Structure of a classical MHC class I molecule that binds "non-classical" ligands

Chicken YF1 genes share a close sequence relationship with classical MHC class I loci but map outside of the core MHC region. To obtain insights into their function, we determined the structure of the YF1*7.1/β₂-microgloblin complex by X-ray crystallography at 1.3 Å resolution. It exhibits the architecture typical of classical MHC class I molecules but possesses a hydrophobic binding groove that contains a non-peptidic ligand. This finding prompted us to reconstitute YF1*7.1 also with various self-lipids. Seven additional YF1*7.1 structures were solved, but only polyethyleneglycol molecules could be modeled into the electron density within the binding groove. However, an assessment of YF1*7.1 by native isoelectric focusing indicated that the molecules were also able to bind nonself-lipids. The ability of YF1*7.1 to interact with hydrophobic ligands is unprecedented among classical MHC class I proteins and might aid the chicken immune system to recognize a diverse ligand repertoire with a minimal number of MHC class I molecules.     

Asymmetric reduction of ketopantolactone using a strictly (<Emphasis Type="Italic">R</Emphasis>)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy

Objectives

To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL].

Results

The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%.

Conclusions

Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

     

TRPV1 activation impedes foam cell formation by inducing autophagy in oxLDL-treated vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) are an important origin of foam cells besides macrophages. The mechanisms underlying VSMC foam cell formation are relatively little known. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1) and autophagy have a potential role in regulating foam cell formation. Our study demonstrated that autophagy protected against foam cell formation in oxidized low-density lipoprotein (oxLDL)-treated VSMCs; activation of TRPV1 by capsaicin rescued the autophagy impaired by oxLDL and activated autophagy–lysosome pathway in VSMCs; activation of TRPV1 by capsaicin impeded foam cell formation of VSMCs through autophagy induction; activation of TRPV1 by capsaicin induced autophagy through AMP-activated protein kinase (AMPK) signaling pathway. This study provides evidence that autophagy plays an important role in VSMC foam cell formation and highlights TRPV1 as a promising therapeutic target in atherosclerosis.     

Dynamic 1H NMR-based extracellular metabonomic analysis of oligodendroglia cells infected with herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) is a large, neurotropic, double-stranded DNA virus that establishes a lifelong latent infection in neurons and glial cells. Previous studies reveal that several metabolic perturbations are associated with HSV-1 infection. However, the extracellular metabolic alterations associated with HSV-1 infection have not been systematically profiled in human cells. Here, a proton nuclear magnetic resonance-based metabonomic approach was applied to differentiate the extracellular metabonomic profiles of HSV-1 infected human oligodendroglia cells (n = 18) and matched control cells (n = 18) at three time points (12, 24, and 36 h post-infection). Resulting spectra were analyzed by chemometric and statistical methods. Metabonomic profiling revealed perturbations in 21 extracellular metabolites. Partial least squares discriminant analysis demonstrated that the whole metabolic patterns enabled statistical discrimination between HSV-1 infected human oligodendroglia cells and control cells. Eight extracellular metabolites, seven of which were amino acids, were primarily responsible for score plot discrimination between HSV-1 infected human oligodendroglia cells and control cells at 36 h post-infection: alanine, glycine, isoleucine, leucine, glutamate, glutamine, histidine, and lactate. HSV-1 infection alters amino acid metabolism in human oligodendroglia cells cultured in vitro. HSV-1 infection may disturb these host cellular pathways to support viral replication. Through elucidating the extracellular metabolic changes incident to HSV-1 infection, this study also provides future directions for investigation into the pathogenic mechanism of HSV-1.     

MiR-133a Is Functionally Involved in Doxorubicin-Resistance in Breast Cancer Cells MCF-7 via Its Regulation of the Expression of Uncoupling Protein 2

The development of novel targeted therapies holds promise for conquering chemotherapy resistance, which is one of the major hurdles in current breast cancer treatment. Previous studies indicate that mitochondria uncoupling protein 2 (UCP-2) is involved in the development of chemotherapy resistance in colon cancer and lung cancer cells. In the present study we found that lower level of miR133a is accompanied by increased expression of UCP-2 in Doxorubicin-resistant breast cancer cell cline MCF-7/Dox as compared with its parental cell line MCF-7. We postulated that miR133a might play a functional role in the development of Doxorubicin-resistant in breast cancer cells. In this study we showed that: 1) exogenous expression of miR133a in MCF-7/Dox cells can sensitize their reaction to the treatment of Doxorubicin, which is coincided with reduced expression of UCP-2; 2) knockdown of UCP-2 in MCF-7/Dox cells can also sensitize their reaction to the treatment of Doxorubicin; 3) intratumoral delivering of miR133a can restore Doxorubicin treatment response in Doxorubicin-resistant xenografts in vivo, which is concomitant with the decreased expression of UCP-2. These findings provided direct evidences that the miR133a/UCP-2 axis might play an essential role in the development of Doxorubicin-resistance in breast cancer cells, suggesting that the miR133a/UCP-2 signaling cohort could be served as a novel therapeutic target for the treatment of chemotherapy resistant in breast cancer.     

Magnesium Deficiency Enhances Hydrogen Peroxide Production and Oxidative Damage in Chick Embryo Hepatocyte In Vitro

Magnesium deficiency and oxidative stress have been identified as correlative factors in many diseases. The origin of free radicals correlated with oxidative damage resulting from Mg-deficiency is unclear at the cellular level. To investigate whether hydrogen peroxide (H₂O₂) is associated in the oxidative stress induced by Mg-deficiency, the effect of Mg²⁺ deficiency (0, 0.4, 0.7 mM) on the metabolism of H₂O₂ was investigated in cultured chick embryo hepatocytes. After being cultured in the media with various concentrations of Mg²⁺ for 1, 2, 4, 6 and 10 days, parameters of H₂O₂ production, catalase activity, lipid peroxidation, intracellular total Mg and cell viability were analyzed. Results demonstrated that long-term incubation of chick embryo hepatocyte in extracellular Mg²⁺-deprivative and Mg²⁺-deficient (0.4 mM) states significantly enhanced the production of H₂O₂ (approximately twofold, respectively) and lipid peroxidation in the cell cultures, while decreasing the cell viability. Additionally, the reversing action of Mg²⁺ re-added to 1.0 mM and the partial reversing action of dimethylthiourea suggested that (i) [Mg²⁺]_e deficiency induced the increase of H₂O₂ production, (ii) [Mg²⁺]_e deficiency decreased catalase activity in chick embryo hepatocyte in vitro, subsequently causing oxidative stress and cell peroxidative damage.     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.     

Fusaric acid induction of programmed cell death modulated through nitric oxide signalling in tobacco suspension cells

Fusaric acid (FA) is a nonhost-selective toxin mainly produced by Fusarium oxysporum, the causal agent of plant wilt diseases. We demonstrate that FA can induce programmed cell death (PCD) in tobacco suspension cells and the FA-induced PCD is modulated by nitric oxide (NO) signalling. Cells undergoing cell death induced by FA treatment exhibited typical characteristics of PCD including cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane plasmolysis, and formation of small cytoplasmic vacuoles. In addition, caspase-3-like activity was activated upon the FA treatment. The process of FA-induced PCD was accompanied by a rapid accumulation of NO in a FA dose-dependent manner. Pre-treatment of cells with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N ^G-monomethyl-arginine monoacetate (L-NMMA) significantly reduced the rate of FA-induced cell death. Furthermore, the caspase-3-like activity and the expression of PAL and Hsr203J genes were alleviated by application of cPTIO or L-NMMA to FA-treated tobacco cells. This indicates that NO is an important factor involved in the FA-induced PCD. Our results also show that pre-treatment of tobacco cells with a caspase-3-specific inhibitor, Ac-DEVD-CHO, can reduce the rate of FA-induced cell death. These results demonstrate that the FA-induced cell death is a PCD and is modulated by NO signalling through caspase-3-like activation.