Products of thiocyanate peroxidation: properties and reaction mechanisms

The lactoperoxidase-catalyzed oxidation of thiocyanate (SCN-) was studied in the pH range 3-8. The ultraviolet spectra of the oxidation products, the hypothiocyanite ion, OSCN- (at pH 8) and hypothiocyanous acid, HOSCN (at pH 3), were recorded. The absorbance maxima for OSCN- and HOSCN were observed at 220 and 240 nm, respectively. The extinction coefficients for OSCN- and HOSCN were determined to be 3870 (at 220 nM) and 95 M-1 X cm-1 (at 240 nM), respectively. Pure solutions of OSCN- (at pH 8) and HOSCN (at pH 3) were stable, but the mixtures of these two species at intermediate pH values were unstable. The decomposition could be divided into two periods, an initial period of rapid increase in oxidizing equivalents and a second period of decomposition. Decomposition during the second period followed first-order kinetics, and the pH-dependence of the apparent first-order rate constant was consistent with a decomposition mechanism which involved HOSCN. The first-order rate constant for this step was estimated to be 6 X 10(-3) s-1 at 37 degrees C.     

Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis

Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry and ELISA. Significantly, deglycosylated CRPs showed a 7–8-fold reduced binding with erythrocytes confirming the role of glycosylated moieties. Scatchard analysis revealed striking differences in the apparent binding constants (10⁴–10⁵ M⁻¹) and number of binding sites (10⁶–10⁷sites/erythrocyte) for CRP on patients’ erythrocytes as compared to normal. Western blotting along with immunoprecipitation analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis, even at physiological concentration of CRP (10 μg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two diseases.     

Kynurenic Acid Triggers Firm Arrest of Leukocytes to Vascular Endothelium under Flow Conditions

Recent studies have demonstrated that kynurenic acid (KYNA), a compound produced endogenously by the interferon-γ-induced degradation of tryptophan by indoleamine 2,3-dioxygenase, activates the previously orphaned G protein-coupled receptor, GPR35. This receptor is expressed in immune tissues, although its potential function in immunomodulation remains to be explored. We determined that GPR35 was most highly expressed on human peripheral monocytes. In an in vitro vascular flow model, KYNA triggered the firm arrest of monocytes to both fibronectin and ICAM-1, via β₁ integrin- and β₂ integrin-mediated mechanisms, respectively. Incubation of monocytes with pertussis toxin prior to use in flow experiments significantly reduced the KYNA-induced monocyte adhesion, suggesting that adhesion is triggered by a G_i-mediated process. Furthermore, KYNA-triggered adhesion of monocytic cells was reduced by short hairpin RNA-mediated silencing of GPR35. Although GPR35 is expressed at slightly lower levels on neutrophils, KYNA induced firm adhesion of these cells to an ICAM-1-expressing monolayer as well. KYNA also elicited neutrophil shedding of surface L-selectin, another indicator of leukocyte activation. Taken together, these data suggest that KYNA could be an important early mediator of leukocyte recruitment.Leukocyte recruitment into tissue compartments is a tightly regulated process orchestrated by chemokines (1). Chemokines convert leukocyte rolling or tethering on the vascular endothelium to firm arrest via the activation of leukocyte surface integrins (2, 3). As chemoattractants, chemokines subsequently play an important role in the directional migration of leukocytes through tissues.Chemoattractant receptors are a subtype of G protein-coupled receptors (GPCRs),³ one of the largest known families of human proteins. Chemoattractant receptors bind a variety of agonists, including proteins such as interleukin-8 (IL-8, CXCL8) (4) and monocyte chemoattractant protein-1 (MCP-1, CCL2) (5), small peptides such as fMLP (6), as well as bioactive lipids including leukotriene B₄ (7). As such, chemoattractant receptors mirror the entire family of GPCRs, which can be activated by ligands ranging in size from metabolites to large proteins (8).Because GPCRs serve as targets for therapeutic intervention, considerable activity has gone into the identification of both putative GPCR genes and the ligands for the resulting receptors (8). Recently, the tryptophan metabolite kynurenic acid (KYNA) was identified as an agonist for the previously “orphaned” receptor GPR35. KYNA was shown to elicit intracellular release of Ca²⁺ in Chinese hamster ovary cells in which GPR35 was co-expressed in the context of a chimeric G protein signaling apparatus. HEK93 cells transfected with GPR35 and G_qo proteins accumulated inositol phosphate upon exposure to KYNA. KYNA also induced internalization of GPR35 on HeLa cells, which is commonly seen following the activation of GPRs with agonists such as chemokines (9).KYNA is produced endogenously as a result of the degradation of tryptophan (Fig. 1). In most tissues, the rate-limiting step in this degradation is the conversion of tryptophan to N-formylkynurenine, a reaction that can be catalyzed by the inducible enzyme indolemine 2,3-dioxygenase (IDO). IDO is induced by interferon-γ, which leads to a substantial increase in the concentration of KYNA and other tryptophan catabolites during inflammatory processes.Open in a separate windowFIGURE 1.Tryptophan catabolism pathway. Schematic diagram of tryptophan catabolism. Tryptophan is converted to N-formylkynurenine by IDO, and is then deformylated to form kynurenine. Kynurenine can be converted to kynurenic acid via kynurenine aminotransferases I and II (KAT), or converted via alternative pathways to anthranilic acid or quinolinic acid, a NAD precursor.Previous work has demonstrated that KYNA acts as a neuroprotective agent by antagonizing both N-methyl-d-aspartate and α7-nicotinic receptors (10–12). Many peripheral tissues, including the heart and vasculature, are also capable of generating KYNA (13, 14), although its role in these tissues has not been well defined. Expression analysis indicates that GPR35 is selectively present in immune and intestinal tissues. From a functional perspective, KYNA treatment inhibited the secretion of tumor necrosis factor-α by mononuclear cells treated with lipopolysaccharide (9). This finding is in general agreement with the prevailing literature suggesting that IDO-mediated tryptophan catabolism appears to play a significant counter-regulatory role in dampening down the activation of the immune system (15). However, the potential spectrum of physiological roles for KYNA in immune modulation remains incompletely characterized.Given the reported high level of GPR35 expression on circulating leukocytes, here we tested the hypothesis that KYNA may play a chemokine-like role in modulating leukocyte-endothelial interactions under physiologically relevant flow conditions as seen in the vasculature. We explored the intracellular signaling pathways by which KYNA may be activating leukocytes, as well as the surface integrins modulating these effects. We report the unanticipated finding that KYNA is sufficient to drive early leukocyte adhesion.     

Molecular basis for Rac1 recognition by guanine nucleotide exchange factors.

Rho GTPases are activated by a family of guanine nucleotide exchange factors (GEFs) known as Dbl family proteins. The structural basis for how GEFs recognize and activate Rho GTPases is presently ill defined. Here, we utilized the crystal structure of the DH/PH domains of the Rac-specific GEF Tiam1 in complex with Rac1 to determine the structural elements of Rac1 that regulate the specificity of this interaction. We show that residues in the Rac1 beta2-beta3 region are critical for Tiam1 recognition. Additionally, we determined that a single Rac1-to-Cdc42 mutation (W56F) was sufficient to abolish Rac1 sensitivity to Tiam1 and allow recognition by the Cdc42-specific DH/PH domains of Intersectin while not impairing Rac1 downstream activities. Our findings identified unique GEF specificity determinants in Rac1 and provide important insights into the mechanism of DH/PH selection of GTPase targets.     

Accumulation of mutations and somatic selection in aging neural stem/progenitor cells

Genomic instability within somatic stem cells may lead to the accumulation of mutations and contribute to cancer or other age-related phenotypes. However, determining the frequency of mutations that differ among individual stem cells is difficult from whole tissue samples because each event is diluted in the total population of both stem cells and differentiated tissue. Here the ability to expand neural stem/progenitor cells clonally permitted measurement of genomic alterations derived from a single initial cell. C57Bl/6 x DBA/2 hybrid mice were used and PCR analysis with strain-specific primers was performed to detect loss of heterozygosity on nine different chromosomes for each neurosphere. The frequency with which changes occurred in neurospheres derived from 2-month- and 2-year-old mice was compared. In 15 neurospheres derived from young animals both parental chromosomes were present for all nine chromosome pairs. In contrast, 16/17 neurospheres from old animals demonstrated loss of heterozygosity (LOH) on one or more chromosomes and seven exhibited a complete deletion of at least one chromosomal region. For chromosomes 9 and 19 there is a significant bias in the allele that is lost where in each case the C57Bl/6 allele is retained in 6/6 neurospheres exhibiting LOH. These data suggest that aging leads to a substantial mutational load within the neural stem cell compartment which can be expected to affect the normal function of these cells. Furthermore, the retention of specific alleles for chromosomes 9 and 19 suggests that a subset of mutational events lead to an allele-specific survival advantage within the neural stem cell compartment.     

NCBI Reference Sequence project: update and current status

The goal of the NCBI Reference Sequence (RefSeq) project is to provide the single best non-redundant and comprehensive collection of naturally occurring biological molecules, representing the central dogma. Nucleotide and protein sequences are explicitly linked on a residue-by-residue basis in this collection. Ideally all molecule types will be available for each well-studied organism, but the initial database collection pragmatically includes only those molecules and organisms that are most readily identified. Thus different amounts of information are available for different organisms at any given time. Furthermore, for some organisms additional intermediate records are provided when the genome sequence is not yet finished. The collection is supplied by NCBI through three distinct pipelines in addition to collaborations with community groups. The collection is curated on an ongoing basis. Additional information about the NCBI RefSeq project is available at http://www.ncbi.nih.gov/RefSeq/.