Monoterpene-flavonoid conjugates from Sarcandra glabra and their autophagy modulating activities.

Fourteen new monoterpene-flavonoid conjugates including four monoterpene-conjugated chalcones (glabratins A-D, 1-4), seven monoterpene-conjugated dihydrochalcones (glabratins E-K, 5-11), and three monoterpene-conjugated flavanones (glabratins L-N, 12-14), together with four known analogues (15-18) were isolated from the aerial parts of Sarcandra glabra. The structures and the absolute configurations of these compounds were elucidated by the spectroscopic data, single-crystal X-ray diffraction, and electronic circular dichroism (ECD) calculations. Compounds 1, 4-6, 9-14, and 18 showed obvious cell autophagy-inducing activities at 25 µM in HEK293 cells. Furthermore, the bioassay results also showed that 18 induced cell autophagy in a dose dependent manner. Our findings revealed a rare class of monoterpene-flavonoid conjugates in nature and firstly reported their autophagy-inducing activities.

Cloning, characterization and expression analysis of metallothioneins from Ipomoea aquatica and their cultivar-dependent roles in Cd accumulation and detoxification.

To explore the possible roles of metallothioneins (MTs) played in cadmium (Cd) accumulation of water spinach, three IaMT genes, IaMT1, IaMT2 and IaMT3 in a high-shoot-Cd (T308) and a low-shoot-Cd accumulation cultivar (QLQ) were cloned, characterized, and quantitated. Gene expression analysis suggested that the expression of the IaMTs was differentially regulated by Cd stress in different cultivars, and T308 showed higher MTs expression overall. Furthermore, only shoot IaMT3 expression was cultivar dependent among the three IaMTs. Antioxidant analysis showed that the high production of IaMTs in T308 should be associated with its high oxidation resistance. The role of IaMTs in protecting against Cd toxicity was demonstrated in vitro via recombinant E. coli strains. The results showed that IaMT1 correlated with neither Cd tolerance nor Cd accumulation of E. coli, while IaMT2 conferred Cd tolerance in E. coli, IaMT2 and IaMT3 increased Cd accumulation in E. coli. These findings help to clarify the roles of IaMTs in Cd accumulation, and increase our understanding of the cultivar-dependent Cd accumulation in water spinach.

Enantioselective Borylation of Aromatic C-H Bonds with Chiral Dinitrogen Ligands.

The borylation of C-H bonds catalyzed by transition metals has been investigated extensively in the past two decades, but no iridium-catalyzed enantioselective borylation of C-H bonds has been reported. We report a set of iridium-catalyzed enantioselective borylations of aromatic C-H bonds. This reaction relies on a set of newly developed chiral quinolyl oxazoline ligands. This process proceeds under mild conditions with good to excellent enantioselectivity, and the borylated products can be converted to enantioenriched derivatives containing new C-O, C-C, C-Cl, or C-Br bonds.

A Chiral Nitrogen Ligand for Enantioselective, Iridium-Catalyzed Silylation of Aromatic C-H Bonds.

Iridium catalysts containing dative nitrogen ligands are highly active for the borylation and silylation of C-H bonds, but chiral analogs of these catalysts for enantioselective silylation reactions have not been developed. We report a new chiral pyridinyloxazoline ligand for enantioselective, intramolecular silylation of symmetrical diarylmethoxy diethylsilanes. Regioselective and enantioselective silylation of unsymmetrical substrates was also achieved in the presence of this newly developed system. Preliminary mechanistic studies imply that C-H bond cleavage is irreversible, but not the rate-determining step.

Effects of different reduced sulfur forms as electron donors in the start-up process of short-cut sulfur autotrophic denitrification.

In this study, two short-cut sulfur autotrophic denitrification (SSADN) reactors were initiated using different reduced sulfur forms as electron donors and their effects on the start-up speed of the SSADN process, NO2--N accumulation characteristics, and microbial community were investigated. Results revealed that during the same period, due to the relatively slow S0 dissolution rate, the NO2--N production rate realized by microorganisms in S0-SSADN (NO2--N production rate (NPR), 174 mg/(L·d)) was significantly slower than S2--SSADN (NPR, 679 mg/(L·d)). The NO2--N accumulation efficiency (NAE) was maintained > 80%, which was significantly higher than S2--SSADN. In the SSADN system using different reduced sulfur forms, the microbial community structure and abundance considerably differed. The main sulfur-oxidizing bacteria (SOB) in S0-SSADN were Sulfurimonas (6.5%) and Thiobacillus (5.3%). The main SOB species in S2--SSADN was Thiomonas (13.6%). Thermomonas played an important role in the two reactors as an important NO3--N denitrifying bacteria species.

Single nucleotide polymorphism of rs430397 in the fifth intron of GRP78 gene and clinical relevance of primary hepatocellular carcinoma in Han Chinese: risk and prognosis.

Large number of data showed that allele variants in certain genes are markers for hepatocellular carcinoma (HCC). GRP78 is a stress-associated protein which is a central regulator of endoplasmic reticulum homeostasis due to its multiple functional roles in the folding, maturation and transport of proteins. A case-control study was conducted on 576 HCC patients, and 539 age- and gender-matched healthy subjects to examine whether rs430397 polymorphism in the fifth intron of GRP78 gene is associated with the development and prognosis of HCC. Polymorphism in rs430397 was analyzed by resequencing and TaqMan real-time PCR. Allele A, genotype AA and combined genotypes (AG+AA) displayed significantly increased risk for HCC (OR = 1.48, 95%CI = 1.07-1.79, p = 0.010; OR = 2.25, 95%CI = 1.08-3.38, p = 0.019; and OR = 1.50, 95%CI = 1.09-1.85, p = 0.012, respectively). Genotypes AA and AG were mainly associated with HBV-related HCC (85.8%; p < 0.00001 versus HBV noncarriers with HCC) and cirrhosis-related HCC (90%; p = 0.011 versus noncirrhosis HCC). Patients carrying the AA genotype had a shorter survival time (median 23.0 months in all cases; median 21.0 months in the cases carrying HBsAg). Like HBV and cirrhosis, the rs430397 is an independent prognostic factor influencing the survival of HCC. In conclusion, allele A and genotypes AA and AG of rs430397 may represent high risk and poor prognosis for HCC.

Improvement of enzyme activity and soluble expression of an alkaline protease isolated from oil-polluted mud flat metagenome by random mutagenesis.

A new protease gene (pro1437)was separated from an oil-polluted Mud flat metagenomic library. Pro1437 belongs to a peptidase M48 superfamily according to the results of sequence analysis, and it showed very low identities compared to other known proteases or peptidases. The error-prone PCR was used to introduce random mutations and improve the expression of pro1437. After two rounds of mutagenesis and screening, a mutant (Pro2T21) with a 6.6-fold higher activity and a 4.8-fold higher expression level than Pro1437 was obtained. Sequence analysis found three amino acid substitutions (A54V, L192H, F224L) in Pro2T21. 3D structure modelling analysis indicated A54V and L192H probably played a crucial role in the improvement of enzymatic activity and soluble expression level of Pro2T21. Furthermore, Pro2T21opti displayed a 5.8-fold higher expression level than the wild type under optimal pH 8.0 at 50°C after codon-optimization. Also, Pro2T21opti represented robust compatibility with several popular laundry detergents, and blood stains on white cloth pieces were completely washed away when endogenous protease-inactivated Tide and Pro2T21opti were used together. Therefore, Pro2T21opti has great potential for use as an additive in detergents after further study.

[Development of transgenic oilseed plants resistant to glyphosate and insects].

In this study, the aroA-M12 gene encoding bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and the Btslm recombinant gene encoding Bacillus thuringiensis (Bt) toxin gene were introduced into a Brassica napus variety, Xiangyou No. 15, via Agrobacterium-mediated transformation using glyphosate as a selectable agent. PCR amplification and Southern blot analyses of T0 transgenic plants showed that the alien genes were transferred and integrated stably into the genome of Xiangyou No. 15. Western blot further confirmed that the alien genes were expressed in these plants. Transgenic plants with tolerance to both glyphosate and the tested pest were demonstrated by bioassays. Our result also clearly shows that the aroA-M12 gene can be used as an efficient selectable marker gene instead of antibiotic resistant markers in plant transformation.

[Inducible expression of a promoter of the gene encoding barley beta-1, 3-glucanase isoenzyme GIII in transgenic rice].

A promoter of the gene encoding beta-1, 3-glucanase isoenzyme GIII was amplified from barley genomic DNA using PCR. The GIII gene promoter, designated P(GIII), was ligated upstream of the gus report gene and pGIII-gus fusion fragment was then cloned into a binary vector pCAMBIA1300 for Agrobacterium-mediated transformation of rice (Oryza sativa L. cv. Taipei 309). PCR analyses indicated that the fusion gene was present in all T0 transgenic plants. The integration of the gene into rice genomic DNA was further confirmed by Southern blot. Histochemical staining and spectrofluorophotometric analyses of transgenic rice leaves showed that the GUS activity was increased after treatment with SA and fungal elicitor. Northern blot analysis also showed expression of such induction. Histochemical staining of T(1) rice seeds displayed marked GUS activity after induction with SA and elicitor, while no GUS activity was detected in untreated T(1) rice seeds. The results presented here strongly suggested that P(GIII) could be pathogen-inducible promoter.

[Glyphosate-resistant cotton (Gossypium hirsutum L.) Transformed with aroAM12 gene via Agrobacterium tumefaciens].

A mutant, aroAM12, exhibiting resistance to glyphosate produced in a previous study using the staggered extension process with aroA genes from Salmonella typhimurium and Eschrichia coli. In this paper, we constructed a vector pGRA1300 carrying aroAM12 gene, comprising transit peptide of Arabidopsis EPSPS, under the control of the CaMV35S promoter and used as selectable marker for cotton plant (Gossypium hirsutum L.) transformation. Transgenic cottons with increased resistance to glyphosate were obtained by cotransformtion of hypocotyl segments with Agrobacterium tumefaciens and selected directly on medium containing glyphosate. Regeneration of glyphosate-resistant calli was carried out on a MS basic medium containing 2,4-D 0.1 mg/L, KT 0.1 mg/L, cefotaxime 500 mg/L and glyphosate 60 micromol/L. Globular embryos were induced and then developed by culturing on MSB (MS salts+B(5) vitamins) medium supplemented with asparagine 1 g/L and glutamine 2 g/L, but not containing hormone, for 40 d. The developed plantlets were then removed and cultured on an MS medium. After about 20 d, the deeply-rooted shoots were in soil. PCR analysis showed that the aroAM12 gene was present in all T(0) transgenic plants. The integration of the aroAM12 gene in the genomic DNA of cotton was further confirmed by Southern blot, which showed that the transgenic plants carried one or two copies of the aroAM12 genes. Western blot analysis showed that a 48-kD band of was detected in all T(0) transgenic plants. There was no apparent corelation between copy numbers and the expression level of the aroAM12 gene. Greenhouse screening for glyphosate resistance was performed to test 65 independent T(0) plants by spraying (three times) with an aqueous suspension at a dose corresponding to 9.317 kg/ha of Roundup (once every 5 d). After 15 d, phenotype examination was carried out of the plants in comparison with untransformed control plants. Under these conditions, it was observed that the plants transformed with pGRA1300 showed high resistance to glyphosate whereas the control plants were all killed. The glyphosate resistance of T(1) generation was measured by spraying with Roundup, the numbers of glyphosate resistance and sensitive phenotypes showed Mendelian segregation ratio.

[hNUDC promotes proliferation and differentiation of megakaryocytopoiesis on human CD341+ cells].

AIM: aTo study the effect of human nuclear distribution C èhNUDCé on human megakaryocyte proliferation and differentiation from cord blood CD34(+) cells in vitro. METHODS: aHuman CD34(+) cells were isolated using the Dynal CD34 Progenitor Cell Selection System from umbilical cord blood. The CD34(+) cells were then cultured in serum free methylcellulose semi-solid media, the morphologic aspects and number of small, medium and large CFU-MK colonies were observed and scored on the day12 by microscopy analysis. The CD34(+) cells were cultured in serum free liquid media, cells were removed on day 10 and formation of CD41(+) in human megakaryocyte and its DNA polyploidization of nuclear were analyzed on a FACsort flowcytometer. RESULTS: ahNUDC supported the formation of small and medium CFU-MK colony in serum free semi-solid media. Furthermore, hNUDC induced a remarkable increase in expression of the megakaryocyte cell surface marker CD41(+) and stimulated the CD41(+) DNA polyploidization more effectively than TPO. CONCLUSION: hNUDC may play an important role in megakaryocyte proliferation and differentiation.

[Construction of a vector conferring herbicide and pest resistance in tobacco plant].

A binary plant expression vector, pCM12-slm, carrying the aroAM12 mutant gene encoding bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and the Bts1m recombinant gene consisting of 331 N-terminal amino acids of CryIAc and 284 C-terminal amino acids of CryIAb has been constructed. The truncated Bts1 gene was fused with the PR1b signal peptide sequence and expressed in tobacco plants under the control of 2E-CaMV35S promoter and the omega (omega) translation enhancer sequence from tobacco mosaic virus. The mutant aroAM12 was fused with the transit sequence of tobacco EPSPS and expressed in tobacco plants under the control of the CaMV35S promoter. Tobacco leaves were transformed with Agrobacterium tumefaciens LBA4404 harboring the pCM12-slm plasmid, and the transgenic plants were selected directly on medium containing the herbicide. Forty glyphosate resistant plants were regenerated, with a transformation frequency of 27%. Transgenic plants were initially assessed for glyphosate resistance by placing leaf discs on shoot induction media containing the herbicide. Rooted plantlets, propagated from selected transgenic tobacco, were transferred to soil in a greenhouse and tested for glyphosate resistance by spraying them with Roundup at a commercial recommended dose. The glyphosate resistance assay indicated that all the transgenic plants showed highly resistant to the herbicide. The PCR assay showed that the aroAM12 gene was present in all of the 40 T0 transfer plants, and Bts1m genes present in 28 of 40 of the transgenic plants. Southern blot analysis further confirmed that the copy number of the transgenes varied from one to three copies in different transgenic plants. Northern blot and immunodot blot showed that the aroAM12 and Bts1m genes were expressed at the transcription and translation levels. Transgenic plants containing both the aroA M12 and Bts1m genes were further assessed for insect resistance. Tobacco leaves of T0 transgenic plants were infested with tobacco bollworm H. assulta larvae for 6 days. The result (table 1) showed that the survival rate of insect larvae was between 0-10%, and the growth of insect larvae was seriously inhibited, suggesting pCM12-slm as a dual functional vector with potential application in breeding of glyphosate and insect resistance transgenic plants.