In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker

This study was undertaken to evaluate oxidative stress in the kidney of diabetic mice by electron spin resonance (ESR) imaging technique. Oxidative stress in the kidney was evaluated as organ-specific reducing activity with the signal decay rates of carbamoyl-PROXYL probe using ESR imaging. The signal decay rates were significantly faster in corresponding image pixels of the kidneys of streptozotocin-induced diabetic mice than in those of controls. This technique further demonstrated that administration of angiotensin II type 1 receptor blocker (ARB), olmesartan (5 mg/kg), completely restored the signal decay rates in the diabetic kidneys to control values. In conclusion, this study provided for the first time the in vivo evidence for increased oxidative stress in the kidneys of diabetic mice and its normalization by ARB as evaluated by ESR imaging. This technique would be useful as a means of further elucidating the role of oxidative stress in diabetic nephropathy.     

Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis

Bif-1, a member of the endophilin B protein family, interacts with Bax and promotes interleukin-3 withdrawal-induced Bax conformational change and apoptosis when overexpressed in FL5.12 cells. Here, we provide evidence that Bif-1 plays a regulatory role in apoptotic activation of not only Bax but also Bak and appears to be involved in suppression of tumorigenesis. Inhibition of endogenous Bif-1 expression in HeLa cells by RNA interference abrogated the conformational change of Bax and Bak, cytochrome c release, and caspase 3 activation induced by various intrinsic death signals. Similar results were obtained in Bif-1 knockout mouse embryonic fibroblasts. While Bif-1 did not directly interact with Bak, it heterodimerized with Bax on mitochondria in intact cells, and this interaction was enhanced by apoptosis induction and preceded the Bax conformational change. Moreover, suppression of Bif-1 expression was associated with an enhanced ability of HeLa cells to form colonies in soft agar and tumors in nude mice. Taken together, these findings support the notion that Bif-1 is an important component of the mitochondrial pathway for apoptosis as a novel Bax/Bak activator, and loss of this proapoptotic molecule may contribute to tumorigenesis.     

An ADP-ribosylation factor GTPase-activating protein Git2-short/KIAA0148 is involved in subcellular localization of paxillin and actin cytoskeletal organization

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein beta-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling.     

B Cells Play Key Roles in Th2-Type Airway Immune Responses in Mice Exposed to Natural Airborne Allergens

Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH^−/− mice were exposed intranasally to a cocktail of allergen extracts, including Alternaria, Aspergillus, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH^−/− mice. The allergen-induced expansion of CD4⁺ T cells was impaired in the lungs and draining lymph nodes of JH^−/− mice. Furthermore, lymphocytes from JH^−/− mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation in vitro. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.     

Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

Background

It has been hypothesized that persistent hepatitis C virus (HCV) infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4), composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation.

Methods

Human C4 was incubated with HCV nonstructural (NS) 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined.

Results

HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease–mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4.

Conclusions

C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.