Tricin inhibits proliferation of human hepatic stellate cells in vitro by blocking tyrosine phosphorylation of PDGF receptor and its signaling pathways

4',5,7-Trihydroxy-3',5'-dimethoxyflavone (Tricin), a naturally occurring flavone, has anti-inflammatory potential and exhibits diverse biological activities including antigrowth activity in several human cancer cell lines and cancer chemopreventive effects in the gastrointestinal tract of mice. The present study aimed to investigate the biological actions of tricin on hepatic stellate cells (HSCs) in vitro, exploring its potential as a treatment of liver fibrosis, since HSC proliferation is closely related to the progression of hepatic fibrogenesis in chronic liver diseases leading to irreversible liver cirrhosis and hepatocellular carcinoma. Tricin inhibited platelet-derived growth factor (PDGF)-BB-induced cell proliferation by blocking cell cycle progression and cell migration in the human HSC line LI90 and culture-activated HSCs. It also reduced the phosphorylation of PDGF receptor β and the downstream signaling molecules ERK1/2 and Akt, which might be due to its tyrosine kinase inhibitor properties rather than inhibition of the direct binding between PDGF-BB and its receptor. Our findings suggest that tricin might be beneficial in HSC-targeting therapeutic or chemopreventive applications for hepatic fibrosis.     

Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors of transforming growth factor-β type I receptor kinase

A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. N-{[5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-2-yl]methyl}butanamide 20, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC(50)=8.2nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC(50)=32nM).     

Isolation and characterization of thermotolerant bacterium utilizing ammonium and nitrate ions under aerobic conditions

A thermotolerant bacterium, identified as Bacillus licheniformis, completely utilized 0.1% (w/v) NH₄NO₃ at 30 and 50°C under aerobic condition. The addition of 0.5 mM Fe²⁺ to the NH₄NO₃ medium markedly promoted the utilization of NH₄⁺ and NO₃⁻. At 50°C, of total nitrogen originally provided, 24% was taken up into the cells and 20% remained in the culture supernatant. Residual nitrogen (56%) was probably removed into the atmosphere. The cell extracts contained enzymes involved in denitrification. GC-MS demonstrated that NH₄ ¹⁵NO₃ had been converted to ¹⁵N₂O. These results indicate that the strain has denitrification ability under aerobic condition.     

Genotypic and phenotypic diversity of rhizobia isolated from Lathyrus japonicus indigenous to Japan

Sixty-one rhizobial strains from Lathyrus japonicus nodules growing on the seashore in Japan were characterized and compared to two strains from Canada. The PCR-based method was used to identify test strains with novel taxonomic markers that were designed to discriminate between all known Lathyrus rhizobia. Three genomic groups (I, II, and III) were finally identified using RAPD, RFLP, and phylogenetic analyses. Strains in genomic group I (related to Rhizobium leguminosarum) were divided into two subgroups (Ia and Ib) and subgroup Ia was related to biovar viciae. Strains in subgroup Ib, which were all isolated from Japanese sea pea, belonged to a distinct group from other rhizobial groups in the recA phylogeny and PCR-based grouping, and were more tolerant to salt than the isolate from an inland legume. Test strains in genomic groups II and III belonged to a single clade with the reference strains of R. pisi, R. etli, and R. phaseoli in the 16S rRNA phylogeny. The PCR-based method and phylogenetic analysis of recA revealed that genomic group II was related to R. pisi. The analyses also showed that genomic group III harbored a mixed chromosomal sequence of different genomic groups, suggesting a recent horizontal gene transfer between diverse rhizobia. Although two Canadian strains belonged to subgroup Ia, molecular and physiological analyses showed the divergence between Canadian and Japanese strains. Phylogenetic analysis of nod genes divided the rhizobial strains into several groups that reflected the host range of rhizobia. Symbiosis between dispersing legumes and rhizobia at seashore is discussed.     

Gene expression profiles of endothelial progenitor cells by oligonucleotide microarray analysis

Among the many tissue stem or progenitor cells recently being unveiled, endothelial progenitor cells (EPCs) have attracted particular attention, not only because of their cardinal role in vascular biology and embryology but also because of their potential use in the therapeutic development of a variety of postnatal diseases, including cardiovascular and peripheral vascular disorders and cancer. The aim of this study is to provide some basic and comprehensive information on gene expression of EPCs to characterize the cells in molecular terms. Here, we focus on EPCs derived from CD34-positive mononuclear cells of human umbilical cord blood. The EPCs were purified and expanded in culture and analyzed by a high-density oligonucleotide microarray and real-time RT-PCR analysis. We identified 169 up-regulated and 107 down-regulated genes in the EPCs compared with three differentiated endothelial cells of human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (LMEC) and human aortic endothelial cells (AoEC). It is expected that the obtained list include key genes which are critical for EPC function and survival and thus potential targets of EPC recognition in vivo and therapeutic modulation of vasculogenesis in cancer as well as other diseases, in which de novo vasculogenesis plays a crucial role. For instance, the list includes Syk and galectin-3, which encode protein tyrosine kinase and β-galactoside-binding protein, respectively, and are expressed higher in EPCs than the three control endothelial cells. In situ hybridization showed that the genes were expressed in isolated cells in the fetal liver at E11.5 and E14.5 of mouse development.     

Where should wild species be sampled? New method based on isolation‐by‐distance objectively gives the answer

Random sampling is an important statistical assumption, but virtually impossible when sampling a wild species as we cannot know where all the individuals exist. While interpopulation or intrataxa sampling methods have been developed, there are currently few intrataxon sampling methods to objectively decide where to sample wild taxa. We suggest a new sampling method which computes appropriate sampling locations from coordinates, assuming geographical autocorrelation of phylogeny within a taxon (isolation‐by‐distance). The computed locations encompass the highest genetic diversity, providing a genetically representative sample. In addition, it can utilize presence/absence information during sampling to reoptimize sampling scheme. Comparing to the single existing method of the similar purpose, the merits of ours is unnecessity of environmental data resulting in easy application, and is theoretically deduced. We tested this method using published phylogeographical data. The test result was generally encouraging, but the method failed where species showed uniform genetic structure or recent distribution expansion which violate the assumption of geographical autocorrelation of phylogeny. Though simple, our method constructs a methodological and statistical foundation for sampling wild species, and is applicable to revising taxonomic study and conservation biology.     

Lead generation of heat shock protein 90 inhibitors by a combination of fragment-based approach, virtual screening, and structure-based drug design

Heat shock protein 90 (Hsp90) is a molecular chaperone which regulates maturation and stabilization of its substrate proteins, known as client proteins. Many client proteins of Hsp90 are involved in tumor progression and survival and therefore Hsp90 can be a good target for developing anticancer drugs. With the aim of efficiently identifying a new class of orally available inhibitors of the ATP binding site of this protein, we conducted fragment screening and virtual screening in parallel against Hsp90. This approach quickly identified 2-aminotriazine and 2-aminopyrimidine derivatives as specific ligands to Hsp90 with high ligand efficiency. In silico evaluation of the 3D X-ray Hsp90 complex structures of the identified hits allowed us to promptly design CH5015765, which showed high affinity for Hsp90 and antitumor activity in human cancer xenograft mouse models.     

Synaptotagmin II and Gangliosides Bind Independently with Botulinum Neurotoxin B but Each Restrains the Other

Botulinum neurotoxin type B (BoNT/B) initiates its toxicity by binding to synaptotagmin II (SytII) and gangliosides GD1a and GT1b on the neural membrane. We synthesized two 27-residue peptides that carry the BoNT/B binding sites on mouse SytII (mSytII 37–63) or human SytII (hSytII 34–60). BoNT/B bound to these peptides, but showed substantially higher binding to mSytII peptide than to hSytII peptide. The mSytII peptide inhibited almost completely BoNT/B binding to synaptosomes (snps) and displayed a high affinity. BoNT/B bound strongly to mSytII peptide and binding was inhibited by the peptide. Binding of BoNT/B to snps was also inhibited (~80 %) by a larger excess of gangliosides GD1a or GT1b. The mSytII peptide inhibited very strongly (at least 80 %) the toxin binding to snps, while the two gangliosides were much less efficient inhibitors requiring much larger excess to achieve similar inhibition levels. Furthermore, gangliosides GD1a or GT1b inhibited BoNT/B binding to mSytII peptide at a much larger excess than the inhibition by mSytII peptide. Conversely, BoNT/B bound well to each ganglioside and binding could be inhibited by the correlate ganglioside and much less efficiently by the mSytII peptide. There was no apparent collaboration between mSytII peptide and either ganglioside. mSytII peptide displayed some protective activity in vivo in mice against a lethal BoNT/B dose. We concluded that SytII peptide and gangliosides bind independently but, with their binding sites on BoNT/B being spatially close, each can influence BoNT/B binding to the other due to regional conformational perturbations or steric interference or both. Ganglioside involvement in BoNT/B binding might help in toxin translocation and endocytosis.     

DNA homology of α-globin gene among five races of carp, Cyprinus carpio

Common DNA fragments of the α-globin gene were observed in five races of adult common carp: mirror, scaly-German, Yamato-Suwa, Yamato-Saku, and Yamato-hybrid carp. There were different hybridization patterns between the Japanese carp and the German carp. Three to four DNA fragments hybridized with α-globin and indicated polymorphysim in the globin gene of carp.

Zusammenfassung

DNA-Homologie des α-Globin-Genes bei fünf Karpfenrassen, Cyprinus carpio
Identische Fragmente des α-Globin-Genes wurden bei fünf Rassen des adulten gemeinen Karpfens (Spiegeikarpfen, deutscher Schuppenkarpfen, Yamato-Suwa-, Yamato-Saku-und Yamato-Hybrid-karpfen) gefunden. Es gab verschiedene Hybridisationsmuster zwischen dem japanischen und dem deutschen Karpfen. Drei bis vier DNA-Fragmente hybridisierten mit α-Globin und wiesen auf einen Polymorphismus des Globingenes des Karpfens hin.

Résumé

Homologie de DNA dans le gène d'α-globine chez cinq races de carpe, Cyprinus carpio
Des fragments communs de DNA dans des gènes d'α-globine ont été observés chez cinq races de carpe commune adulte: carpe miroir, allemande écailleuse, Yamato-Suwa, Yamato-Saku et Yamato-hybride. Il existait différentes combinaisons d'hybridation entre la carpe allemande et la carpe japonaise. Trois à quatre fragments de DNA hybridisaient avec l'α-globine et indiquaient un polymorphisme dans les gènes de globine de carpe.