A case of maxillary sarcoma in a chimpanzee (Pan troglodytes)

Oral malignancy is rare in chimpanzees. A 34‐year‐old female chimpanzee (Pan troglodytes) at Kumamoto Sanctuary, Japan, had developed it. Treatment is technically difficult for chimpanzees while malignant neoplasm is seemingly rising in captive populations. Widespread expert discussion, guidelines for treatment, especially for great apes in terminal stages is urgently needed.     

A series of novel indazole derivatives of Sirt 1 activator as osteogenic regulators

A series of indazole derivatives were identified as Sirt 1 activators though high-throughput screening. Optimization of each substituent on the indazole ring led to the identification of compound 13. Compound 13 appeared to give the best Sirt 1 activity of the compounds tested and also showed osteogenesis activity in a cell assay. Sirt 1 activators are therefore potential candidates for the treatment of osteoporosis.     

The participation of nitric oxide in peritoneal exudate cell cytotoxicity of mice by Fusobacterium nucleatum

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum(Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram-negative rod periodontal pathogen. NO could not be induced by heat-killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.     

Bisulfighter: accurate detection of methylated cytosines and differentially methylated regions

Analysis of bisulfite sequencing data usually requires two tasks: to call methylated cytosines (mCs) in a sample, and to detect differentially methylated regions (DMRs) between paired samples. Although numerous tools have been proposed for mC calling, methods for DMR detection have been largely limited. Here, we present Bisulfighter, a new software package for detecting mCs and DMRs from bisulfite sequencing data. Bisulfighter combines the LAST alignment tool for mC calling, and a novel framework for DMR detection based on hidden Markov models (HMMs). Unlike previous attempts that depend on empirical parameters, Bisulfighter can use the expectation-maximization algorithm for HMMs to adjust parameters for each data set. We conduct extensive experiments in which accuracy of mC calling and DMR detection is evaluated on simulated data with various mC contexts, read qualities, sequencing depths and DMR lengths, as well as on real data from a wide range of biological processes. We demonstrate that Bisulfighter consistently achieves better accuracy than other published tools, providing greater sensitivity for mCs with fewer false positives, more precise estimates of mC levels, more exact locations of DMRs and better agreement of DMRs with gene expression and DNase I hypersensitivity. The source code is available at http://epigenome.cbrc.jp/bisulfighter.     

Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV.     

Change in enantioselectivity in bufuralol 1''-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6

The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate. Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors. The enantiomeric BF 1'-hydroxylase activities of the mutants were compared with those of the wild type. When enantiomeric BF 1'-hydroxylase activities at a substrate concentration of 100 microM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF > S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF < or = S-BF), whereas the product diastereoselectivity of (1'R-OH-BF < 1'-S-OH-BF) was similar between the wild type and the mutant. The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type. The kinetics demonstrated that apparent K(m) values were similar among the recombinant enzymes, and V(max) values clearly reflected the selectivity described above. These results indicate that Phe-120 has a key role in the enantioselective BF 1'-hydroxylation by CYP2D6.     

Type I IFN-mediated enhancement of anti-leukemic cytotoxicity of gammadelta T cells expanded from peripheral blood cells by stimulation with zoledronate

BACKGROUND: In order to establish efficient gammadelta T-cell based tumor immunotherapy, we explored a method to enhance the cytotoxicity of gammadelta T cells against leukemia cells by stimulating gammadelta T cells with type I IFN. METHODS: Gammadelta T cells were expanded from normal PBMC by culturing with zoledronate and a low concentration of IL-2 for 2 weeks. For the activation of gammadelta T cells, gammadelta T cells were cultured with type I IFN (HLBI, IFN-alpha2b and IFN-beta) for 1-3 days. The cytotoxicity of HLBI-activated gammadelta T cells against leukemia cell lines and fresh leukemia cells was evaluated by 51Cr-release assay. RESULTS: Gammadelta T cells, which were expanded and purified with magnetic beads using an anti-gammadelta TCR MAb, were demonstrated to be cytotoxic against leukemia cell lines of both lymphoid and myeloid origin and fresh myeloid leukemia cells. By culturing expanded gammadelta T cells with type I IFN, the expression of the activation marker CD69 was increased and the cytometric bead array showed an elevated production of IFN-gamma by gammadelta T cells. In addition, the cytotoxicity of gammadelta T cells against leukemia cells was definitely enhanced by culturing gammadelta T cells with HLBI. DISCUSSION: The present study has demonstrated that type I IFN could enhance the anti-leukemic cytotoxicity of expanded gammadelta T cells, which implies that in vitro bisphosphonate (such as zoledronate)-expanded and type I IFN-activated gammadelta T cells could be applied to immunotherapy for hematologic malignancies such as leukemia and lymphoma.