DJ-1, a Parkinson's disease (PD)–associated gene, has been shown to protect against oxidative stress in Drosophila. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in DJ-1-deficient flies is not fully understood. Here we showed that Daxx-like protein (DLP), a Drosophila homologue of the mammalian Death domain-associated protein (Daxx), was upregulated under oxidative stress conditions in the loss-of-function mutants of Drosophila DJ-1β, a Drosophila homologue of DJ-1. DLP overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK)/Drosophila forkhead box subgroup O (dFOXO) pathway, whereas loss of DLP increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of DJ-1β mutants were dramatically rescued by DLP deficiency, suggesting that enhanced expression of DLP contributes to the DJ-1β mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in DLP expression in DJ-1β mutants and that dFOXO activity was increased in the heads of DJ-1β mutants. In addition, subcellular localization of DLP appeared to be influenced by DJ-1 expression so that cytosolic DLP was increased in DJ-1β mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed DJ-1β under oxidative stress conditions; and, furthermore, targeted expression of DJ-1β to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1β protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of DJ-1-associated PD.

BACKGROUND AND PURPOSEHepatitis C virus (HCV) infection is responsible for various chronic inflammatory liver diseases. Here, we have identified a naturally occurring compound with anti-HCV activity and have elucidated its mode of antiviral action. EXPERIMENTAL APPROACHLuciferase reporter and real-time RT-PCR assays were used to measure HCV replication. Western blot, fluorescence-labelled HCV replicons and infectious clones were employed to quantitate expression levels of viral proteins. Resistant HCV mutant mapping, in vitro NS3 protease, helicase, NS5B polymerase and drug affinity responsive target stability assays were also used to study the antiviral mechanism. KEY RESULTSA resveratrol tetramer, vitisin B from grapevine root extract showed high potency against HCV replication (EC 50 = 6 nM) with relatively low cytotoxicity (EC 50 >10 μM). Combined treatment of vitisin B with an NS5B polymerase inhibitor (sofosbuvir) exhibited a synergistic or at least additive antiviral activity. Analysis of a number of vitisin B-resistant HCV variants suggested an NS3 helicase as its potential target. We confirmed a direct binding between vitisin B and a purified NS3 helicase in vitro. Vitisin B was a potent inhibitor of a HCV NS3 helicase (IC 50 = 3 nM). In vivo, Finally, we observed a preferred tissue distribution of vitisin B in the liver after i.p. injection in rats, at clinically attainable concentrations. CONCLUSION AND IMPLICATIONSVitisin B is one of the most potent HCV helicase inhibitors identified so far. Vitisin B is thus a prime candidate to be developed as the first HCV drug derived from natural products.

bLeishmaniasis is a disease caused by pathogenic Leishmania parasites; current treatments are toxic and expensive, and drug resistance has emerged. While pentamidine, a diamidine-type compound, is one of the treatments, its antileishmanial mechanism of action has not been investigated in depth. Here we tested several diamidines, including pentamidine and its analog DB75, against Leishmania donovani and elucidated their antileishmanial mechanisms. We identified three promising new antileishmanial diamidine compounds with 50% effective concentrations (EC 50 s) of 3.2, 3.4, and 4.5 M, while pentamidine and DB75 exhibited EC 50 s of 1.46 and 20 M, respectively. The most potent antileishmanial inhibitor, compound 1, showed strong DNA binding properties, with a shift in the melting temperature (⌬T m ) of 24.2°C, whereas pentamidine had a ⌬T m value of 2.1°C, and DB75 had a ⌬T m value of 7.7°C. Additionally, DB75 localized in L. donovani kinetoplast DNA (kDNA) and mitochondria but not in nuclear DNA (nDNA). For 2 new diamidines, strong localization signals were observed in kDNA at 1 M, and at higher concentrations, the signals also appeared in nuclei. All tested diamidines showed selective and dose-dependent inhibition of kDNA, but not nDNA, replication, likely by inhibiting L. donovani topoisomerase IB. Overall, these results suggest that diamidine antileishmanial compounds exert activity by accumulating toward and blocking replication of parasite kDNA.

Parkin is the most prevalent genetic factor in the onset of autosomal recessive juvenile parkinsonism (AR-JP), and mutations in parkin has been reported to cause motor defects, which result from dopamine deficiency caused by dopaminergic neuronal cell death. Activation of c-Jun N-terminal kinase (JNK) has also been implicated in neuronal cell death in Parkinson's disease (PD). Moreover, Drosophila models for AR-JP, loss of function mutants of Drosophila parkin, also show dopaminergic neural degeneration associated with hyperactivation of JNK, increased apoptosis, and mitochondrial defects. However, the molecular mechanism by which Parkin protects cells from apoptosis remains unclear. In the present study, we tested whether Drosophila Parkin suppressed the JNK signaling pathway in developing tissues. Ectopically expressed parkin strongly suppressed the constitutively active form of Hemipterous (Hep(CA)), a Drosophila JNK kinase that induces an eye degeneration phenotype and apoptosis in the eye imaginal disc. Moreover, parkin also suppressed extra vein formation induced by Basket (Bsk), a Drosophila JNK. Interestingly, the bsk mRNA level was markedly reduced by parkin over-expression, suggesting that the effect of parkin on the phenotype induced by activation of JNK signaling was achieved by transcriptional regulation. Furthermore, we found that the expression level of JNK target genes was reduced by parkin over-expression. Taken together, these results suggest that Drosophila Parkin suppresses JNK signaling by reducing bsk transcription.

dWe report the results of a screen of a library of 925 potential prenyl synthase inhibitors against Trypanosoma brucei farnesyl diphosphate synthase (TbFPPS) and against T. brucei, the causative agent of human African trypanosomiasis. The most potent compounds were lipophilic analogs of the bone resorption drug zoledronate, some of which had submicromolar to low micromolar activity against bloodstream form T. brucei and selectivity indices of up to ϳ300. We evaluated the effects of two such inhibitors on survival and parasitemia in a T. brucei mouse model of infection and found that survival increased by up to 16 days. We also investigated the binding of three lipophilic bisphosphonates to an expressed TbFPPS using crystallography and investigated the thermodynamics of binding using isothermal titration calorimetry.

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Background Replacement therapy is the most common treatment for reduction of bleeding and control of episodic bleeding in individuals with hemophilia. Despite the proven effectiveness of factor replacement therapy, repeated intravenous administration is a heavy burden to individuals with hemophilia. Objectives To reduce the burden, therapeutic agents that can be subcutaneously administered need to be developed, and an anti–tissue factor pathway inhibitor (TFPI) antibody may be a suitable candidate for this purpose. Methods MG1113 is an IgG4 monoclonal antibody that binds to Kunitz‐2 domain (KD2) of TFPI. To confirm the coagulation potential of MG1113, several tests were conducted using factor VIII (FVIII)‐ or IX (FIX)‐deficient plasma. For the ex vivo spiking test, platelet‐poor plasma samples from 14 individuals with hemophilia were spiked with MG1113. The in vivo efficacy was determined using blood loss tests, modified prothrombin time (mPT), and free TFPI quantification after intravenous or subcutaneous administration of MG1113 into hemophilia A (HA)‐induced rabbits. Results Radiographic crystallography demonstrated the specific binding site between MG1113 and KD2. In FVIII‐deficient plasma and the plasma of individuals with hemophilia, peak thrombin and endogenous thrombin levels were increased by MG1113 in a concentration‐dependent manner. Rotational thromboelastometry assay revealed that clotting time, clot formation time, and maximum clot firmness were normalized in MG1113‐treated blood of patients. Intravenous or subcutaneous injection of MG1113 into HA‐induced rabbits resulted in rebalancing of blood loss, mPT, and free TFPI levels. Conclusions These results indicate that subcutaneous administration of MG1113 neutralizes the function of TFPI and regulates bleeding in individuals with hemophilia.

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