In vitro stability and in vivo anti-inflammatory efficacy of synthetic jasmonates

A chlorinated methyl jasmonate analog (J7) was elaborated as an in vitro anti-inflammatory lead. However, its in vitro efficacy profile was not reproduced in a subsequent in vivo evaluation, presumably due to its rapid enzymatic hydrolysis in a biological system. In an attempt to improve the metabolic stability of the lead J7 by replacement of its labile methyl ester with reasonable ester groups, several analogs resistant to enzymatic hydrolysis were synthesized. In vivo evaluation of the stability-improved analogs showed that these compounds displayed higher efficacy than the lead J7, suggesting that these new jasmonate analogs may serve as potential anti-inflammatory leads.     

An essential role for a mammalian SWI/SNF chromatin-remodeling complex during male meiosis

Germ cell development and gametogenesis require genome-wide transitions in epigenetic modifications and chromatin structure. These changes include covalent modifications to the DNA and histones as well as remodeling activities. Here, we explore the role of the mammalian SWI/SNF chromatin-remodeling complex during spermatogenesis using a conditional allele of the ATPase subunit, brahma-related gene 1 (Brg1, or Smarca4). Not only do BRG1 levels peak during the early stages of meiosis, genetic ablation of Brg1 in murine embryonic gonocytes results in arrest during prophase of meiosis I. Coincident with the timing of meiotic arrest, mutant spermatocytes accumulate unrepaired DNA and fail to complete synapsis. Furthermore, mutant spermatocytes show global alterations to histone modifications and chromatin structure indicative of a more heterochromatic genome. Together, these data demonstrate a requirement for BRG1 activity in spermatogenesis, and suggest a role for the mammalian SWI/SNF complex in programmed recombination and repair events that take place during meiosis.     

Purification and characterization of recombinant human endothelin receptor type A

Human endothelin receptor type A (ET(A)) is a G-protein coupled receptor that mediates vasoconstriction of blood vessels. To determine the structural characteristics and signaling mechanism of ET(A), we have expressed recombinant ET(A) as a fusion protein with p9 envelope protein from phi6 bacteriophage. The His-tag-labeled p9-ET(A) fusion protein was highly expressed in the membrane fraction of Escherichia coli and purified to homogeneity by single affinity chromatography after solubilization with detergents. Purified p9-ET(A) appeared as an oligomer and presented mainly as an α-helical structure. The protein also showed specific binding to endothelin-1 (ET-1) and the alpha subunit of G(q) protein with apparent K(D) values of 17 and 20 nM, respectively. An antagonist of ET(A), bosentan, prevented the interaction between p9-ET(A) and ET-1 in a concentration-dependent manner. These results indicate that recombinant p9-ET(A) has a competent conformation for interactions with ET-1 and the alpha subunit of G(q) protein.     

Enterovirus 71 VPg Uridylation Uses a Two-Molecular Mechanism of 3D Polymerase

VPg uridylylation is essential for picornavirus RNA replication. The VPg uridylylation reaction consists of the binding of VPg to 3D polymerase (3D^pol) and the transfer of UMP by 3D^pol to the hydroxyl group of the third amino acid Tyr of VPg. Previous studies suggested that different picornaviruses employ distinct mechanisms during VPg binding and uridylylation. Here, we report a novel site (Site-311, located at the base of the palm domain of EV71 3D^pol) that is essential for EV71 VPg uridylylation as well as viral replication. Ala substitution of amino acids (T313, F314, and I317) at Site-311 reduced the VPg uridylylation activity of 3D^pol by >90%. None of the Site-311 mutations affected the RNA elongation activity of 3D^pol, which indicates that Site-311 does not directly participate in RNA polymerization. However, mutations that abrogated VPg uridylylation significantly reduced the VPg binding ability of 3D^pol, which suggests that Site-311 is a potential VPg binding site on enterovirus 71 (EV71) 3D^pol. Mutation of a polymerase active site in 3D^pol and Site-311 in 3D^pol remarkably enables trans complementation to restore VPg uridylylation. In contrast, two distinct Site-311 mutants do not cause trans complementation in vitro. These results indicate that Site-311 is a VPg binding site that stabilizes the VPg molecule during the VPg uridylylation process and suggest a two-molecule model for 3D^pol during EV71 VPg uridylylation, such that one 3D^pol presents the hydroxyl group of Tyr3 of VPg to the polymerase active site of another 3D^pol, which in turn catalyzes VPg→VPg-pU conversion. For genome-length RNA, the Site-311 mutations that reduced VPg uridylylation were lethal for EV71 replication, which indicates that Site-311 is a potential antiviral target.     

Unexpectedly complex gradation of coral population structure in the Nansei Islands,Japan

To establish effective locations and sizes of potential protected areas for reef ecosystems, detailed information about source and sink relationships between populations is critical, especially in archipelagic regions. Therefore, we assessed population structure and genetic diversity of Acropora tenuis, one of the dominant stony coral species in the Pacific, using 13 microsatellite markers to investigate 298 colonies from 15 locations across the Nansei Islands in southwestern Japan. Genetic diversity was not significant among sampling locations, even in possibly peripheral locations. In addition, our results showed that there are at least two populations of A. tenuis in the study area. The level of genetic differentiation between these populations was relatively low, but significant between many pairs of sampling locations. Directions of gene flow, which were estimated using a coalescence‐based approach, suggest that gene flow not only occurs from south to north, but also from north to south in various locations. Consequently, the Yaeyama Islands and the Amami Islands are potential northern and southern sources of corals. On the other hand, the Miyako Islands and west central Okinawa Island are potential sink populations. The Kerama Islands and the vicinity of Taketomi Island are potential contact points of genetic subdivision of coral populations in the Nansei Islands. We found that genetic population structure of A. tenuis in the Nansei Islands is more complex than previously thought. These cryptic populations are very important for preserving genetic diversity and should be maintained.     

Crystal structure of a secreted lipase from Gibberella zeae reveals a novel “double-lock” mechanism

Fusarium graminearum (sexual stage: Gibberella zeae) is the causative agent of Fusarium Head Blight (FHB), which is one of the most destructive plant disease of cereals, accounting for high grain yield losses, especially for wheat and maize. Like other fungal pathogens, several extracellular enzymes secreted by G. zeae are known to be involved in host infection. Among these secreted lipases, G. zeae lipase (GZEL), which is encoded by the FGL1 gene, was demonstrated to be crucial to G. zeae pathogenicity. However, the precise mechanism of GZEL remains unclear due to a lack of detailed structural information. In this study, we report the crystal structure of GZEL at the atomic level. The structure of GZEL displays distinct structural differences compared to reported homologues and indicates a unique “double lock” enzymatic mechanism. To gain insight into substrate/inhibitor recognition, we proposed a model of GZEL in complex with substrate and the lipase inhibitor ebelactone B (based on the reported structures of GZEL homologues), which defines possible substrate binding sites within the catalytic cleft and suggests an “anti sn-l” binding mode. These results pave the way to elucidating the mechanism of GZEL and thus provide clues for the design of anti-FHB inhibitors.     

Fgf9 induces proliferation and nuclear localization of FGFR2 in Sertoli precursors during male sex determination

Recently, we demonstrated that loss of Fgf9 results in a block of testis development and a male to female sex-reversed phenotype; however, the function of Fgf9 in sex determination was unknown. We now show that Fgf9 is necessary for two steps of testis development just downstream of the male sex-determining gene, Sry: (1) for the proliferation of a population of cells that give rise to Sertoli progenitors; and (2) for the nuclear localization of an FGF receptor (FGFR2) in Sertoli cell precursors. The nuclear localization of FGFR2 coincides with the initiation of Sry expression and the nuclear localization of SOX9 during the early differentiation of Sertoli cells and the determination of male fate.     

Structure–anti-MRSA activity relationship of macrocyclic bis(bibenzyl) derivatives

We synthesized a series of macrocyclic bis(bibenzyl) derivatives, including riccardin-, isoplagiochin- and marchantin-class structures, and evaluated their antibacterial activity towards methicillin-resistant Staphylococcus aureus (anti-MRSA activity). The structure–activity relationships and the results of molecular dynamics simulations indicated that bis(bibenzyl)s with potent anti-MRSA activity commonly have a 4-hydroxyl group at the D-benzene ring and a 2-hydroxyl group at the C-benzene ring in the hydrophilic part of the molecule, and an unsubstituted phenoxyphenyl group in the hydrophobic part of the molecule containing the A–B-benzene rings. Pharmacological characterization of the bis(bibenzyl) derivatives and 2-phenoxyphenol fragment 25, previously proposed as the minimum structure of riccardin C 1 for anti-MRSA activity, indicated that they have different action mechanisms: the bis(bibenzyl)s are bactericidal, while 25 is bacteriostatic, showing only weak bactericidal activity.