A Human CD4+ T Cell Epitope in the Influenza Hemagglutinin Is Cross-Reactive to Influenza A Virus Subtypes and to Influenza B Virus

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.     

Sphingosine 1-phosphate protects primary human keratinocytes from apoptosis via nitric oxide formation through the receptor subtype S1P3

Although the lipid mediator sphingosine 1-phosphate (S1P) has been identified to induce cell growth arrest of human keratinocytes, the sphingolipid effectively protects these epidermal cells from apoptosis. The molecular mechanism of the anti-apoptotic action induced by S1P is less characterized. Apart from S1P, endogenously produced nitric oxide (NO^?) has been recognized as a potent modulator of apoptosis in keratinocytes. Therefore, it was of great interest to elucidate whether S1P protects human keratinocytes via a NO^?-dependent signalling pathway. Indeed, S1P induced an activation of endothelial nitric oxide synthase (eNOS) in human keratinocytes leading to an enhanced formation of NO^?. Most interestingly, the cell protective effect of S1P was almost completely abolished in the presence of the eNOS inhibitor L-NAME as well as in eNOS-deficient keratinocytes indicating that the sphingolipid metabolite S1P protects human keratinocytes from apoptosis via eNOS activation and subsequent production of protective amounts of NO^?. It is well established that most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Therefore, the involvement of S1P-receptor subtypes in S1P-mediated eNOS activation has been examined. Indeed, this study clearly shows that the S1P₃ is the exclusive receptor subtype in human keratinocytes which mediates eNOS activation and NO^? formation in response to S1P. In congruence, when the S1P₃ receptor subtype is abrogated, S1P almost completely lost its ability to protect human keratinocytes from apoptosis.     

First crystal structure of an endo-inulinase,INU2, from Aspergillus ficuum: Discovery of an extra-pocket in the catalytic domain responsible for its endo-activity

Endo-inulinase is a member of glycosidase hydrolase family 32 (GH32) degrading fructans of the inulin type with an endo-cleavage mode and is an important class of industrial enzyme. In the present study, we report the first crystal structure of an endo-inulinase, INU2, from Aspergillus ficuum at 1.5 Å. It was solved by molecular replacement with the structure of exo-inulinase as search model. The 3D structure presents a bimodular arrangement common to other GH32 enzymes: a N-terminal 5-fold β-propeller catalytic domain with four β-sheets and a C-terminal β-sandwich domain organized in two β-sheets with five β-strands. The structural analysis and comparison with other GH32 enzymes reveal the presence of an extra pocket in the INU2 catalytic site, formed by two loops and the conserved motif W-M(I)-N-D(E)-P-N-G. This cavity would explain the endo-activity of the enzyme, the critical role of Trp40 and particularly the cleavage at the third unit of the inulin(-like) substrates. Crystal structure at 2.1 Å of INU2 complexed with fructosyl molecules, experimental digestion data and molecular modelling studies support these hypotheses.     

PEGylation of protein-based MRI contrast agents improves relaxivities and biocompatibilities

Magnetic resonance imaging (MRI) has emerged as a leading diagnostic technique in clinical and preclinical settings. However, the application of MRI to assess specific disease markers for diagnosis and monitoring drug effect has been severely hampered by the lack of desired contrast agents with high relaxivities, and optimized in vivo retention time. We have reported the development of protein-based MRI contrast agents (ProCA1) by rational design of Gd^3 + binding sites into a stable protein resulting in significantly increased longitudinal (r₁) and transverse (r₂) relaxivities compared to Gd-DTPA. Here, we report a further improvement of protein contrast agents ProCA1 for in vivo imaging by protein modification with various sizes of polyethylene glycol (PEG) chain. PEGylation results in significant increases of both r₁ and r₂ relaxivities (up to 200%), and these high relaxivities persist even at field strengths up to 9.4 T. In addition, our experimental results demonstrate that modified contrast agents have significant improvement of in vivo MR imaging and biocompatibilities including dose efficiency, protein solubility, blood retention time and decreased immunogenicity. Such improvement can be important to the animal imaging and pre-clinical research at high or ultra-high field where there is an urgent need for molecular imaging probes and optimized contrast agent.     

CB(1) antagonism restores hepatic insulin sensitivity without normalization of adiposity in diet-induced obese dogs

The endocannabinoid system is highly implicated in the development of insulin resistance associated with obesity. It has been shown that antagonism of the CB(1) receptor improves insulin sensitivity (S(I)). However, it is unknown whether this improvement is due to the direct effect of CB(1) blockade on peripheral tissues or secondary to decreased fat mass. Here, we examine in the canine dog model the longitudinal changes in S(I) and fat deposition when obesity was induced with a high-fat diet (HFD) and animals were treated with the CB(1) antagonist rimonabant. S(I) was assessed (n = 20) in animals fed a HFD for 6 wk to establish obesity. Thereafter, while HFD was continued for 16 additional weeks, animals were divided into two groups: rimonabant (1.25 mg·kg(-1)·day(-1) RIM; n = 11) and placebo (n = 9). Euglycemic hyperinsulinemic clamps were performed to evaluate changes in insulin resistance and glucose turnover before HFD (week -6) after HFD but before treatment (week 0) and at weeks 2, 6, 12, and 16 of treatment (or placebo) + HFD. Magnetic resonance imaging was performed to determine adiposity- related changes in S(I). Animals developed significant insulin resistance and increased visceral and subcutaneous adiposity after 6 wk of HFD. Treatment with RIM resulted in a modest decrease in total trunk fat with relatively little change in peripheral glucose uptake. However, there was significant improvement in hepatic insulin resistance after only 2 wk of RIM treatment with a concomitant increase in plasma adiponectin levels; both were maintained for the duration of the RIM treatment. CB(1) receptor antagonism appears to have a direct effect on hepatic insulin sensitivity that may be mediated by adiponectin and independent of pronounced reductions in body fat. However, the relatively modest effect on peripheral insulin sensitivity suggests that significant improvements may be secondary to reduced fat mass.     

Abdominal fat analyzed by DEXA scan reflects visceral body fat and improves the phenotype description and the assessment of metabolic risk in mice

Clinical studies have demonstrated a strong relationship between visceral fat content and metabolic diseases, such as type 2 diabetes and liver steatosis. Obese mouse models are an excellent tool to study metabolic diseases; however, there are limited methods for the noninvasive measurement of fat distribution in mice. Although micromagnetic resonance imaging and microcomputed tomography are the "gold standards" in the measurement of fat distribution, more economical and accessible methods are required. Dual energy X-ray absorptiometry (DEXA) is an effective method in characterizing fat content; however, it cannot discriminate between visceral and subcutaneous fat depots. We demonstrate that an evaluation of abdominal fat content measured by DEXA through the selection of one localized abdominal area strongly correlates with visceral fat content in C57BL/6J mice. We found that DEXA is able to measure fat pad volume ex vivo with high accuracy; however, the measurement of visceral fat in vivo shows an overestimation caused by subcutaneous tissue interference. The overestimation is almost constant for a wide range of values, and thus it is possible to correct the data for a more accurate estimation of visceral fat content. We demonstrate the utility of this technique in characterizing phenotypes of several obese mouse models (ob/ob, db/db, MC4R-KO, and DIO) and evaluating the effect of treatments on visceral fat content in longitudinal studies. Additionally, we also establish abdominal obesity as a potential biomarker for metabolic abnormalities (liver fat accumulation, insulin resistance/diabetes) in mice, similar to that described in humans.     

Glutathione in plants: an integrated overview

Plants cannot survive without glutathione (γ-glutamylcysteinylglycine) or γ-glutamylcysteine-containing homologues. The reasons why this small molecule is indispensable are not fully understood, but it can be inferred that glutathione has functions in plant development that cannot be performed by other thiols or antioxidants. The known functions of glutathione include roles in biosynthetic pathways, detoxification, antioxidant biochemistry and redox homeostasis. Glutathione can interact in multiple ways with proteins through thiol-disulphide exchange and related processes. Its strategic position between oxidants such as reactive oxygen species and cellular reductants makes the glutathione system perfectly configured for signalling functions. Recent years have witnessed considerable progress in understanding glutathione synthesis, degradation and transport, particularly in relation to cellular redox homeostasis and related signalling under optimal and stress conditions. Here we outline the key recent advances and discuss how alterations in glutathione status, such as those observed during stress, may participate in signal transduction cascades. The discussion highlights some of the issues surrounding the regulation of glutathione contents, the control of glutathione redox potential, and how the functions of glutathione and other thiols are integrated to fine-tune photorespiratory and respiratory metabolism and to modulate phytohormone signalling pathways through appropriate modification of sensitive protein cysteine residues.