Physicochemical Properties of the Mammalian Molecular Chaperone HSP60.

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.

High-Performance Anion Exchange Membrane Water Electrolyzers Enabled by Highly Gas Permeable and Dimensionally Stable Anion Exchange Ionomers.

Designing suitable anion exchange ionomers is critical to improving the performance and in situ durability of anion exchange membrane water electrolyzers (AEMWEs) as one of the promising devices for producing green hydrogen. Herein, highly gas-permeable and dimensionally stable anion exchange ionomers (QC6xBA and QC6xPA) are developed, in which bulky cyclohexyl (C6) groups are introduced into the polymer backbones. QC650BA-2.1 containing 50 mol% C6 composition shows 16.6 times higher H2 permeability and 22.3 times higher O2 permeability than that of QC60BA-2.1 without C6 groups. Through-plane swelling of QC650BA-2.1 decreases to 12.5% from 31.1% (QC60BA-2.1) while OH- conductivity slightly decreases (64.9 and 56.2 mS cm-1 for QC60BA-2.1 and QC650BA-2.1, respectively, at 30 °C). The water electrolysis cell using the highly gas permeable QC650BA-2.1 ionomer and Ni0.8Co0.2O in the anode catalyst layer achieves two times higher performance (2.0 A cm-2 at 1.69 V, IR-included) than those of the previous cell using in-house ionomer (QPAF-4-2.0) (1.0 A cm-2 at 1.69 V, IR-included). During 1000 h operation at 1.0 A cm-2, the QC650BA-2.1 cell exhibits nearly constant cell voltage with a decay rate of 1.1 µV h-1 after the initial increase of the cell voltage, proving the effectiveness of the highly gas permeable and dimensionally stable ionomer in AEMWEs.

Gender difference in allergic airway remodelling and immunoglobulin production in mouse model of asthma.

BACKGROUND AND OBJECTIVE: Epidemiological studies have shown that the prevalence of adult asthma and severe asthma is higher in women. It has also been reported that female mice are more susceptible than males to the development of allergic airway inflammation and airway hyperresponsiveness (AHR). The influence of gender difference in the pathogenesis of severe asthma, especially airway remodelling in an animal model, has been studied rarely. We investigated gender difference in the development of airway remodelling using a long-term antigen-challenged mouse asthma model. METHODS: Following ovalbumin (OVA)/alum intraperitoneal injection, male or female mice (BALB/c) were challenged with aerosolized 1% OVA on 3 days/week for 5 weeks, and differences in AHR, airway inflammation and airway remodelling between the sexes were investigated. RESULTS: In OVA-sensitized and OVA-challenged (OVA/OVA) female mice, eosinophils, lymphocytes, T-helper type 2 cytokines and growth factors in bronchoalveolar lavage fluid were increased compared with OVA/OVA male mice. Histological features of airway remodelling were also increased in OVA/OVA female mice. Serum total and OVA-specific immunoglobulin E (IgE) and serum IgA were significantly elevated in OVA/OVA female mice. CONCLUSIONS: These results indicate that female mice experience more airway remodelling compared with male mice. These results suggest the involvement of sex hormones and gender differences in cellular functions in airway remodelling.

Characterization of YIPF3 and YIPF4, cis-Golgi Localizing Yip domain family proteins.

The Yip1 domain family (YIPF) proteins are homologues of yeast Yip1p and Yif1p, which are proposed to function in ER to Golgi transport. Here, we report the characterization of YIPF3 and YIPF4, homologues of human Yif1p and Yip1p, respectively. Immunofluorescence and immuno-electron microscopy showed that both YIPF3 and YIPF4 are clearly concentrated in the cis-Golgi. While YIPF4 was detected as a single mobility form consistent with its predicted molecular weight, three different mobility forms of YIPF3 were detected by western blotting. Biochemical and immunofluorescence experiments strongly indicated that YIPF3 is synthesized in the ER as a N-glycosylated form (40 kDa), is then O-glycosylated in the Golgi apparatus to become a lower mobility form (46 kDa) and finally becomes a higher mobility form cleaved at its C-terminal luminal domain (36 kDa). YIPF3 and YIPF4 form a complex in the Golgi apparatus, and this was suggested to be important for their proper localization and function. The knockdown of YIPF3 or YIPF4 in HeLa cells induced fragmentation of the Golgi apparatus, suggesting their involvement in the maintenance of the Golgi structure.

The pathophysiological roles of PI3Ks and therapeutic potential of selective inhibitors in allergic inflammation.

Phosphoinositide 3-kinases (PI3Ks) are known to be involved in a variety of cellular responses such as cell survival, proliferation, differentiation and cell migration. Recently, PI3Ks have been associated with the pathogenesis of asthma because various immune cells regulate allergic responses. Among the three classes of PI3Ks, the roles of PI3K gamma and PI3K delta in allergic responses have attracted particular attention. In a previous report, allergic airway hyperresponsiveness (AHR), inflammation and airway remodeling in an ovalbumin-induced asthma model were decreased in PI3K gamma-deficient mice compared with wild-type mice. In addition, AHR and inflammation were attenuated by administration of a selective PI3K delta inhibitor in a murine model of asthma. These results indicate that PI3K gamma and PI3K delta may be new therapeutic targets for asthma. However, PI3K gamma and PI3K delta may differ in terms of the mechanism of regulation. In this review, we focus on the roles of PI3K gamma and PI3K delta in the pathogenesis of asthma and discuss the mechanistic differences between PI3K gamma and PI3K delta.

Retinoic acids are potent inhibitors of spontaneous human eosinophil apoptosis.

Retinoic acids (RAs), which are active metabolites of vitamin A, are known to enhance Th2-type immune responses in vitro, but the role of RAs in allergic inflammatory cells remains unclear. In this study, we demonstrated that purified peripheral blood eosinophils expressed nuclear receptors for RAs at the mRNA and protein levels. Eosinophils cultured with all-trans RA (ATRA) and 9-cis-RA showed dramatically induced cell survival and nuclear hypersegmentation, and the efficacy of RAs (10(-6)M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Pharmacological manipulation with receptor-specific agonists and antagonists indicated that the antiapoptotic effect of RAs was mediated through ligand-dependent activation of both retinoid acid receptors and retinoid X receptors (mainly retinoid acid receptors). Furthermore, using a gene microarray and a cytokine Ab array, we discovered that RAs induced vascular endothelial growth factor, M-CSF, and MCP-1 secretion, although they were not involved in eosinophil survival. RA-induced eosinophil survival appears to be associated with down-regulation of caspase 3 and inhibition of its enzymatic activity. These findings indicate an important role of RAs in homeostasis of granulocytes and provide further insight into the cellular and molecular pathogenesis of allergic reactions.

Allergic airway hyperresponsiveness, inflammation, and remodeling do not develop in phosphoinositide 3-kinase gamma-deficient mice.

BACKGROUND: Bronchial asthma is characterized by chronic airway inflammation caused by inflammatory cells. Phosphoinositide 3-kinases (PI3Ks) are known to play a prominent role in fundamental cellular responses of various inflammatory cells, including proliferation, differentiation, and cell migration. PI3Ks therefore are expected to have therapeutic potential for asthma. Although some investigations of the involvement between the pathogenesis of asthma and PI3K have been performed, it is unknown whether PI3Kgamma, a PI3K isoform, is involved in the pathogenesis of asthma. OBJECTIVE: We investigated the role of PI3Kgamma in allergen-induced allergic airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling with PI3Kgamma-deficient mice. METHODS: After ovalbumin (OVA) sensitization, wild-type (WT) and PI3Kgamma-deficient mice were exposed to aerosolized OVA 3 days per week for 5 weeks. RESULTS: In OVA-sensitized and OVA-challenged (OVA/OVA) PI3Kgamma-deficient mice, levels of airway inflammation, AHR, and airway remodeling were significantly decreased compared with those in OVA/OVA WT mice. On the other hand, no significant differences were detected in serum OVA-specific IgE and IgG1 levels and CD4/CD8 balance in bronchoalveolar lavage fluid between OVA/OVA WT mice and OVA/OVA PI3Kgamma-deficient mice. To determine in which phase of allergic responses PI3Kgamma plays a role, we transferred splenocytes from OVA-sensitized WT or PI3Kgamma-deficient mice to naive mice of either genotype. Similar increased levels of eosinophils were induced in both WT recipient mice but not in both PI3Kgamma-deficient recipient mice. CONCLUSION: PI3Kgamma might be involved in allergic airway inflammation, AHR, and airway remodeling by regulating the challenge/effector phase of allergic responses.

Anti-allergic inflammatory effects of hepatocyte growth factor.

Hepatocyte growth factor (HGF) is known to influence a number of cell types and regulate various biological activities including cytokine production, cell migration, proliferation and survival. Thus, HGF is now recognized to be a key factor in the prevention and attenuation of disease progression. We have reported that HGF reduces allergic airway inflammation, airway hyperresponsiveness, remodeling and development of Th2 cytokines as well as growth factors such as transforming growth factor-beta in vivo. In vitro, HGF directly attenuates chemotaxis of eosinophils in the absence of Th2 cytokines and modulates mitogen-activated protein kinases, which play an important role in eosinophil migration. In this review, we discuss the physiological role of HGF in allergic inflammation and its mechanism of anti-inflammatory effects, including the regulation of eosinophil functions.

Mechanism of up-regulation of human Toll-like receptor 3 secondary to infection of measles virus-attenuated strains.

PolyI:C, a synthetic double-stranded (ds)RNA, and viruses act on cells to induce IFN-beta which is a key molecule for anti-viral response. Although dsRNA is a virus-specific signature and a ligand for human Toll-like receptor 3 (TLR3), largely uncharacterized multiple pathways associate virus-mediated IFN-beta induction. Here, we demonstrated that laboratory-adapted but not wild-type strains of measles virus (MV) up-regulated TLR3 expression both in dendritic cells and epithelial cell line A549. The kinetics experiments with the laboratory MV strain revealed that TLR3 was induced late compared to IFN-beta and required new protein synthesis. Furthermore, neutralizing antibodies against IFN-beta or IFNAR (Interferon-alpha/beta receptor) suppressed MV-induced TLR3 induction, indicating that type I IFN, IFN-alpha/beta, is critical for MV-mediated TLR3 induction. Yet, a recently identified virus-inducible IFN, the IFN-lambda, did not contribute to TLR3 expression. A virus-responsive element that up-regulates TLR3 was identified in the TLR3-promoter region by reporter gene experiments. The ISRE, a recently reported site for IFN-beta induction, but not STAT binding site, located around -30bp of TLR3 promoter responded to MV to induce TLR3 expression. This further indicates the importance of type I IFN for TLR3 up-regulation in the case of viral infection. In HeLa and MRC5 cells, augmented production of IFN-beta was observed in response to dsRNA when TLR3 had been induced beforehand. Thus, the MV-induced expression of TLR3 may reflect amplified IFN production that plays a part in host defense to viral infection.

Subcellular localization of Toll-like receptor 3 in human dendritic cells.

Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-kappaB and IFN-beta promoter. Type I IFNs (IFN-alpha/beta) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-beta secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-alpha and -beta in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c(+) blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3.

Mycobacterium bovis BCG cell wall-specific differentially expressed genes identified by differential display and cDNA subtraction in human macrophages.

We have analyzed the gene expression profile of monocytes in response to a highly purified cell wall fraction of Mycobacterium bovis BCG, a clinically approved adjuvant known as BCG cell wall skeleton (BCG-CWS). It is composed of mycolic acid, arabinogalactan, and peptidoglycan and confers Toll-like receptor 2 (TLR2)- and TLR4-dependent signaling that induces monocytes to differentiate into antigen-presenting cells (APCs). Here we report differential gene expression analysis with BCG-CWS-stimulated versus nonstimulated monocytes. BCG-CWS exerted massive induction of genes regulated by TLR signaling. Marked gene regulatory characteristics in BCG-CWS-stimulated cells compared to lipopolysaccharide (LPS)-stimulated cells follow. (i) Spliced mRNAs encoding soluble forms of TREM-1 and TREM-2 (recently discovered inflammatory-signal-amplifying receptors) were regulated by BCG-CWS, resulting in their differential expression. (ii) The genes for zinc-iron transporter protein (ZIP)-like family proteins HKE-1 and LIV-1 were induced exclusively by BCG-CWS. (iii) Interleukin-23 (IL-23), rather than IL-12p70, was induced by BCG-CWS, while interferon-inducible genes were induced only by LPS. By Northern and reverse transcription-PCR analyses, we confirmed the differential expression of more than 30 BCG-CWS regulatory genes, and their expression was compared with that of LPS and other known TLR ligands. A battery of genes responded rapidly and for a short time to LPS but for a long time to BCG-CWS. Structural analysis of the identified novel or hypothetical proteins revealed that some are potential candidates as signaling mediators or transcriptional regulators. Hence, BCG-CWS may profoundly modulate APC responses in a way distinct from that of LPS, leading to possible advantages for its adjuvant-active therapeutic potential.