP signaling. They also suggest that there may be differences between mice and humans with respect to the mechanism by which IL-1β up-regulates hepcidin.     

Na-H Exchanger Isoform-2 (NHE2) Mediates Butyrate-dependent Na+ Absorption in Dextran Sulfate Sodium (DSS)-induced Colitis

Diarrhea associated with ulcerative colitis (UC) occurs primarily as a result of reduced Na⁺ absorption. Although colonic Na⁺ absorption is mediated by both epithelial Na⁺ channels (ENaC) and Na-H exchangers (NHE), inhibition of NHE-mediated Na⁺ absorption is the primary cause of diarrhea in UC. As there are conflicting observations reported on NHE expression in human UC, the present study was initiated to identify whether NHE isoforms (NHE2 and NHE3) expression is altered and how Na⁺ absorption is regulated in DSS-induced inflammation in rat colon, a model that has been used to study UC. Western blot analyses indicate that neither NHE2 nor NHE3 expression is altered in apical membranes of inflamed colon. Na⁺ fluxes measured in vitro under voltage clamp conditions in controls demonstrate that both HCO₃⁻-dependent and butyrate-dependent Na⁺ absorption are inhibited by S3226 (NHE3-inhibitor), but not by HOE694 (NHE2-inhibitor) in normal animals. In contrast, in DSS-induced inflammation, butyrate-, but not HCO₃⁻-dependent Na⁺ absorption is present and is inhibited by HOE694, but not by S3226. These observations indicate that in normal colon NHE3 mediates both HCO₃⁻-dependent and butyrate-dependent Na⁺ absorption, whereas DSS-induced inflammation activates NHE2, which mediates butyrate-dependent (but not HCO₃⁻-dependent) Na⁺ absorption. In in vivo loop studies HCO₃⁻-Ringer and butyrate-Ringer exhibit similar rates of water absorption in normal rats, whereas in DSS-induced inflammation luminal butyrate-Ringer reversed water secretion observed with HCO₃⁻-Ringer to fluid absorption. Lumen butyrate-Ringer incubation activated NHE3-mediated Na⁺ absorption in DSS-induced colitis. These observations suggest that the butyrate activation of NHE2 would be a potential target to control UC-associated diarrhea.