Effects of Danhong injection, a traditional Chinese medicine, on nine cytochrome P450 isoforms in vitro.

Danhong injection (DHI) is made from Salvia miltiorrhiza Bunge. and Carthamus tinctorius L. extract and is widely used in the clinical treatment of cardiovascular and cerebrovascular diseases. This study aimed to evaluate the effect of DHI on cytochrome P450 (CYP450) enzymes in vitro to predict drug-drug interactions based on CYP450 as combination therapy. To assess the inhibitory effect of DHI on CYP450, we detected the IC50 value of DHI on CYP450 in vitro by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Simultaneously, the induction effect of DHI on CYP450s was also evaluated. The relative induction ratios of DHI on CYP1A2, CYP2B6 and CYP3A4 activity were calculated by LC-MS/MS. The expression level of CYP3A4 mRNA was determined by reverse transcription PCR (RT-PCR). The LC-MS/MS data showed DHI intensively inhibit CYP2A6 activity and the intensity of inhibition was followed by CYP2C8, CYP3A4, CYP2C19, CYP2B6, CYP2D6, CYP1A2, CYP2E1 and CYP2C9 in vitro. The results of RT-PCR showed that there is a certain induction of DHI on CYP3A4 mRNA in human primary hepatocytes in vitro. The study suggested that drug-drug interactions might occur in clinical co-administration of drugs owing to the CYP2A6 inhibition and CYP3A4 induction.

Effects of diosmetin on nine cytochrome P450 isoforms, UGTs and three drug transporters in vitro.

Diosmetin (3', 5, 7-trihydroxy-4'-methoxyflavone), a natural flavonoid from traditional Chinese herbs, has been used in various medicinal products because of its anticancer, antimicrobial, antioxidant, estrogenic and anti-inflammatory activity. However, flavonoids could affect the metabolic enzymes and cause drug-drug interactions (DDI), reducing the efficacy of co-administered drugs and potentially resulting in serious adverse reactions. To evaluate its potential to interact with co-administered drugs, the IC50 value of phase I cytochrome P450 enzymes (CYPs), phase II UDP-glucuronyltransferases (UGTs) and hepatic uptake transporters (organic cation transporters (OCTs), organic anion transporter polypeptides (OATPs) and Na+-taurocholate cotransporting polypeptides (NTCPs)) were examined in vitro by LC-MS/MS. Diosmetin showed strong inhibition of CYP1A2 in a concentration-dependent manner. The intensity of the inhibitory effect was followed by CYP2C8, CYP2C9, CYP2C19 and CYP2E1. For CYP2A6, CYP2B6, CYP2D6 and CYP3A4, diosmetin was found to have no significant inhibitory effects, and the induction effect on CYPs was not significant. For UGTs, diosmetin had a minimal inhibitory effect. In addition, the inhibitory effects of diosmetin on OATP and OCT1 were weak, and it had little effect on NTCP. This finding indicated that drug-drug interactions induced by diosmetin may occur through co-administration of drugs metabolized by CYP1A2.

Transcription factor gene TuGTγ-3 is involved in the stripe rust resistance in Triticum urartu.

Wheat stripe rust, caused by Puccinia striiformis West. f. sp. tritici Eriks. ＆Henn. (Pst), is a serious fungal disease. Identification of new genes associate with stripe rust resistance is important for developing disease resistant wheat cultivars and studying the mechanism of disease resistance. Trihelix is a plant specific transcription factor family, which is involved in regulation of growth and development, morphogenesis, and response to stresses. So far, no study reports on the relationship between the Trihelix family and wheat stripe rust. In this study, a gene in the GTγ subfamily of Trihelix family, designated TuGTγ-3, was cloned from Triticum urartu Tum. (2n=2x=14, AA). The results of sequencing demonstrated that TuGTγ-3 gene consisted of a complete open reading frame (ORF), and its coding sequence was 1329 bp in length, which encoded a protein with 442 amino acids. The predicted molecular weight of this protein was 50.31 kDa and the theoretical isoelectric point was 6.12. Bioinformatic analysis revealed that TuGTγ-3 protein had a monopartite nuclear localization signal (GLPMQKKMRYT), and had neither transmembrane domain nor signal peptide. The conserved trihelix domain, the fourth α-helix and the CC domain were located in the regions of Q115?R187, F234?Y241 and K362?K436, respectively. Dissection of secondary structure showed that TuGTγ-3 protein comprised of 43.89% α-helix, 9.51% extended strand, 9.95% ß-turn and 36.65% random coil structures. Based on the BLAST search against the genome database of common wheat from IWGSC, TuGTγ-3 was located on the long arm of chromosome 5A. Transient expression experiment using onion inner epidermal cell showed that the fusion protein TuGTγ-3-GFP distributed mainly in nuclear and slightly in cytoplasm. Expression profiles in different organs indicated that expression level of TuGTγ-3 was much higher in leaves than that in roots or leaf sheaths, and the expression in leaves was extremely up-regulated by infection of the Pst race CYR32. Furthermore, the BSMV-VIGS experiment demonstrated that the transcription factor TuGTγ-3 positively regulated resistance to stripe rust in T. urartu.

Xiaoaiping injection enhances paclitaxel efficacy in ovarian cancer via pregnane X receptor and its downstream molecules.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoaiping injection, a traditional Chinese medical injection extracted from root of Marsdenia tenacissima (Roxb.) Moon, has been exclusively used on curing malignant tumor in China and as adjuvant therapeutic agent for chemotherapeutics, including paclitaxel. AIM OF THE STUDY: The goal of this study was to investigate the synergistic inhibitory efficacy of Xiaoaiping injection and paclitaxel on ovarian cancer. The mechanism may be associated with nuclear receptor pregnane X receptor (PXR) regulating its downstream molecules. MATERIALS AND METHODS: In vitro, MTT assay, flow cytometry and Hoechst dyeing were used to evaluate the SK-OV-3 cell proliferation, apoptosis and cell cycle respectively. The mRNA and protein expression of PXR and its downstream CYP450 enzymes, transporters and Bcl-2 families were measured by qRT-PCR and Western blot. Rhodamine 123 efflux experiment was conducted to detect the P-gp efflux ability. PXR plasmid and PXR siRNA were transiently transfected into SK-OV-3 cells respectively to establish PXR-overexpressed or PXR-interfered cells. In vivo, xenograft tumor mice model was established by SK-OV-3 cells to estimate the antitumor effect of Xiaoaiping injection combined with paclitaxel. The expressions of PXR and its downstream molecules in tumor tissues were determined to further clarify the potential mechanism. RESULTS: Xiaoaiping injection significantly enhanced the anti-proliferation, pro-apoptosis effect of paclitaxel on SK-OV-3 cells. The synergetic effect was displayed by Xiaoaiping injection inhibiting paclitaxel-induced PXR and CAR expression, which subsequently inhibited CYP450 enzymes CYP2C8 and CYP3A4, transporter P-gp and anti-apoptotic proteins Bcl-2 and Bcl-xl in SK-OV-3 cells. In PXR-overexpressed cells, Xiaoaiping injection down-regulated the expression of PXR and its downstream molecules. The result of xenograft tumor model showed that Xiaoaiping injection combined with paclitaxel enhanced anti-tumor effect on ovarian cancer in vivo. CONCLUSIONS: Xiaoaiping injection enhances anti-tumor effect of paclitaxel by inhibiting cell proliferation, inducing apoptosis process. The mechanism may be associated with Xiaoaiping injection inhibiting PXR and its downstream metabolic enzymes CYP2C8, CYP3A4, transporter P-gp and anti-apoptosis protein Bcl-2.

Rapid EST isolation from chromosome 1R of rye.

BACKGROUND: To obtain important expressed sequence tags (ESTs) located on specific chromosomes is currently difficult. Construction of single-chromosome EST library could be an efficient strategy to isolate important ESTs located on specific chromosomes. In this research we developed a method to rapidly isolate ESTs from chromosome 1R of rye by combining the techniques of chromosome microdissection with hybrid specific amplification (HSA). RESULTS: Chromosome 1R was isolated by a glass needle and digested with proteinase K (PK). The DNA of chromosome 1R was amplified by two rounds of PCR using a degenerated oligonucleotide 6-MW sequence with a Sau3AI digestion site as the primer. The PCR product was digested with Sau3AI and linked with adaptor HSA1, then hybridized with the Sau3AI digested cDNA with adaptor HSA2 of rye leaves with and without salicylic acid (SA) treatment, respectively. The hybridized DNA fragments were recovered by the HSA method and cloned into pMD18-T vector. The cloned inserts were released by PCR using the partial sequences in HSA1 and HSA2 as the primers and then sequenced. Of the 94 ESTs obtained and analyzed, 6 were known sequences located on rye chromosome 1R or on homologous group 1 chromosomes of wheat; all of them were highly homologous with ESTs of wheat, barley and/or other plants in Gramineae, some of which were induced by abiotic or biotic stresses. Isolated in this research were 22 ESTs with unknown functions, probably representing some new genes on rye chromosome 1R. CONCLUSION: We developed a new method to rapidly clone chromosome-specific ESTs from chromosome 1R of rye. The information reported here should be useful for cloning and investigating the new genes found on chromosome 1R.

[Genome constitution of Agropyron elongatum 4x by biochemical and SSR markers].

We analyzed the electrophoritic patterns of 16 kinds of isozyme and three kinds of storage protein in Agropyron elongatum 2x and Ag. elongatum 4x by isoelectric focusing (IEF) and SDS-PAGE, respectively. The results showed three types of electrophoritic patterns. In the first type, the zymogram phenotypes are identical between Ag. elongatum 2x and Ag. elongatum 4x. Two kinds of isozyme belong to this type, which accounts for 10.5% of all the biochemical markers analyzed. In the second type, Ag. elongatum 4x showed a phenotype which is made up of combinations of the all of Ag. elongatum 2x bands and the specific bands. Ten kinds of isozyme and three kinds of storage protein fall into this class, which is the most part and accounts for 63.2% of all the markers analyzed. In the third type, Ag. elongatum 2x and Ag. elongatum 4x showed some identical bands and their own specific bands. Five kinds of isozyme were classified to this group,which accounts for 26.3% of all the markers analyzed. The results above suggested that Ag. elongatum 4x is a allotetraploid composed of one genome originated from Ag. elongatum 2x and another unknown genome which is apparently distinct from the St, J and N genomes of relative species. Otherwise, SSR primers were used to analyze the relation of Ag. elongatum 2x and Ag. elongatum 4x. The amplification results of most primers showed that Ag. elongatum 2x and Ag. elongatum 4x have some identical bands and Ag. elongatum 4x have some specific bands. This result validated the conclusion from biochemical marker analyses that Ag. elongatum 4x is a allotetraploid.

[Genetic analysis and molecular markers of a novel stripe rust resistance gene YrHua in wheat originated from Psathyrostachys huashanica Keng].

The H9020-17-5,a common wheat-Psathyrostachys huashanica Keng translocation line, possesses excellent resistance to wheat stripe rust. Genetic analysis of F2 and BC1 populations derived from H9020-17-5 x Mingxian169 indicated that resistance to stripe rust in H9020-17-5 was a dominant character controlling by single gene originated from Ps. huashanica. This resistance gene originated from Ps. huashanica was first reported in the present study and named as YrHua. In order to map the resistance gene YrHua, AFLP approach was employed to analyze the 119 individuals of H9020-17-5 x Mingxian169 F2 population which were inoculated by stripe rust isolate CY30. As a result,two markers, PM14(301) and PM42(249) were found to be linked to the resistance gene YrHua,and the genetic distances between the markers and target gene were 5.4 cM and 2.7 cM, respectively. For the convenience of marker-assisted selection in wheat breeding, one of the two AFLP markers was converted to PCR marker using a pair of special primers based on the DNA sequence of PM14 (301) and the polymorphism of restriction site. Our research results provided a useful tool for marker-assisted selection and laid a foundation of fine mapping and map based cloning of YrHua gene.

[Efficient production of wheat alien translocation lines and characterization by molecular cytogenetics].

The Triticum aestivum-Leymus mollis and T. aestivum-Thinopyrum intermedium translocation lines were induced by gametocidal chromosome 3C derived from Aegilops triuncialis and gamma-ray irradiated pollens of a T. aestivum-Th. intermedium addition line TAI-14 with a lower dosage (10Gy), respectively. By genomic in situ hybridization (GISH) analysis, three T. aestivum-L. mollis translocation lines (WM-10, WM-43 and WM-47) and three deletion lines (WM-18, WM-43 and WM-44) were selected from 59 F2 plants derived from a cross combination of T. aestivum-L. mollis substitution line M8724-8-13 x T. aestivum-Ae. triunicalis 3C chromosome addition line. The frequency of translocation lines produced and total frequency of chromosome structural variation occurred were 5.08% and 8.47%, respectively. Two of the three translocation lines, WM-10 and WM-43 all were heterozygous translocation lines carrying one T. aestivum-L. mollis Robertsonian translocation chromosome, but the translocation chromosome in the two lines were different in morphology. The other one, WM-47 was a double heterozygous translocation with two different translocation chromosomes. By the C-banding, one of the three translocation lines was identified, the translocation chromosome consisted of 7DL of wheat and a chromosome arm of L. mollis. In addition, wheat chromosome deletions were observed in some plants. In another cross combination involved in common wheat and T. aestivum-Th. intermedium addition line, two alien terminal non-Robertsonian translocation lines (WI-21 and WI-68) were identified from 69 F2 plants by C-banding and GISH, and the percentage of translocation line was 2.90%. By C-banding analysis, the translocation chromosomes involved in wheat chromosomes 3A and 4A in the two lines, respectively. These results indicate that inducing wheat alien translocation line by gametocidal chromosome and irradiated pollens all are efficient methods.

[Preliminary gene-mapping of photoperiod-temperature sensitive genic male sterility in wheat (Triticum aestivum L.)].

The photoperiod-temperature sensitive genic male sterile (PTSGMS) line in wheat is important for the utilization of heterosis. The wheat line, BAU3338, is an excellent PTSGMS material identified in the recent years. In this study, its PTSGMS genes were mapped using molecular markers, SSR and ISSR. The result of molecular analysis showed that the two PTSGMS loci were identified and designated as ptms1 and ptms2, respectively. In addition, the genetic effect analysis indicated that the locus effect of ptms1 was 2-3 times larger than that of ptms2.

Cloning, sequence and expression analysis of a zinc finger protein gene in wheat.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed on total RNA of resistant-line TAM104R and susceptible-line TAM104S using 33 primers designed according to conservative domain of plant R-genes and the sequence of barley powdery mildew resistance genes Mlo, Mla1, Mla6 and wheat leaf rust resistance gene Lrk10. A polymorphic cDNA fragment TaZF was cloned and sequenced. The Open Reading Frame (ORF) of TaZF is comprised of 822 base pairs which encodes a zinc finger-like DNA or RNA-binding protein with 273 amino acids and molecular weight of 31 kD. TaZF is more than one copy gene in TAM104R genome based on southern blot. It is constitutively expressed, but level of expression was enhanced in tissue infected by Erysiphe Graminis. There is no intron in TaZF genome DNA.

[Evolution of partial promoter region of HMW glutenin genes from E and E1 genome of Agropyron elongatum].

The partial promoter regions of HMW glutenin subunit genes were cloned form the genomes E (in diploid Agropyron elongatum) and E1 (in tetraploid Agropyron elongatum) by PCR approach. There was only one nucleotide acid difference in the promoter sequences of x-type subunits between the two genomes; moreover, the promoter sequences of the two y-type subunits were completely identical. Although these promoter regions were very similar to each other, differences still existed in sequence size and the kind of nucleotide acid between the x-type and y-type subunits. It was speculated that the E1 genome in tetraploid Agropyron elongatum was probably originated from E genome in diploid species. The comparisons of these subunits with some of those from A, B, D and G genome of Triticeae demonstrated that the sequences of their partial promoter regions were conserved and shared a high homology more than 90%. The phylogenetic analysis based on the sequences in this region indicated that the y-type HMW glutenin subunits of Agropyron elongatum species were different from other subunits, whereas the x-type subunits of them were most closely related to that from the B genome.

Genome-wide analysis of the MADS-box gene family in Brachypodium distachyon.

MADS-box genes are important transcription factors for plant development, especially floral organogenesis. Brachypodium distachyon is a model for biofuel plants and temperate grasses such as wheat and barley, but a comprehensive analysis of MADS-box family proteins in Brachypodium is still missing. We report here a genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. We identified 57 MADS-box genes and classified them into 32 MIKC(c)-type, 7 MIKC*-type, 9 Mα, 7 Mß and 2 Mγ MADS-box genes according to their phylogenetic relationships to the Arabidopsis and rice MADS-box genes. Detailed gene structure and motif distribution were then studied. Investigation of their chromosomal localizations revealed that Brachypodium MADS-box genes distributed evenly across five chromosomes. In addition, five pairs of type II MADS-box genes were found on synteny blocks derived from whole genome duplication blocks. We then performed a systematic expression analysis of Brachypodium MADS-box genes in various tissues, particular floral organs. Further detection under salt, drought, and low-temperature conditions showed that some MADS-box genes may also be involved in abiotic stress responses, including type I genes. Comparative studies of MADS-box genes among Brachypodium, rice and Arabidopsis showed that Brachypodium had fewer gene duplication events. Taken together, this work provides useful data for further functional studies of MADS-box genes in Brachypodium distachyon.

A new strategy to produce a defensin: stable production of mutated NP-1 in nitrate reductase-deficient Chlorella ellipsoidea.

Defensins are small cationic peptides that could be used as the potential substitute for antibiotics. However, there is no efficient method for producing defensins. In this study, we developed a new strategy to produce defensin in nitrate reductase (NR)-deficient C. ellipsoidea (nrm-4). We constructed a plant expression vector carrying mutated NP-1 gene (mNP-1), a mature α-defensin NP-1 gene from rabbit with an additional initiator codon in the 5'-terminus, in which the selection markers were NptII and NR genes. We transferred mNP-1 into nrm-4 using electroporation and obtained many transgenic lines with high efficiency under selection chemicals G418 and NaNO(3). The mNP-1 was characterized using N-terminal sequencing after being isolated from transgenic lines. Excitingly, mNP-1 was produced at high levels (approximately 11.42 mg/l) even after 15 generations of continuous fermentation. In addition, mNP-1 had strong activity against Escherichia coli at 5 µg/ml. This research developed a new method for producing defensins using genetic engineering.