Evidence for a role of vertebrate Rad52 in the repair of topoisomerase II-mediated DNA damage

DNA topoisomerase II (Top2) inhibitors are useful as anticancer agents, mostly by virtue of their ability to induce DNA double-strand breaks (DSBs). These DSBs are repaired almost exclusively by Rad52-dependent homologous recombination (HR) in yeast. However, we have recently shown that in vertebrate cells such lesions are primarily repaired by nonhomologous end-joining, but not HR. This finding, taken together with previous observations that disruption of RAD52 does not severely affect HR in vertebrate cells, makes it highly unlikely that Rad52 contributes to the repair of Top2-mediated DNA damage. However, in this paper we show that chicken cells lacking Rad52 do exhibit increased sensitivity to the Top2 inhibitor VP-16. Remarkably, the level of hypersensitivity of RAD52-null cells was comparable to that of RAD54-null cells, albeit only at high doses. Our data thus provide the first demonstration of a major repair defect associated with loss of Rad52 in vertebrate cells.     

Augmentation of signaling through BCR containing IgE but not that containing IgA due to lack of CD22-mediated signal regulation

The B cell membrane molecules CD22 and CD72 contain ITIMs in their cytoplasmic portion, and negatively regulate signaling through BCR. Various lines of evidence suggest that ligation of BCR containing IgG (IgG-BCR) transmits augmented signaling due to lack of CD22-mediated signal regulation. However, the signaling capacities of BCR containing IgA and IgE remain largely undefined. In this study, we demonstrate that both IgE-BCR and IgG-BCR, but not IgA-BCR, transmit augmented signaling compared with IgM-BCR. Ligation of IgE-BCR does not induce signaling events required for CD22-mediated signal inhibition, and restoration of these signaling events by coligation of CD22 with BCR abrogates signal augmentation. Furthermore, the cytoplasmic portion of IgE but not that of IgA is sufficient for suppressing CD22-mediated signal inhibition. These findings strongly suggest that the cytoplasmic portion of IgE but not that of IgA reverses CD22-mediated signal inhibition, leading to augmentation of signaling through IgE-BCR but not IgA-BCR. Augmented IgE-BCR signaling appears to play a role in production of large amounts of IgE during helminth infection, whereas regulated signaling through IgA-BCR may be crucial for constitutive production of IgA for mucosal immunity.     

Inhibition of fungal ABC transporters by unnarmicin A and unnarmicin C, novel cyclic peptides from marine bacterium

Novel inhibitors of fungal ATP-binding cassette transporters were obtained by screening compounds and crude extracts from marine-derived fungi and bacteria using disk diffusion assays of Saccharomyces cerevisiae strains overexpressing a variety of fungal multi-drug efflux pumps. The cyclodepsipeptides unnarmicin A and unnarmicin C were able to sensitize cells overexpressing azole drug pumps ScPdr5p, CaCdr1p, CgCdr1p, and CgPdh1p to sub-MIC concentrations of fluconazole without affecting the growth of CaCdr2p and CaMdr1p overexpressing cells. Unnarmicin A and unnarmicin C were potent inhibitors of rhodamine 6G efflux of CaCdr1p expressing cells with IC₅₀ values of 3.61 and 5.65 μM, respectively. They inhibited the in vitro CaCdr1p ATPase activity at IC₅₀ values of 0.495 and 0.688 μM, respectively. And most importantly, they were able to sensitize azole-resistant Candida albicans clinical isolates to fluconazole. Unnarmicin A and unnarmicin C are candidate efflux pump inhibitors with the potential to be used as adjuvants for antifungal chemotherapy.     

Biochemical investigation and gene expression analysis of the immunostimulatory functions of an edible Salacia extract in rat small intestine

Roots and bark from plants belonging to genus Salacia of the family Hippocrateaceae (Salacia reticulata, Salacia oblonga, etc.) have been used for traditional Ayurvedic medicine, particularly for the treatment of diabetes. In our study, we evaluated the gene expression profiles in the small intestinal epithelium of rats that were given a Salacia plant extract to gain insight into its effects on the small intestine. In detail, DNA microarray analysis was performed to evaluate the gene expression profiles in the rat ileal epithelium. The intestinal bacterial flora was also studied using T-RFLP (Nagashima method) in these rats. Expressions of many immune-related genes, especially Th1-related genes associated with cell-mediated immunity, were found to increase in the small intestinal epithelium and the intestinal bacterial flora became similar to those in the case with Salacia plant extract administration. Our study thus revealed that Salacia plant extract exerts bioregulatory functions by boosting intestinal immunity.     

Reduced BMP Signaling Results in Hindlimb Fusion with Lethal Pelvic/Urogenital Organ Aplasia: A New Mouse Model of Sirenomelia

Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4^flox/flox) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4^flox/flox conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.     

Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization

We investigated the enzymatic properties and immunohistochemical localization of cuticular hemocyanin, a known oxygen transporter in the prawn Penaeus japonicus. The molecular weight of hemocyanin purified from the cuticle was estimated to be 67-77 k using SDS-PAGE, and the purified protein was effectively converted into a phenoloxidase-like enzyme by an SDS-treatment. The activated enzyme catalyzed the o-hydroxylation of monophenols and the oxidation of o-diphenols and was inhibited by typical inhibitors of phenoloxidase. These characteristics were nearly identical to the enzymatic properties of hemolymph hemocyanin. Immunological detection showed a diffuse distribution of hemocyanin over the exocuticle and endocuticle, and a higher signal level was observed in the latter. Based on these results, roles of hemocyanin in various physiological processes such as immune response and sclerotization of the cuticle were discussed.     

Morphology of Gambierdiscus scabrosus sp. nov. (Gonyaulacales): a new epiphytic toxic dinoflagellate from coastal areas of Japan

A new epiphytic dinoflagellate is described, G ambierdiscus scabrosus sp. nov., from tidal pools and rocky shores along the coastal areas of Japan. Cells are 63.2 ± 5.7 μm in depth, 58.2 ± 5.7 μm in width, and 37.3 ± 3.5 μm in length. The plate formula of G . scabrosus is Po, 4′, 0a, 6′′, 6c, ?s, 5′′′, 0p, and 2′′′′. Morphologically, G . scabrosus resembles G . belizeanus as follows: anterioposteriorly compressed cell shape, narrow 2′′′′ plate, and areolated surface. Despite this similarity, the cells of G . scabrosus can be distinguishable by the presence of the asymmetric shaped 3′′ plate and the rectangular shaped 2′ plate.     

Involvement of IL-18 in the Expansion of Unique Hepatic T Cells with Unconventional Cytokine Profiles during Schistosoma mansoni Infection

Infection with schistosomes invokes severe fibrotic granulomatous responses in the liver of the host. Schistosoma mansoni infection induces dramatic fluctuations in Th1 or Th2 cytokine responses systemically; Th1 reactions are provoked in the early phase, whilst Th2 responses become dominant after oviposition begins. In the liver, various unique immune cells distinct from those of conventional immune competent organs or tissues exist, resulting in a unique immunological environment. Recently, we demonstrated that S. mansoni infection induces unique CD4⁺ T cell populations exhibiting unconventional cytokine profiles in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. They produce both IFN-γ and IL-4 or both IFN-γ and IL-13 simultaneously. Moreover, T cells secreting triple cytokines IFN-γ, IL-13 and IL-4 were also induced. We term these cells Multiple Cytokine Producing Hepatic T cells (MCPHT cells). During the transition phase, when MCPHT cells increase, IL-18 secretion was up-regulated in the liver and sera. In S. mansoni-infected IL-18-deficient mice, expansion of MCPHT cells was curtailed. Thus our data suggest that IL-18 produced during S. mansoni infection play a role in the expansion of MCPHT cells.     

Synthesis of all stereoisomers of 3-hydroxypipecolic acid and 3-hydroxy-4,5-dehydropipecolic acid and their evaluation as glycosidase inhibitors

A highly practicable synthesis of both enantiomers of 3-hydroxypipecolic acid derivatives 1, 2, 3, 4 is described. Screening of these molecules for glycosidase inhibition has been examined. Compound 3 was shown to be a potent inhibitor of beta-N-acetylglucosaminidase as well as Escherichia coli beta-glucuronidase.