Site-Selective modification of vitamin D analogue (Deltanoid) through a resin-based version of organoselenium 2,3-sigmatropic rearrangement.

A site-selective modification of a vitamin D analogue (Deltanoid) through a two-step 2,3-sigmatropic rearrangement of organoselenium resin to prepare the key intermediate of calcipotriol has been developed. The polystyrene-supported selenium resins used here not only facilitate separation of product but also assist the crucial 2,3-sigmatropic rearrangement to introduce an important functional group (1α-hydroxyl) with high stereo- and regioselectivity.

Regulation of Nrf2- and AP-1-mediated gene expression by epigallocatechin-3-gallate and sulforaphane in prostate of Nrf2-knockout or C57BL/6J mice and PC-3 AP-1 human prostate cancer cells.

AIM: To examine the regulatory crosstalk between the transcription factors Nrf2 and AP-1 in prostate cancer (PCa) by dietary cancer chemopreventive compounds (-)epigallocatechin-3-gallate (EGCG) from green tea and sulforaphane (SFN) from cruciferous vegetables. METHODS: We performed (i) in vitro studies including luciferase reporter gene assays, MTS cell viability assays, and quantitative real-time PCR (qRT-PCR) in PC-3 AP-1 human PCa cells, (ii) in vivo temporal (3 h and 12 h) microarray studies in the prostate of Nrf2-deficient mice that was validated by qRT-PCR, and (iii) in silico bioinformatic analyses to delineate conserved Transcription Factor Binding Sites (TFBS) in the promoter regions of Nrf2 and AP-1, as well as coregulated genes including ATF-2 and ELK-1. RESULTS: Our study shows that AP-1 activation was attenuated by the combinations of SFN (25 micromol/L) and EGCG (20 or 100 micromol/L) in PC-3 cells. Several key Nrf2-dependent genes were down-regulated (3-fold to 35-fold) after in vivo administration of the combination of EGCG (100 mg/kg) and SFN (45 mg/kg). Conserved TFBS signatures were identified in the promoter regions of Nrf2, AP-1, ATF2, and ELK-1 suggesting a potential regulatory mechanism of crosstalk between them. CONCLUSION: Taken together, our present study of transcriptome profiling the gene expression changes induced by dietary phytochemicals SFN and EGCG in Nrf2-deficient mice and in PC-3 cells in vitro demonstrates that the effects of SFN+EGCG could be mediated via concerted modulation of Nrf2 and AP-1 pathways in the prostate.

Properties of Recombinant Aeromonas punctata Prolyl Endopeptidase.

The properties of recombinant Aeromonas punctata prolyl endopeptidase(apPEP) were studied using specific substrate and peptides. Results show that the optimum catalytic temperature and pH was 34 degrees and 8.4, the stability of the apPEP was in the range of 4-32 degrees and pH 6.0-10.0, and its K(m) was 0.03 mmol/L based on the Z-Gly-Pro-betaNA. The apPEP was not sensitive to PMSF, TLCK, TPCK, Trypsion inhibitor, EDTA, tetrathionate and some metal ions, but was sensitive to SDS and Zn(2 ), and was completely inhibited by DFP. Oxytocin and calcitonin could be specifically hydrolyzed by apPEP at the carboxyl site of proline residue, but the hydrolysis efficiency of calcitonin by the enzyme was less than for oxytocin and for Z-Gly-Pro-betaNA.

Cloning and Nucleotide Sequencing of Prolyl Endopeptidase Gene from Aeromonas punctata subsp. punctata.

Prolyl endopeptidase activity was found in Aeromonas punctata subsp. Punctata. The genomic DNA was partially digested with EcoRI and the recovered 8-16 kb DNA fragments were inserted into the EcoRI site of plasmid pUC19, and were transformated into Escherichia coli DH5alpha. The resulted clones were screened by using Benzyloxycarbonyl-Gly-Pro-beta-naphthylamide, the specific substrate of prolyl endopeptidase and a positive clone was obtained. The 12 kb insertion fragment of recombinant plasmid was digested with HincII and subcloned. The PEP gene was found in the 3.5 kb HincII/EcoRI fragment. Nucleotide sequence of the gene was completely sequenced by Auto Sequencer. The complete gene consisted of 2 073 bp corresponding to 690 amino acid residues with a calculated molecular weight of 76 467 Da. The amino acid sequence was 92.3% 53.2% 33.5% 33.2% and 20.5% homologous to those of Aeromonas hydrophila, Flavobacterium meningosepticum, porcine brain, human lymphocytes and Pyrococcus furiosus respectively. From a survey of sequence homology with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser(538) Asp(622) and His(657).