Replication-competent recombinant vesicular stomatitis virus encoding hepatitis C virus envelope proteins

Although in vitro replication of the hepatitis C virus (HCV) JFH1 clone of genotype 2a (HCVcc) has been developed, a robust cell culture system for the 1a and 1b genotypes, which are the most prevalent viruses in the world and resistant to interferon therapy, has not yet been established. As a surrogate virus system, pseudotype viruses transiently bearing HCV envelope proteins based on the vesicular stomatitis virus (VSV) and retrovirus have been developed. Here, we have developed a replication-competent recombinant VSV with a genome encoding unmodified HCV E1 and E2 proteins in place of the VSV envelope protein (HCVrv) in human cell lines. HCVrv and a pseudotype VSV bearing the unmodified HCV envelope proteins (HCVpv) generated in 293T or Huh7 cells exhibited high infectivity in Huh7 cells. Generation of infectious HCVrv was limited in some cell lines examined. Furthermore, HCVrv but not HCVpv was able to propagate and form foci in Huh7 cells. The infection of Huh7 cells with HCVpv and HCVrv was neutralized by anti-hCD81 and anti-E2 antibodies and by sera from chronic HCV patients. The infectivity of HCVrv was inhibited by an endoplasmic reticulum alpha-glucosidase inhibitor, N-(n-nonyl) deoxynojirimycin (Nn-DNJ), but not by a Golgi mannosidase inhibitor, deoxymannojirimycin. Focus formation of HCVrv in Huh7 cells was impaired by Nn-DNJ treatment. These results indicate that the HCVrv developed in this study can be used to study HCV envelope proteins with respect to not only the biological functions in the entry process but also their maturation step.     

Brain neurotransmitter receptor-binding characteristics in rats after oral administration of haloperidol, risperidone and olanzapine

The present study was conducted to characterize the binding of neurotransmitter receptors (dopamine D(2), serotonin 5-HT(2), histamine H(1), adrenaline alpha(1) and muscarine M(l) receptors) in the rat's brain after the oral administration of haloperidol, risperidone, and olanzapine. Haloperidol at 1 and 3 mg/kg displayed significant activity to bind the D(2) receptor (increase in the Kd value for [(3)H]raclopride binding) in the corpus striatum with little change in the activity toward the 5-HT(2) receptor (binding parameters for [(3)H]ketanserin). In contrast, risperidone (0.1-3 mg/kg) showed roughly 30 times more affinity for the 5-HT(2) receptor than D(2) receptor. Also, olanzapine (1-10 mg/kg) was most active toward the H(1) receptor in the cerebral cortex, corpus striatum, and hippocampus, was less active in binding 5-HT(2) and D(2) receptors, and showed the least affinity for alpha(1) and M(1) receptors. In conclusion, haloperidol and risperidone administered orally selectively bind D(2) and 5-HT(2) receptors, respectively, in the rat brain, while olanzapine binds H(1), 5-HT(2), and D(2) receptors more than alpha(1) and M(1) receptors.     

A Calcium-Receptor Agonist Induces Gustatory Neural Responses in Bullfrogs

The effect of calcium-sensing receptor (CaR) agonists on frog gustatory responses was studied using glossopharyngeal nerve recording and whole-cell patch-clamp recording of isolated taste disc cells. Calcimimetic NPS R-467 dissolved in normal saline solution elicited a large transient response in the nerve. The less active enantiomer of the compound, NPS S-467 induced only a small neural response. The EC₅₀ for NPS R-467 was about 20 μM. Cross-adaptation experiments were performed to examine the effect of 30 μM NPS R-467 and 100 μM quinine on the gustatory neural response. The magnitude of the R-467-induced response after adaptation to quinine was approximately equal to that after adaptation to normal saline solution, indicating that the receptor site for NPS R-467 is different from the site for quinine. NPS R-467 (100 μM) also induced an inward current accompanied with conductance increase and large depolarization in two (13%) of 15 rod cells, and a sustained decrease in outward current and small depolarization in six (40%) other rod cells. NPS S-467 (100 μM) induced a sustained decrease in outward current and depolarization in five (50%) of 10 rod cells. Another calcimimetic cinacalcet (100 μM) induced an inward current accompanied with conductance increase in three (27%) of 11 rod cells. The results suggest that NPS R-467 induces neural responses through cell responses unrelated to a resting K⁺ conductance decrease.     

Manipulation of host innate immune responses by the malaria parasite

It has long been known that malaria infection causes host immune modulation by various mechanisms. However, the role of Toll-like receptors (TLRs) in mediating innate immune responses to parasite-derived components during the blood stages of malaria has only recently been described. TLRs might have an important role in pathogenesis during malaria infection, as supported by genetic analyses in mice and humans. Moreover, recent findings revealed that sporozoites can partially differentiate in lymph nodes and that liver stages induce the formation of previously unknown parasite-filled vesicles (merosomes) that could function as immune escape machinery. Elucidation of the mechanisms by which the host innate immune system responds to, and/or is manipulated by, Plasmodium infection will hopefully lead to discoveries of potential targets that will ultimately prevent and/or intervene in malaria infection.     

Identification of a key pathway required for the sterile inflammatory response triggered by dying cells

Dying cells stimulate inflammation, and this response is thought to contribute to the pathogenesis of many diseases. Very little has been known, however, about how cell death triggers inflammation. We found here that the acute neutrophilic inflammatory response to cell injury requires the signaling protein myeloid differentiation primary response gene 88 (Myd88). Analysis of the contribution of Myd88-dependent receptors to this response revealed only a minor reduction in mice doubly deficient in Toll-like receptor 2 (Tlr2) and Tlr4 and normal responses in mice lacking Tlr1, Tlr3, Tlr6, Tlr7, Tlr9, Tlr11 or the interleukin-18 receptor (IL-18R). However, mice lacking IL-1R showed a markedly reduced neutrophilic inflammatory response to dead cells and tissue injury in vivo as well as greatly decreased collateral damage from inflammation. This inflammatory response required IL-1alpha, and IL-1R function was required on non-bone-marrow-derived cells. Notably, the acute monocyte response to cell death, which is thought to be important for tissue repair, was much less dependent on the IL-1R-Myd88 pathway. Also, this pathway was not required for the neutrophil response to a microbial stimulus. These findings suggest that inhibiting the IL-1R-Myd88 pathway in vivo could block the damage from acute inflammation that occurs in response to sterile cell death, and do so in a way that might not compromise tissue repair or host defense against pathogens.     

Stereoselective hexobarbital 3'-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476

We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3'-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3'-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB>(S)-HB and metabolite diastereoselectivity of 3'(R)<3'(S) in 3'-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3'-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB<(S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3'-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3'(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19.     

Molecular cloning and expression of two novel beta-N-acetylglucosaminidases from silkworm Bombyx mori

Beta-N-acetylglucosaminidase is a major glycosidase involved in several physiological processes, such as fertilization, metamorphosis, glycoconjugate degradation, and glycoprotein biosynthesis in insects. A search using the Bombyx mori cDNA database revealed the existence of two putative beta-N-acetylglucosaminidase genes. Their full-length cDNAs were cloned by rapid amplification of cDNA ends and polymerase chain reaction using specific primers, and named BmGlcNAcase1 and BmGlcNAcase2. A BLAST search revealed that BmGlcNAcase1 and BmGlcNAcase2 are homologous to a beta-subunit homolog encoded by Drosophila melanogaster HEXO2 and the Spodoptera frugiperda beta-N-acetylglucosaminidase gene respectively. The recombinant proteins of BmGlcNAcase1 and BmGlcNAcase2 without putative transmembrane domains were expressed in the yeast Pichia pastoris. Both enzymes showed broad substrate specificity, and cleaved terminal N-acetylglucosamine residues from the alpha-3 and alpha-6 branches of a biantennary N-glycan substrate, and also hydrolyzed chitotriose to chitobiose.     

Toll-like receptor 9 acts at an early stage in host defence against pneumococcal infection

Toll-like receptor 9 (TLR9) induces an inflammatory response by recognition of unmethylated CpG dinucleotides, mainly present in prokaryotic DNA. So far, TLR9-deficient mice have been shown to be more sensitive than wild-type mice to viral, but not to bacterial infections. Here, we show that mice deficient in TLR9 but not in TLR1, TLR2, TLR4 and TLR6 or IL-1R/IL-18R are more susceptible to a respiratory tract bacterial infection caused by Streptococcus pneumoniae. Intranasal challenge studies revealed that TLR9 plays a protective role in the lungs at an early stage of infection prior to the entry of circulating inflammatory cells. Alveolar as well as bone marrow-derived macrophages deficient in either TLR9 or the myeloid adaptor differentiation protein MyD88 were impaired in pneumococcal uptake and in pneumococcal killing. Our data suggest that in the airways, pneumococcal infection triggers a TLR9 and MyD88-dependent activation of phagocytic activity from resident macrophages leading to an early clearance of bacteria from the lower respiratory tract.     

Homeostatic defects in interleukin 18-deficient mice contribute to protection against the lethal effects of endotoxin

Toll-like receptor-4-lipopolysaccharide (LPS)-mediated inflammation is used to delineate signals involved in cross-talk between antigen-presenting cells (APCs) and lymphocytes such as natural killer (NK) cells. Following APC stimulation and cytokine release, NK cells produce interferon (IFN)-γ. High levels of LPS induce endotoxicosis, a systemic inflammatory disease in which IFN-γ causes significant morbidity and mortality. Several studies have highlighted the role of interleukin (IL)-18, IL-1β, IL-17A and IFN-γ in the development of endotoxicosis, but whether these cytokines interact with each other is yet to be determined. Our data demonstrate that IL-18 and IL-17A have important roles in NK cell IFN-γ production during endotoxicosis. Importantly, we provide the first evidence that IL-18 also has a role in IL-17A production by T-cell receptor (TCR)-δ cells. Furthermore, we demonstrate that IL-18-deficient mice have a defect in γδ T-cell homeostasis and IL-1β production, both of which can contribute to the development of disease through induction of IL-17A. These results reveal novel requirements for IL-18 in innate immune cell homeostasis and activation, demonstrating that the role of IL-18 in innate immunity occurs at a level other than activation.