Isolation, evaluation of bioactivity and structure determination of amethinol A, a prototypic amethane diterpene from Isodon amethystoides bearing a six/five/seven-membered carbon-ring system.

We report the isolation of a novel diterpene, designated as `amethane', from Isodon amethystoides (Lamiaceae). The diterpene [amethinol A; systematic name: (4aR,4bR,7R,10aS)-4b,7-dihydroxy-7-isopropyl-1,1-dimethyl-9-oxododecahydrobenzo[a]azulene-4a(2H)-carboxylic acid], possesses a unique skeleton containing a six/five/seven-membered tricyclic system. Intermolecular O-H...O close contacts were found to the carboxyl, carbonyl and hydroxy groups, connecting molecules into a two-dimensional structure. A possible biosynthetic pathway has been proposed. In addition, the compound was evaluated for its biological activities against different disease targets, and was found to significantly attenuate RORγt-dependent autoimmune responses.

Molecular cloning and characterization of 5-enolpyruvylshikimate-3-phosphate synthase gene from Convolvulus arvensis L.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS), the target enzyme for glyphosate inhibition, catalyzes an essential step in the shikimate pathway for aromatic amino acid biosynthesis. The full-length cDNA of 1,751 nucleotides (CaEPSPS, Genbank accession number: EU698030) from Convolvulus arvensis was cloned and characterized. The CaEPSPS encodes a polypeptide of 520 amino acids with a calculated molecular weight of 55.5 kDa and an isoelectric point of 7.05. The results of homology analysis revealed that CaEPSPS showed highly homologous with EPSPS proteins from other plant species. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in stems, leaves and roots, with lower expression in roots. CaEPSPS expression level could increase significantly with glyphosate treatment, and reached its maximum at 24 h after glyphosate application. We fused CaEPSPS to the CaMV 35S promoter and introduced the chimeric gene into Arabidopsis. The resultant expression of CaEPSPS in transgenic Arabidopsis plants exhibited enhanced tolerance to glyphosate in comparison with control.

Selective estrogen receptor modulator BC-1 activates antioxidant signaling pathway in vitro via formation of reactive metabolites.

AIM: Benzothiophene compounds are selective estrogen receptor modulators (SERMs), which are recently found to activate antioxidant signaling. In this study the molecular mechanisms of antioxidant signaling activation by benzothiophene compound BC-1 were investigated. METHODS: HepG2 cells were stably transfected with antioxidant response element (ARE)-luciferase reporter (HepG2-ARE cells). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in HepG2-ARE cells was suppressed using siRNA. The metabolites of BC-1 in rat liver microsome incubation were analyzed using LC-UV and LC-MS. RESULTS: Addition of BC-1 (5 µmol/L) in HepG2-ARE cells resulted in a 17-fold increase of ARE-luciferase activity. Pretreatment with the estrogen receptor agonist E2 (5 µmol/L) or antagonist ICI 182,780 (5 µmol/L) did not affect BC-1-induced ARE-luciferase activity. However, transfection of the cells with anti-Nrf2 siRNA suppressed this effect by 79%. Addition of BC-1 in rat microsome incubation resulted in formation of di-quinone methides and o-quinones, followed by formation of GSH conjugates. BC-1 analogues with hydrogen (BC-2) or fluorine (BC-3) at the 4' position did not form the di-quinone methides. Both BC-2 and BC-3 showed comparable estrogenic activity with BC-1, but did not induce ARE-luciferase activity in HepG2-ARE cells. CONCLUSION: Benzothiophene compound BC-1 activates ARE signaling via reactive metabolite formation that is independent of estrogen receptors.

[Molecular Cloning of Smooth Muscle Calponin h1 in SHeep.].

It was found that the level of Calponin h1 (CaP h1) mRNA was significantly up-regulated by Estrogen in the myometrium of sheep towards the end pregnancy. Although the CaP h1 has been widely used as a reference gene to observe the changes of expression level of other genes, the full-length gene in sheep has not been obtained. With the oligo nucleotide primers according to human, mouse and pig CaP h1 mRNA, the full-length cDNA of CaP h1 was cloned by 5'- and 3'-RACE (Genbank accession number = AY327118). The cDNA was 1499bp in length and contained a complete open reading frame of 891 bp, encoding a protein of 297 amino acid residues. 5'-and 3'-UTR was 79 bp and 529bp, respectively. With PCR-SSP approaches,the genomic DNA of sheep CaP h1 was obtained .It showed that the gene has 7 exons and 6 introns, spanning over 8kb(Genbank accession number of introns : AY771807,AY771808, AY771809, AY771810.) Homologous comparison indicated that the cDNA sequences are highly conserved across the species. The highest homology was found in wild pig (92%), followed by human (88%), rat (81%), mouse (81%) and chicken (79%). The intron sequence and length showed a large variation among species (>50%).

[Identification and characterization of alternativly splicing variants for murine mater gene].

Mater encoding an oocyte-specific autoantigen,and is associated with premature autoimmune ovarian dysgenesis (AOD) in mouse. Based on RT-PCR, cDNA cloning, screening, sequencing and analysis, we have detected a total of four Mater splice variants, designated as variant B, E, F and G. All these splicing forms are in frame in terms of expected protein products. Among these, B was consistent with the previous report, whereas E, F, G belong to novel splice variants that have not been reported previously. Variant E lacks exon 6, variant F both lacks exon 10 and retains a part of intron 8, variant G lacks part of exon 14, and variant H lacks part of exon 13. The cDNA sequences at all the exon-intron boundaries confirms to the "GT-AG" splicing rule. Variant B, E, F exist in all the four strains. Variant G exists only in SWR/J. According to the cDNA sequences of these four splice variants, amimo acid sequences of the corresponding expected protein isoforms were deduced, and their potential functional effects were predicted in this thesis. Further identification and characterization of these expected protein isoforms would provide valuable information for their functional importance.