Genetic variations estimated from PCR-RFLP analysis of two morphs of the freshwater goby <Emphasis Type="Italic">Rhinogobius</Emphasis> in the Lake Biwa water system

The freshwater goby Rhinogobius is the most abundant fish in the shore area of Lake Biwa, Japan. Recently, it has been reported that two morphs of Rhinogobius inhabit this lake. In this study, genetic variations in Rhinogobius sp. OR (Orange-type) and Rhinogobius sp. BW (Biwa-type) in the Lake Biwa water system have been investigated using polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial DNA, including the variable D-loop region. Samples of Rhinogobius sp. OR were collected from the middle sites of three rivers, two outlets, and two lakeshore sites, whereas samples of Rhinogobius sp. BW were taken from two lakeshore sites. Rhinogobius sp. OR and Rhinogobius sp. BW did not share any haplotypes, suggesting that PCR-RFLP analysis is effective for distinguishing between these species of goby. The haplotype diversity of Rhinogobius sp. OR (0.214–0.543) was lower than that of Rhinogobius sp. BW (0.543–0.682). There were no significant differences in haplotype frequencies between Rhinogobius sp. BW groups from the two localities. In addition, haplotype frequencies in Rhinogobius sp. OR did not differ significantly among samples from the middle sites of rivers, the outlets, and the shores. These results indicate that in Rhinogobius sp. OR there is frequent gene flow among fish inhabiting different sites, and that this species of goby consists of a single population throughout the Lake Biwa water system.     

Molecular dynamics and interactions for creation of stimulation-induced stabilized rafts from small unstable steady-state rafts

We have evaluated the sizes and lifetimes of rafts in the plasma membrane from the existing literature, with a special attention paid to their intrinsically broad distributions and the limited time and space scales that are covered by the observation methods used for these studies. Distinguishing the rafts in the steady state (reserve rafts) from those after stimulation or unintentional crosslinking of raft molecules (stabilized receptor-cluster rafts) is critically important. In resting cells, the rafts appear small and unstable, and the consensus now is that their sizes are smaller than the optical diffraction limit (250 nm). Upon stimulation, the raft-preferring receptors are clustered, inducing larger, stabilized rafts, probably by coalescing small, unstable rafts or cholesterol-glycosphingolipid complexes in the receptor clusters. This receptor-cluster-induced conversion of raft types may be caused by suppression of alkyl chain isomerization and the lipid lateral diffusion in the cluster, with the aid of exclusion of cholesterol from the bulk domain and the boundary region of the majority of transmembrane proteins. We critically inspected the possible analogy to the boundary lipid concept. Finally, we propose a hypothesis for the coupling of GPI-anchored receptor signals with lipid-anchored signaling molecules in the inner-leaflet raft.     

Efficient asymmetric syntheses, determination of absolute configurations and biological activities of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine as a novel potent dopamine uptake inhibitor in the central nervous system

An efficient asymmetric synthesis of the chiral N-(3-chloro-2-hydroxypropyl)anilines (2a and 2b) was achieved through the regioselective ring-opening reaction of chiral epichlorohydrin with aniline. This was applied to an asymmetric synthesis of the enantiomers of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 1 as a novel potent dopamine uptake inhibitor. Both enantiomers as trihydrochlorides, 4a.3HCl and 4b.3HCl, could be synthesized in good total yields and optical purities of 100% ee in three steps synthesis, respectively. The absolute configurations of 4a.3HCl and 4b.3HCl were determined using the modified Mosher's method with the related compounds, the intermediates (2a and 2b) and the free bases (4a and 4b). The analytical results indicated that 4a.3HCl and 4b.3HCl have the (S)- and (R)-configuration, respectively, and a series of reactions to provide them proceeded without the apparent influence on the stereochemistry at the chiral centers. In in vitro pharmacological evaluations, 4a.3HCl and 4b.3HCl showed potent dopamine transporter binding affinities, high dopamine, moderate serotonin, and weak norepinephrine uptake inhibitory activities, and 4a.3HCl exhibited a more potent and selective dopamine uptake inhibition over the serotonin or norepinephrine uptake inhibition as compared with 4b.3HCl. An ex vivo evaluation revealed that the oral administrations of both enantiomers at a dose of 30 mg/kg in rats displayed apparent dopamine uptake inhibitory activities and 4a.3HCl had a stronger tendency to inhibit dopamine uptake compared with 4b.3HCl.     

Synthesis and pharmacological properties of benzamide derivatives as selective serotonin 4 receptor agonists

A series of 4-amino-5-chloro-2-methoxy-N-(piperidin-4-ylmethyl)benzamides with a polar substituent group at the 1-position of the piperidine ring was synthesized and evaluated for its effect on gastrointestinal motility. The benzoyl, phenylsulfonyl, and benzylsulfonyl derivatives accelerated gastric emptying and increased the frequency of defecation. One of them, 4-amino-N-[1-[3-(benzylsulfonyl)propyl]piperidin-4-ylmethyl]-5-chloro-2-methoxybenzamide (13a, Y-36912), was a selective 5-HT4 receptor agonist offering potential as a novel prokinetic with reduced side effects derived from 5-HT3- and dopamine D2 receptor-binding affinity. In the oral route of administration, this compound enhanced gastric emptying and defecation in mice, and has a possibility as a prokinetic agent, which is effective on both the upper and the lower gastrointestinal tract.     

应用ESR检测探讨急性热暴露大鼠肝脏氧化还原状态的动态变化

目的:通过电子顺磁自旋共振技术(ESR)动态观察大鼠在过热条件下肝脏的氧化还原状态.方法:将52只雄性Wistar大鼠随机分成4组:①加温组:麻醉后进行整体加温到直肠温达(43.0±0.5)℃,持续15 min;②对照组:只进行麻醉处理;③,MPG预处理组:用抗氧化剂MPG预处理后,再进行与上述①同样条件的加温处理;④非MPG预处理组:在③中用生理盐水代替MPG.经过以上处理后在不同时间点取肝脏制备组织匀浆,测定ESR波谱.结果:与对照组比较,加温组热暴露处理后记录的ESR波谱振幅-时间直线斜率增大,2 h达最大值.以后逐渐恢复,24 h接近对照组水平.经抗氧化剂预处理上述反应减弱.结论:过热能诱导肝脏产生活性氧,增强其氧化还原反应.     

Targeting apoptotic tumor cells to Fc gamma R provides efficient and versatile vaccination against tumors by dendritic cells

Dendritic cells (DCs) loaded with tumor-associated Ags (TAAs) act as potent adjuvant that initiates antitumor immune responses in vivo. However, TAA-based DC vaccination requires prior identification of TAAs. Apoptotic tumor cells (ATCs) can be an excellent source for DC loading because their potential uncharacterized Ags would be efficiently presented to T cells without any prior characterization and isolation of these Ags. However, ATCs alone are considered to be inefficient for activating antitumor immunity, possibly because of their inability to induce DC maturation. In this study, the aim was to enhance antitumor immune response by taking advantage of ATCs that have been opsonized with IgG (ATC-immune complexes, ATC-ICs) so as to target them to FcR for IgG (FcgammaRs) on DCs. It was found that when compared with ATCs, ATC-ICs were efficiently internalized by DCs via FcgammaRs, and this process induced maturation of DCs, which was more efficient than that of ATCs. Importantly, ATC-IC loading was shown to be more efficient than ATCs alone in its capacity for inducing antitumor immunity in vivo, in terms of cytotoxic T cell induction and tumor rejection. These results show that using ATC-ICs may overcome the limitations and may enhance the immune response of current ATC-based DC vaccination therapy.     

ARF tumor suppressor induces mitochondria-dependent apoptosis by modulation of mitochondrial Bcl-2 family proteins

A tumor suppressor gene product, ARF, sensitizes cells to apoptosis in the presence of appropriate collateral signals. In this study, we analyzed the mechanism of ARF-dependent apoptosis and demonstrated that ARF induces mitochondria-dependent apoptosis in p53 wild-type, ARF/p16-null cells. We also found that ARF evokes cytochrome c release from mitochondria, decreases mitochondrial membrane potential, and activates pro-caspase-9 to induce apoptosis. Our findings suggest that this apoptotic cellular modulation is brought about by up-regulation of the proapoptotic Bcl-2 family proteins Bax and Bim and down-regulation of antiapoptotic Bcl-2 in mitochondrial fractions. Additionally, ARF seems to down-regulate Bcl-2 in a p53-dependent manner while up-regulating Bax/Bim via a p53-independent pathway.     

Protein region important for regulation of lipid metabolism in angiopoietin-like 3 (ANGPTL3): ANGPTL3 is cleaved and activated in vivo

Angiopoietin-like 3 (ANGPTL3) is a secreted protein that is mainly expressed in the liver and regulates lipid metabolism by inhibiting the lipolysis of triglyceriderich lipoproteins. Using deletion mutants of human ANGPTL3, we demonstrated that the N-terminal coiled-coil domain-containing fragment-(17-207) and not the C-terminal fibrinogen-like domain-containing fragment-(207-460) increased the plasma triglyceride levels in mice. We also found that the N-terminal region 17-165 was required to increase plasma triglyceride levels in mice and that a substitution of basic amino acid residues in the region 61-66 of the fragment showed no increase in the plasma triglyceride levels and no inhibition of lipolysis by lipoprotein lipase. In addition, when we analyzed ANGPTL3 in human plasma, we detected cleaved fragments of ANGPTL3. By analyzing recombinant ANGPTL3 in mouse plasma, we found that it was cleaved at two sites, Arg221 downward arrow Ala222 and Arg224 downward arrow Thr225, which are located in the linker region between the coiled-coil domain and the fibrinogen-like domain. Furthermore, a cleavage-resistant mutant of ANGPTL3 was determined to be less active than wild-type ANGPTL3 in increasing mouse plasma triglyceride levels but not in inhibiting lipoprotein lipase activity. These findings suggest that the cleavage of ANGPTL3 is important for the activation of ANGPTL3 in vivo.     

Detection and identification of Escherichia coli,Shigella, and Salmonella by microarrays using the gyrB gene

Commonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for identifying enteric bacteria. However, it may not always be applicable for distinguishing closely related bacteria. Therefore, we selected gyrB genes that encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as target genes. The molecular evolution rate of gyrB genes is higher than that of 16S rRNA, and gyrB genes are distributed universally among bacterial species. Microarray technology includes the methods of arraying cDNA or oligonucleotides on substrates such as glass slides while acquiring a lot of information simultaneously. Thus, it is possible to identify the enteric bacteria easily using microarray technology. We devised a simple method of rapidly identifying bacterial species through the combined use of gyrB genes and microarrays. Closely related bacteria were not identified at the species level using 16S rRNA sequence analysis, whereas they were identified at the species level based on the reaction patterns of oligonucleotides on our microarrays using gyrB genes.