Endoplasmic reticulum stress and its regulator XBP-1 contributes to dendritic cell maturation and activation induced by high mobility group box-1 protein

High mobility group box-1 protein (HMGB1) had been proved to induce maturation and activation of dendritic cell (DC), however, the endogenous changes and mechanisms underlying are unknown. Since endoplasmic reticulum stress (ERS) activates an adaptive unfolded protein response (UPR) that facilitates cellular survival and repair, we hypothesized that HMGB1 may regulate the function of DC by modulating ERS. In our study, HMGB1 stimulation induced significant ERS responses in DCs in a time- and dose-dependent manner, demonstrated by the up-regulation of a number of ERS markers. Gene silence of XBP-1 in splenic DCs decreased the levels of CD80, CD86 as well as major histocompatibility complex (MHC)-II expression and cytokine secretion after HMGB1 treatment, when compared with untransfected or nontargeting-transfected DCs (all P<0.05). Moreover, XBP-1 silenced DCs after treatment with HMGB1 failed to stimulate notable proliferation and differentiation of T cells, unlike normal DCs or nontargeting-transfected DCs (all P<0.05). Gene silence of XBP-1 resulted in down-regulation of the receptor for advanced glycation end products (RAGE) expression on the surface of splenic DCs induced by HMGB1 stimulation (P<0.05). These findings demonstrate an important role for ERS and its regulator XBP-1 in HMGB1-induced maturation and activation of DCs.     

Threonine-insensitive Homoserine Dehydrogenase from Soybean: GENOMIC ORGANIZATION, KINETIC MECHANISM, AND IN VIVO ACTIVITY*

Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K_i = 160–240 mm) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K_i∼150 μm). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.     

Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli

Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23, also known as chemokine-like receptor 1 (CMKLR1). It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. After maturation, Chemerin attracts dendritic cells and macrophages through binding to ChemR23. We report a new method for expression and purification of mature recombinant human Chemerin (rhChemerin) using a prokaryotic system. After being expressed in bacteria, rhChemerin in inclusion bodies was denatured using 6 M guanidine chloride. Soluble rhChemerin was prepared by the protein-specific renaturation solution under defined conditions. It was subsequently purified using ion-exchange columns to more than 95% purity with endotoxin level <1.0 EU/μg. We further demonstrated its biological activities for attracting migration of human dendritic cells and murine macrophages in vitro using established chemotaxis assays.     

Echolocation calls of Rhinolophus ferrumequinum in relation to habitat type and environmental factors

The present experiment was carried out in Luotong Mountain Natural Reserve in Jilin Province of China in 2007. We recorded and analyzed the echolocation calls of Rhinolophus ferrumequinum in different habitats by using Avisoft Bioacoustics USG 116 and Avisoft-SASLAB PRO (Avisoft Bioacoustics, Berlin, Germany). Our results showed that R. ferrumequinum foraged in diverse habitats in the study area, and their echolocation calls were significantly variable in different habitats (One-Way ANOVA, P < 0.05). Vegetative, climatic and topographical factors were selected by using the principal component analysis and the correlations between the parameters of echolocation calls and these environmental factors were analyzed. The results indicated that although R. ferrumequinum always emitted FM/CF/FM echolocation calls in different habitats, the parameters of echolocation calls varied with variable environmental factors. Significant negative correlation existed between FM1 bandwidth and arbor height (r = ?0.948, P < 0.05), FM₂ bandwidth and arbor height (r = ?0.825; P < 0.05), FM1 starting frequency and canopy area (r = ?0.967, P < 0.05), FM₂ ending frequency and canopy area (r = ?0.958, P < 0.05), FM1 starting frequency and air relative humidity (r = ?0.776, P < 0.05), FM₂ ending frequency and air relative humidity (r = ?0.875, P < 0.05), peak frequency and air relative humidity (r = ?0.794, P < 0.05), pulse duration and average shrub height (r = ?0.911, P < 0.05), and inter-pulse interval and average shrub height (r = ?0.990, P < 0.05). Significant positive correlation existed between peak frequency and number of plants (r = 0.756, P < 0.05), and pulse duration and height below the canopy (r = 0.870, P < 0.05). Our results suggested that many kinds of ecological factors (such as vegetation factor, climatic factor and topographical factor) affected the structure of echolocation calls and made them diverse in different habitats, i.e., echolocation calls of bats had phenotypic flexibility and eco-adaptability. These characteristics determined the degree of available habitats and natural resources for R. ferrumequinum.     

The configuration,solvatochromism and metallo‐responses of two novel cyano‐containing oligo(phenylene‐vinylene) derivatives

Two novel cyano‐containing oligo(phenylenevinylene) (OPV) derivatives have been designed and synthesized. Photophysical and sensing properties of the two compounds were studied. Such studies reveal the intramolecular charge transfer process between cyano groups and OPV core. The results showed that the alkyl difference of substituted OPV leads to the changes of molecular configuration and metallo‐response of two compounds. Copyright © 2010 John Wiley & Sons, Ltd.     

A Polymorphism Within the 3′UTR of <Emphasis Type="Italic">NLRP3</Emphasis> is Associated with Susceptibility for Ischemic Stroke in Chinese Population

Stroke was regarded as a severe disorder with high morbidity and high mortality worldwide, ischemic stroke (IS) accounts for 85 to 90 % of new increased stroke cases. Partial mechanisms were elucidated by genetic factors including genomic instability such as single nucleotide polymorphism (SNP). Previous reports demonstrated that inflammation was involved in IS, NLRP3 [nucleotide-binding domain (NOD)-like receptor protein 3], acting as a specific inflammatory gene, however, its function and influence on IS was not well clarified. In this study, a case–control study including 1102 IS patients and 1610 healthy controls was conducted to investigate the association between IS susceptibility with a SNP (rs10754558) in 3′UTR of NLRP3. Logistic regression analysis showed that the heterozygote and the homozygote GG confer a significantly increased risk of CRC after controlling for other covariates (adjusted OR = 1.52, 95 % C.I. 1.19–1.97, P = 0.002; adjusted OR = 2.22, 95 % C.I. 2.18–3.67, P < 0.001, respectively). Carriage of G allele was associated with a greatly increased risk of developing the disease (OR = 1.69, 95 % C.I. 1.31–1.83, P < 0.001). Stratification analysis found that hypertension had interaction with rs10754558 to modulate IS risk. Further in vitro assay revealed that rs10754558 can affect mRNA level of NLRP3, suggesting its possible functional significance. Our data suggested that genetic polymorphisms in NLRP3 may influence IS risk in Chinese population. Replication of our studies in other populations and further functional studies are required for complete comprehension of the roles of NLRP3 polymorphisms in IS risk.     

A new screening method based on yeast-expressed human dipeptidyl peptidase IV and discovery of novel inhibitors

Dipeptidyl peptidase (DPP) IV inhibitors provide a new strategy for the treatment of type 2 diabetes. Human DPP-IV gene was cloned from differentiated Caco-2 cells and expressed in Pichia pastoris. The recombinant enzyme was used in a new system for screening of DPP-IV inhibitors. By high throughput screening, a novel compound (W5188) was identified from 75,000 compounds with an IC₅₀ of 6.5 μM. This method is highly reproducible and reliable for discovery of DPP-IV inhibitors as shown by Z′ value of 0.73 and S/N ratio of 6.89.