Utilization of [11C]phosgene for radiosynthesis of N-(2-{3-[3,5-bis(trifluoromethyl)]phenyl[11C]ureido}ethyl)glycyrrhetinamide, an inhibitory agent for proteasome and kinase in tumors

N-(2-{3-[3,5-Bis(trifluoromethyl)]phenylureido}ethyl)glycyrrhetinamide (2), an ureido-substituted derivative of glycyrrhetinic acid (1), has been reported to display potent inhibitory activity for proteasome and kinase, which are overexpressed in tumors. In this study, we labeled this unsymmetrical urea 2 using [(11)C]phosgene ([(11)C]COCl(2)) as a labeling agent with the expectation that [(11)C]2 could become a positron emission tomography ligand for the imaging of proteasome and kinase in tumors. The strategy for the radiosynthesis of [(11)C]2 was to react hydrochloride of 3,5-bis(trifluoromethyl)aniline (4·HCl) with [(11)C]COCl(2) to possibly give isocyanate [(11)C]6, followed by the reaction of [(11)C]6 with N-(2-aminoethyl)glycyrrhetinamide (3).     

Taxonomic study of the Burmoniscus ocellatus complex (Crustacea, Isopoda, Oniscidea) in Japan shows genetic diversification in the southern Ryukyus, southwestern Japan

To clarify the taxonomic status of the Burmoniscus ocellatus complex in Japan, we carried out morphological observations and phylogenetic analyses of specimens collected from Yonagunijima, Iriomotejima, Ishigakijima, and Miyakojima Islands of the southern Ryukyus and from Okinawajima Island of the central Ryukyus in southwestern Japan. Observations of the holotypes of Aphiloscia iriomotensis ( Nunomura, 1986 ), Ap. ishigakiensis ( Nunomura, 1986 ), and Ap. yonakuniensis ( Nunomura, 1986 ), in addition to the specimens newly collected from the five islands, indicated that these specimens belong to the genus Burmoniscus. Analyses of five morphological characters of 268 specimens collected from the five islands showed that the body size of Okinawajima specimens is distinctly smaller than those of the specimens from the southern Ryukyus. The ranges of the five morphological characters tended to overlap among the specimens from Yonagunijima, Iriomotejima, Ishigakijima and Miyakojima Islands; these morphological characters were congruent with those of 6. ocellatus (Verhoeff, 1928). The phylogenetic analyses were based on three regions of mitochondrial DNA-COI, 12S rRNA, and 16S rRNA-and indicated that the specimens collected from the southern Ryukyus constitute a monophyletic group, which is clearly distinct from the clade composed of the Okinawajima specimens. These results strongly suggest that Ap. iriomotensis, Ap. ishigakiensis, and Ap. yonakuniensis are synonymous with B. ocellatus, a species widely distributed in the southern Ryukyus. On the other hand, the species from Okinawajima Island in the central Ryukyus is considered to be an undescribed Burmoniscus species, which is closely related to but clearly distinct from S. ocellatus.     

NaCl-dependent formation of the highly crystalline phase in sufficiently hydrated dimyristoylphosphatidylglycerol bilayers

We investigated the low-temperature phase behavior of dimyristoylphosphatidylglycerol (DMPG) bilayers in the presence of high concentration of NaCl (≥100 mM). Differential scanning calorimetry showed that the highly crystalline (HC) phase grew after an initial delay period when DMPG bilayers were sufficiently hydrated and incubated at 1 °C in the presence of more than 100 mM NaCl. The HC phase formation reached a plateau, the level of which depended on NaCl concentration; all the lipids were unable to be in the HC phase at the plateau stage without a quite high concentration of NaCl. Since electron microscopic observations suggested that the HC phase formed coexists with the precursor phases in a closed vesicle, elastic constrain and/or shortage of free sodium ions in the inside of the closed vesicle may prevent the complete transition into the HC phase.     

Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells

Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6–7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6–12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.     

Network Properties of Robust Immunity in Plants

Two modes of plant immunity against biotrophic pathogens, Effector Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), are triggered by recognition of pathogen effectors and Microbe-Associated Molecular Patterns (MAMPs), respectively. Although the jasmonic acid (JA)/ethylene (ET) and salicylic acid (SA) signaling sectors are generally antagonistic and important for immunity against necrotrophic and biotrophic pathogens, respectively, their precise roles and interactions in ETI and PTI have not been clear. We constructed an Arabidopsis dde2/ein2/pad4/sid2-quadruple mutant. DDE2, EIN2, and SID2 are essential components of the JA, ET, and SA sectors, respectively. The pad4 mutation affects the SA sector and a poorly characterized sector. Although the ETI triggered by the bacterial effector AvrRpt2 (AvrRpt2-ETI) and the PTI triggered by the bacterial MAMP flg22 (flg22-PTI) were largely intact in plants with mutations in any one of these genes, they were mostly abolished in the quadruple mutant. For the purposes of this study, AvrRpt2-ETI and flg22-PTI were measured as relative growth of Pseudomonas syringae bacteria within leaves. Immunity to the necrotrophic fungal pathogen Alternaria brassicicola was also severely compromised in the quadruple mutant. Quantitative measurements of the immunity levels in all combinatorial mutants and wild type allowed us to estimate the effects of the wild-type genes and their interactions on the immunity by fitting a mixed general linear model. This signaling allocation analysis showed that, contrary to current ideas, each of the JA, ET, and SA signaling sectors can positively contribute to immunity against both biotrophic and necrotrophic pathogens. The analysis also revealed that while flg22-PTI and AvrRpt2-ETI use a highly overlapping signaling network, the way they use the common network is very different: synergistic relationships among the signaling sectors are evident in PTI, which may amplify the signal; compensatory relationships among the sectors dominate in ETI, explaining the robustness of ETI against genetic and pathogenic perturbations.     

Module-specific antibodies against human connective tissue growth factor: utility for structural and functional analysis of the factor as related to chondrocytes

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24/CCN2) shows diverse functions in the process of endochondral ossification. It promotes not only the proliferation and differentiation of chondrocytes and osteoblasts in vitro, but also angiogenesis in vivo. The ctgf gene is a member of the gene family called CCN, and it encodes the characteristic 4-module structure of this family, with the protein containing IGFBP, VWC, TSP and CT modules. We raised several monoclonal antibodies and polyclonal antisera against CTGF, and located the epitopes in the modules by Western blotting. For mapping the epitopes, Brevibacillus-produced independent modules were utilized. As a result, at least 1 antibody or antiserum was prepared for the detection of each module in CTGF. Western blotting with these antibodies is expected to be useful for the analysis of CTGF fragmentation. Moreover, we examined the effects of these monoclonal antibodies on the biological functions of CTGF. One out of 3 humanized monoclonal antibodies was found to neutralize efficiently the stimulatory effect of CTGF on chondrocytic cell proliferation. This particular antibody bound to the CT module. In contrast, surprisingly, all of the 3 antibodies recognizing IGFBP, VWC and CT modules stimulated proteoglycan synthesis in chondrocytic cells. Together with previous findings, these results provide insight into the structural-functional relationships of CTGF in executing multiple functions.     

Molecular evaluation of phylogenetic significances in the highly divergent karyotypes of the genus Gonocephalus (Reptilia: Agamidae) from tropical Asia

The Oriental large-bodied crested dragons of the genus Gonocephalus are known to include two distinct karyomorphs. To evaluate their phylogenetic significances, we conducted phylogenetic analyses of the genus together with other agamid genera on the basis of 862 base positions of mitochondrial 12S and 16S rRNA genes. Results suggested the presence of two distinct lineages within Gonocephalus, of which one, represented by G. robinsonii that has a 2n = 32 karyotype, was closer to other Oriental agamid genera than to the other congeneric lineage. Monophyly of the latter, characterized by unique chromosomal arrangement among agamid genera (2n = 42 karyotype), was confirmed. It is thus likely that states of morphological characters shared between the two lineages are derived through convergence, or represent symplesiomorphy. Our results also suggest that the karyological similarity between G. robinsonii and several Australian agamids, pointed out in a previous study, is actually attributable to homoplasy rather than their recent common ancestry.     

Intracranial administration of transforming growth factor-beta3 increases fat oxidation in rats

The effects of intracranial transforming growth factor (TGF)-beta3 on spontaneous motor activity and energy metabolism were examined in rats. After injection of TGF-beta3 into the cisterna magna of the rat, spontaneous motor activity decreased significantly for 1 h. The intracranial injection of TGF-beta3 produced an immediate decrease in respiratory exchange ratio (RER). No significant changes were observed in energy expenditure. TGF-beta3 induced a significant increase in total fat oxidation and a decrease in total carbohydrate oxidation. Furthermore, the serum substrates associated with fat metabolism were significantly altered in rats injected with TGF-beta3. Both lipoprotein lipase activity in skeletal muscle and the concentration of serum ketone bodies increased, suggesting that the increase in fat oxidation caused by TGF-beta3 may have occurred in the liver and muscle. Intracranial injection of TGF-beta3 appeared to evoke a switch in the energy substrates accessed in energy expenditure. These results suggest that the release of TGF-beta3 in the brain by exercise is a signal for regulating energy consumption.