Suppressive effect of topoisomerase inhibitors on JC polyomavirus propagation in human neuroblastoma cells

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system, in immunocompromised patients. Because no drugs have been approved for treating PML, many antiviral agents are currently being investigated for this purpose. The inhibitory effects of the topoisomerase I inhibitors topotecan and β‐lapachone were assessed by investigating viral replication, propagation and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using the human neuroblastoma cell line IMR‐32 transfected with the JCPyV plasmid and RT‐ PCR combined with Dpn I treatment. Dpn I digests the input plasmid DNA containing methylated adenosine, but not newly replicated JCPyV DNA, in IMR‐32 cells. It was found that JCPyV replicates less in IMR‐32 cells treated with topotecan or β‐lapachone than in untreated cells. Moreover, drug treatment of JCI cells, which are IMR‐32 cells persistently infected with JCPyV, led to a reduction in the amount of JCPyV DNA and population of VP1‐positive cells. These results demonstrate that topotecan and β‐lapachone affects JCPyV propagation in human neuroblastoma cell lines, suggesting that topotecan and β‐lapachone could potentially be used to treat PML.     

Novel Feline Leukemia Virus Interference Group Based on the env Gene

Feline leukemia virus (FeLV) subgroups have emerged in infected cats via the mutation or recombination of the env gene of subgroup A FeLV (FeLV-A), the primary virus. We report the isolation and characterization of a novel env gene, TG35-2, and report that the TG35-2 pseudotype can be categorized as a novel FeLV subgroup. The TG35-2 envelope protein displays strong sequence identity to FeLV-A Env, suggesting that selection pressure in cats causes novel FeLV subgroups to emerge.     

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid

Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.     

Improving the standards for gut microbiome analysis of fecal samples: insights from the field biology of Japanese macaques on Yakushima Island

Fecal DNA-based 16S ribosomal RNA (rRNA) gene sequencing using next-generation sequencers allows us to understand the dynamic gut microbiome adaptation of animals to their specific habitats. Conventional techniques of fecal microbiome analysis have been developed within the broad contexts defined by human biology; hence, many of these techniques are not immediately applicable to wild nonhuman primates. In order to establish a standard experimental protocol for the analysis of the gut microbiomes of wild animals, we selected the Japanese macaques (Macaca fuscata yakui) on Yakushima Island. We tested different protocols for each stage of fecal sample processing: storage, DNA extraction, and choice of the sequencing region in the bacterial 16S rRNA gene. We also analyzed the gut microbiome of captive Japanese macaques as the control. The comparison of samples obtained from identical macaques but subjected to different protocols showed that the tested storage methods (RNAlater and lysis buffer) produced effectively the same composition of bacterial operational taxonomic units (OTUs) as the standard frozen storage method, although the relative abundance of each OTU was quantitatively affected. Taxonomic assignment of the detected bacterial groups was also significantly affected by the region being sequenced, indicating that sequencing regions and the corresponding polymerase chain reaction (PCR) primer pairs for the 16S rRNA gene should be carefully selected. This study improves the current standard methods for microbiome analysis in wild nonhuman primates. Japanese macaques were shown to be a suitable model for understanding microbiome adaptation to various environments.     

Genetic identification of factors for extracellular cellulose accumulation in the thermophilic cyanobacterium Thermosynechococcus vulcanus: proposal of a novel tripartite secretion system

Cells of the thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN (NIES‐2134) aggregate and produce extracellular cellulose under induced conditions of blue light and low temperature, and both aggregation and cellulose production require the cellulose synthase Tll0007 (XcsA) and photosensory diguanylate cyclases. However, overexpression of both the cellulose synthase and a constitutively active diguanylate cyclase was not sufficient to induce cellulose‐mediated cell aggregation under normal growth conditions. Synteny analysis and gene knockout revealed that two putative genes, hlyD‐like tlr0903 (xcsB) and endoglucanase‐like tlr1902 (xcsC), are linked to tll0007, although they are located apart from tll0007 in the T. vulcanus genome. Gene knockdown revealed that tlr1605 (tolC) was essential for the cellulose‐mediated cell aggregation. Low temperature induced marked upregulation of tlr0903, and overexpression of both tlr0903 (but not tlr1902) and diguanylate cyclase resulted in the strong cell aggregation and cellulose accumulation under normal conditions. Based on these and phylogenetic analysis, we propose that the cyanobacterial extracellular cellulose production is due to a novel variant of the bacterial tripartite secretion system.     

Discovery of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazoles as novel class of corticotropin releasing factor 1 receptor antagonists

A new class of corticotropin releasing factor 1 (CRF₁) receptor antagonists characterized by a tricyclic core ring was designed and synthesized. Novel tricyclic derivatives 2a–e were designed as CRF₁ receptor antagonists based on conformation analysis of our original 2-anilinobenzimidazole CRF₁ receptor antagonist. The synthesized tricyclic derivatives 2a–e showed CRF₁ receptor binding activity with IC₅₀ values of less than 400?nM, and the 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2e was selected as a lead compound with potent in vitro CRF₁ receptor binding activity (IC₅₀?=?7.1?nM). To optimize the pharmacokinetic profiles of lead compound 2e, we explored suitable substituents on the 1-position and 6-position, leading to the identification of compound 42c-R, which exhibited potent CRF₁ receptor binding activity (IC₅₀?=?58?nM) with good oral bioavailability (F?=?68% in rats). Compound 42c-R exhibited dose-dependent inhibition of [¹²⁵I]-CRF binding in the frontal cortex (5 and 10?mg/kg, p.o.) as well as suppression of locomotor activation induced by intracerebroventricular administration of CRF in rats (10?mg/kg, p.o.). These results suggest that compound 42c-R successfully binds CRF₁ receptors in the brain and exhibits the potential to be further examined for clinical studies.     

Structure-based design,synthesis, and biological evaluation of imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors

We identified novel potent inhibitors of p38 MAP kinase using structure-based design strategy. X-ray crystallography showed that when p38 MAP kinase is complexed with TAK-715 (1) in a co-crystal structure, Phe169 adopts two conformations, where one interacts with 1 and the other shows no interaction with 1. Our structure-based design strategy shows that these two conformations converge into one via enhanced protein-ligand hydrophobic interactions. According to the strategy, we focused on scaffold transformation to identify imidazo[1,2-b]pyridazine derivatives as potent inhibitors of p38 MAP kinase. Among the herein described and evaluated compounds, N-oxide 16 exhibited potent inhibition of p38 MAP kinase and LPS-induced TNF-α production in human monocytic THP-1 cells, and significant in vivo efficacy in rat collagen-induced arthritis models. In this article, we report the discovery of potent, selective and orally bioavailable imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors with pyridine N-oxide group.     

Cdc14 Phosphatase Promotes TORC1-Regulated Autophagy in Yeast

Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast.