Hepatitis C virus (HCV) is a positive-strand RNA virus, and classified within the Flaviridae family. Atg7-knockdown decreases the amount of HCV replicon RNA, when HCV JFH1 RNA and HCV subgenomic replicon are transfected into Huh7.5 cells. However, when infectious naive HCV particles are directly infected into Huh7.5.1 cells, it is still unclear whether Atg7-knockdown decreases the production of intracellular HCV-related proteins, HCV mRNA and infectious HCV particles. When Atg7 protein in HCV-infected Huh7.5.1 cells was knocked down by RNA-interference, the levels of intracellular HCV core, NS3, NS5A proteins, HCV mRNA and secreted albumin remained unchanged compared with those in the control HCV-infected cells. However, the level of infectious HCV particles released in the medium was decreased by the Atg7-knockdown. Similar results were obtained when Beclin 1 was knocked down by RNA-interference. The colocalization of endogenous LC3-puncta with HCV core, HS5A proteins and lipid droplets was also investigated. However, little endogenous LC3-puncta colocalized with HCV core, NS5A proteins or lipid droplets. These results suggested that autophagy contributed to the effective production of HCV particles, but little to the intracellular production of HCV-related proteins, HCV mRNA and the secretory pathway, in a naive HCV particles-infection system.

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A cytosolic form of LC3 is conjugated to phosphatidylethanolamine by Atg7, an E1-like enzyme, and Atg3, an E2-like enzyme, during autophagy. To monitor intracellular autophagosomes and autolysosomes, GFP-LC3 is a useful tool. However, GFP-LC3 can aggregate without being conjugated to phosphatidylethanolamine, especially when GFP-LC3 is transiently expressed (Kuma A, Matsui M, Mizushima N. Autophagy 2007; 3:323-8). Therefore, as a negative control, we investigated a mutant human LC3DeltaG protein in which the C-terminal Gly(120) essential for LC3-lipidation is deleted, and generated a set of expression plasmids for wild-type human LC3 and mutant LC3DeltaG fused to either CFP, GFP, YFP, or HcRed at the N terminus. We found that the mutant LC3DeltaG protein does not react with human Atg7 and Atg3, indicating that LC3-lipidation does not occur, and few puncta containing mutant LC3DeltaG form under starvation conditions. As observed with wildtype HcRed-LC3, mutant HcRed-LC3DeltaG also co-localizes with polyQ150-aggregates suggesting that the colocalization of HcRed- LC3 to polyQ150-aggregates is independent of LC3-lipidation. These mutant LC3DeltaG proteins will be useful negative controls in recognizing non-specific fluorescent protein-LC3 aggregates.

Both psoriasis and atopic dermatitis (AD) are not only associated with an impaired stratum corneum barrier, but also with abnormal expression of the tight junction (TJ) proteins. Because host defense peptides, including LL-37, are overexpressed in lesional psoriatic skin but are downregulated in lesional AD skin, we hypothesized that LL-37 might regulate the TJ function in keratinocytes. We demonstrated that LL-37 selectively increased the expression of several claudins and occludin, and enhanced their membrane distribution. Furthermore, LL-37 elevated the transepithelial electrical resistance while reducing the paracellular permeability of keratinocyte layers, and this activity was weakened by the claudin inhibitor ochratoxin A. A characterization of the molecular mechanism underlying the regulation of the TJ barrier by LL-37 revealed that LL-37 induced the activation of the Rac1, atypical PKC, glycogen synthase kinase-3 and PI3K pathways, and the specific inhibition of these pathways reversed the LL-37-mediated regulation of TJ function. In addition, LL-37 enhanced the expression of differentiation markers under the control of ochratoxin A, suggesting an association between LL-37-induced TJ function and keratinocyte differentiation. These data provide novel evidence that, in addition to its antimicrobial and other immunoregulatory functions, LL-37 contributes to cutaneous immunity by strengthening the skin's barrier function.

A new potential molecular marker, N-cyclohexyl-2benzothiazolamine (NCBA), was discovered in various environmental matrices (i.e., road dusts, runoff-and riverwater particles, river sediments, aerosols) taken in Tokyo, Japan. Concurrent determination of this compound together with 2-(4-morpholinyl)benzothiazole (24MoBT), previously proposed marker of road dust, demonstrated that both compounds are widely distributed in the urban environment (∼ng/g to ∼µg/g), derived from vehicle tire tread, and transported in the environments in the same way. To assess utilities of NCBA and 24MoBT as molecular markers for vehicle-derived contaminants, these compounds were analyzed in a sediment core from the Chidorigafuchi Moat of Imperial Palace, situated in the center of Tokyo. Remarkable is that NCBA existed at higher concentrations than 24MoBT near the surface (0-6 cm depth) and bottom parts (16-24 cm depth) of the region where BTs were detected but lower in the middle parts (6-16 cm depth). Dating of the core by using 137 Cs and tetrapropylenebased alkylbenzenes (TABs) revealed the two changeovers coincide well with changes in the production history of vulcanization accelerators containing the compounds. The dated downcore profile of ∑BTs (sum of 24MoBT and NCBA) showed positive correlation with the traffic data in Tokyo Metropolitan Area. These results indicate the usefulness of NCBA and 24MoBT as time markers for recent sections of sediment cores and as molecular markers for reconstructing the history of traffic-induced contamination.

Background: Adriamycin (ADR) nephrosis in mice has been extensively studied and has enabled a greater understanding of the processes underlying the progression of renal injury. Dendrin is a novel component of the slit diaphragm with proapoptotic signaling properties, and it accumulates in the podocyte nucleus in response to glomerular injury in mice. The present study re-evaluated chronic progressive nephropathy in ADR mice and the localization of dendrin in mice and in human glomerulopathy. Methods: To investigate the localization of dendrin, a mouse model of nephrosis and glomerulosclerosis was used, in which ADR was injected once. WT-1-positive cells and apoptotic cells were counted in vivo and in vitro. To check the expression of dendrin in ADR mice, immunostaining and Western blot were performed. A survey of dendrin staining was performed on human kidney biopsy specimens. Results: The injection of ADR induced proteinuria, podocyte loss and glomerulosclerosis. It also caused the relocation of dendrin from the slit diaphragm to the podocyte nucleus. We demonstrated the location of dendrin to podocyte nuclei in several cases of human glomerulopathy. The mean occurrence of dendrin-positive nucleus per glomerulus increased in several cases of human glomerulopathy. Conclusions: These findings suggest that the relocation of dendrin to the podocyte nuclei is useful as a novel marker of podocyte injury in human glomerulopathy.

Compared with C-TACE, B-TACE significantly improved cancer nodule control, and it was satisfactory in terms of safety. B-TACE is an effective procedure that enhances the effects of catheterization with miriplatin.