Fitness-related traits of entomopoxviruses isolated from Adoxophyes honmai (Lepidoptera: Tortricidae) at three localities in Japan

Three entomopoxviruses (EPVs) isolated from diseased Adoxophyes honmai larvae at different localities (Tsukuba, Itsukaichi, and Miyazaki) in Japan were compared for biochemical identity and key parameters of virus fitness, fatal infection, speed of kill, and virus yield. When the structural peptides of occlusion bodies (OBs) and occlusion-derived viral particles were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no difference in banding patterns was observed. However, DNA restriction endonuclease analysis showed that the three isolates were genotypically different, but many commonly sized DNA fragments were observed. Five tortricid species, A. honmai, Adoxophyes orana, Adoxophyesdubia, Homona magnanima, and Archips insulanus were susceptible to all isolates. No significant differences in the key viral fitness parameters were detected among the isolates in A. orana. However, the Miyazaki isolate had a different effect on H. magnanima; it allowed infected insects to survive longer and develop to a larger size, but had a lower yield of OBs per larva at any given time to death. OB yields per unit cadaver weight for the Miyazaki isolate, which indicate the conversion rate of the insect to virus, were lower over time compared to the other two isolates. The implications for selecting a candidate isolate to control tortricid pests are discussed.     

Collaborative action of NF-kappaB and p38 MAPK is involved in CpG DNA-induced IFN-alpha and chemokine production in human plasmacytoid dendritic cells

CpG DNA induces plasmacytoid dendritic cells (pDC) to produce type I IFN and chemokines. However, it has not been fully elucidated how the TLR9 signaling pathway is linked to these gene expressions. We examined the mechanisms involving the TLR9 and type I IFN signaling pathways, in relation to CpG DNA-induced IFN-alpha, IFN regulatory factor (IRF)-7, and chemokines CXCL10 and CCL3 in human pDC. In pDC, NF-kappaB subunits p65 and p50 were constitutively activated. pDC also constitutively expressed IRF-7 and CCL3, and the gene expressions seemed to be regulated by NF-kappaB. CpG DNA enhanced the NF-kappaB p65/p50 activity, which collaborated with p38 MAPK to up-regulate the expressions of IRF-7, CXCL10, and CCL3 in a manner independent of type I IFN signaling. We then examined the pathway through which IFN-alpha is expressed. Type I IFN induced the expression of IRF-7, but not of IFN-alpha, in a NF-kappaB-independent way. CpG DNA enabled the type I IFN-treated pDC to express IFN-alpha in the presence of NF-kappaB/p38 MAPK inhibitor, and chloroquine abrogated this effect. With CpG DNA, IRF-7, both constitutively and newly expressed, moved to the nuclei independently of NF-kappaB/p38 MAPK. These findings suggest that, in CpG DNA-stimulated human pDC, the induction of IRF-7, CXCL10, and CCL3 is mediated by the NF-kappaB/p38 MAPK pathway, and that IRF-7 is activated upstream of the activation of NF-kappaB/p38 MAPK in chloroquine-sensitive regulatory machinery, thereby leading to the expression of IFN-alpha.     

Exome sequencing reveals a homozygous SYT14 mutation in adult-onset, autosomal-recessive spinocerebellar ataxia with psychomotor retardation

Autosomal-recessive cerebellar ataxias (ARCAs) are clinically and genetically heterogeneous disorders associated with diverse neurological and nonneurological features that occur before the age of 20. Currently, mutations in more than 20 genes have been identified, but approximately half of the ARCA patients remain genetically unresolved. In this report, we describe a Japanese family in which two siblings have slow progression of a type of ARCA with psychomotor retardation. Using whole-exome sequencing combined with homozygosity mapping, we identified a homozygous missense mutation in SYT14, encoding synaptotagmin XIV (SYT14). Expression analysis of the mRNA of SYT14 by a TaqMan assay confirmed that SYT14 mRNA was highly expressed in human fetal and adult brain tissue as well as in the mouse brain (especially in the cerebellum). In an in vitro overexpression system, the mutant SYT14 showed intracellular localization different from that of the wild-type. An immunohistochemical analysis clearly showed that SYT14 is specifically localized to Purkinje cells of the cerebellum in humans and mice. Synaptotagmins are associated with exocytosis of secretory vesicles (including synaptic vesicles), indicating that the alteration of the membrane-trafficking machinery by the SYT14 mutation may represent a distinct pathomechanism associated with human neurodegenerative disorders.     

Dipeptidyl peptidase-4 inhibitor ameliorates early renal injury through its anti-inflammatory action in a rat model of type 1 diabetes

Introduction

Dipeptidyl peptidase-4 (DPP-4) inhibitors are incretin-based drugs in patients with type 2 diabetes. In our previous study, we showed that glucagon-like peptide-1 (GLP-1) receptor agonist has reno-protective effects through anti-inflammatory action. The mechanism of action of DPP-4 inhibitor is different from that of GLP-1 receptor agonists. It is not obvious whether DPP-4 inhibitor prevents the exacerbation of diabetic nephropathy through anti-inflammatory effects besides lowering blood glucose or not. The purpose of this study is to clarify the reno-protective effects of DPP-4 inhibitor through anti-inflammatory actions in the early diabetic nephropathy.

Materials and methods

Five-week-old male Sprague–Dawley (SD) rats were divided into three groups; non-diabetes, diabetes and diabetes treated with DPP-4 inhibitor (PKF275-055; 3 mg/kg/day). PKF275-055 was administered orally for 8 weeks.

Results

PKF275-055 increased the serum active GLP-1 concentration and the production of urinary cyclic AMP. PKF275-055 decreased urinary albumin excretion and ameliorated histological change of diabetic nephropathy. Macrophage infiltration was inhibited, and inflammatory molecules were down-regulated by PKF275-055 in the glomeruli. In addition, nuclear factor-κB (NF-κB) activity was suppressed in the kidney.

Conclusions

These results indicate that DPP-4 inhibitor, PKF275-055, have reno-protective effects through anti-inflammatory action in the early stage of diabetic nephropathy. The endogenous biological active GLP-1 might be beneficial on diabetic nephropathy besides lowering blood glucose.     

A novel alternative spliced Mpv17-like protein isoform localizes in cytosol and is expressed in a kidney- and adult-specific manner

Mpv17-like protein (M-LP) has been identified as a new protein that shows high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early onset glomerulosclerosis. We previously showed that the originally identified M-LP isoform, designated M-LPL, is, like Mpv17, localized in peroxisomes, and that transfection with M-LPL up-regulates expression of the manganese superoxide dismutase (SOD2) gene [R. Iida, T. Yasuda, E. Tsubota, H. Takatsuka, M. Masuyama, T. Matsuki, K. Kishi, M-LP, Mpv17-like protein, has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene. J. Biol. Chem. 278 (2003) 6301-6306.]. We report here the identification of a novel alternative splicing product of the M-LP gene, designated M-LPS. A comparison of the genomic sequence with the cDNA sequences and an analysis of 5'-flanking regions revealed that the two isoforms are generated by alternative usage of two promoters. M-LPS consists of the C-terminal half of M-LPL (90 amino acids) and therefore lacks the peroxisome targeting signal of membrane protein that exists near the N-terminus of M-LPL. Expression of green fluorescent protein-tagged M-LPS in COS-7 cells demonstrated that M-LPS localizes in the cytosol. In mice, M-LPS is expressed exclusively in kidneys after the age of 6 weeks. Moreover, quantitative real-time PCR analysis revealed that transfection with M-LPS up-regulates expression of the SOD2 gene and down-regulates expression of the cellular glutathione peroxidase (Gpx1) and plasma glutathione peroxidase (Gpx3) genes. Taken together, these results suggest different functional attributes of the two M-LP isoforms during aging and development.     

Intestinal bacteria affect growth of Bacillus thuringiensis in larvae of the oriental tea tortrix, Homona magnanima diakonoff (Lepidoptera: tortricidae)

Spores and parasporal crystals of a Bacillus thuringiensis serovar aizawai were fed to fifth instar larvae of the oriental tea tortrix, Homona magnanima, that had been reared aseptically or that had been reared normally. Viable cell numbers of B. thuringiensis and other bacteria in H. magnanima larvae were estimated by homogenization of samples and dilution plating on peptone-polymyxin agar medium for B. thuringiensis cells and on nutrient agar medium for the other bacterial cells. B. thuringiensis did not grow in the larval cadavers of normally reared H. magnanima while bacteria other than B. thuringiensis grew rapidly. In contrast, B. thuringiensis within the larval cadavers of aseptically reared H. magnanima grew and increased 20 times. The bacteria other than B. thuringiensis from the sample homogenates of normally reared larvae that were fed on B. thuringiensis-treated diets had the same characteristics as the bacteria isolated from the guts of healthy H. magnanima larvae, which were putatively identified as Streptococcus spp. and Staphylococcus spp., typical intestinal bacteria of insects. The results strongly suggest that intestinal bacteria influence the growth of B. thuringiensis in the larvae.     

Endothelin-3 stimulates inositol 1,4,5-trisphosphate production and Ca2+ influx to produce biphasic dopamine release from rat striatal slices

Summary 1. Real-time monitoring of dopamine (DA) release from rat striatal slices demonstrated that endothelin (ET)-3 (0.1–10M) produced a biphasic DA release consisting of transient and sustained components. When extracellular Ca²⁺ was removed, the sustained but not transient response remarkably decreased.2. ET-3 (1–10M) stimulated an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), which also consisted of two components. The external Ca²⁺ depletion inhibited primarily the sustained component of the Ca²⁺ response to ET-3.3. ET-3 increased inositol 1,4,5-trisphosphate (IP3) concentrations in striatal slices. This response peaked at 10 to 20 sec and returned to the basal level 2 min after stimulation, an event which was in good accord with a prompt and transient phase of both cytosolic Ca²⁺ activity and DA release evoked by ET-3.4. Thus, ET-3 produces a transient and a sustained release of DA from striatal slices by stimulating intracellular Ca²⁺ mobilization via IP3 formation and extracellular Ca²⁺ influx, respectively.