Genome structure and variation of Reynoutria japonica Houtt. chloroplast genome / 生物工程学报

Reynoutria japonica Houtt., belonging to Polygoneae of Polygonaceae, is a Chinese medicinal herb with the functions of draining dampness and relieving jaundice, clearing heat and detoxifying, dispersing blood stasis and relieving pain, and relieving cough and resolving phlegm. In this study, we carried out high-throughput sequencing for the chloroplast genome sequences of five cultivars of R. japonica and analyzed the genome structure and variations. The chloroplast genomes of the five R. japonica cultivars had two sizes (163 376 bp and 163 371 bp) and a typical circular tetrad structure composed of a large single-copy (LSC) region of 85 784 bp, a small single-copy (SSC) region of 18 616 bp, and a pair of inverted repeat (IR) regions (IRa/IRb) which are spaced apart. A total of 161 genes were obtained by annotation, which consisted of 106 protein-coding genes, 10 rRNA-coding genes, and 45 tRNA-coding genes. The total GC content was 36.7%. Specifically, the GC content in the LSC, SSC, and IR regions were 34.8%, 30.7%, and 42.7%, respectively. Comparison of the whole chloroplast genome among the five cultivars showed that trnk-UUU, rpoC1, petD, rpl16, ndhA, and rpl12 in coding regions had sequence variations. In the phylogenetic tree constructed for the 11 samples of Polygoneae, the five cultivars of R. japonica clustered into one clade near the root and was a sister group of Fallopia multiflora (Thunb.).

Microbial synthesis of salidroside / 生物工程学报

Salidroside, as one of the main active ingredients of Rhodiala plant, has the effects of anti-hypoxia, anti-radiation, anti-fatigue, anti-tumor, hypoglycemia and improving immunity. With the increasing demand for salidroside and the decreasing of plant resources, microbial production of salidroside has attracted much attention due to its advantages of short period and easy controlling. At present, microbial production of salidroside is still at the basic research stage. In order to make it easier for researchers to understand the advances of microbial synthesis of salidroside, the biosynthesis pathways, uridine diphosphate glucosyltransferases, wild strain/natural enzymes and engineered strain/recombinant enzymes were reviewed.

Research advances in the role and mechanism of peroxisome proliferator-activated receptor alpha in liver diseases / 临床肝胆病杂志

Peroxisome proliferator-activated receptor alpha (PPARα) is a member of the nuclear receptor superfamily and plays a central regulatory role in lipid metabolism. Recent studies have found that PPARα agonists play a key role in the prevention and treatment of nonalcoholic fatty liver disease, which has also been reported in other liver diseases. This article reviews the role and mechanism of PPARα in nonalcoholic fatty liver disease, alcoholic liver disease, viral hepatitis, cholestatic liver disease, drug-induced liver injury, and hepatocellular carcinoma, so as to provide a reference for the research on the new application of conventional drugs associated with PPARα.

Experimental study on bacteriostatic effect of recombinant human lactoferrin on Helicobacter pylori / 重庆医学

Objective To evaluate the bacteriostatic effect of recombinant human lactoferrin(rhLF) on Helicobacter(H .) py‐lori and its influence on CagA ,Ure and gastric mucosal IL‐8 .Methods The minimum inhibitory concentration(MIC)and the influ‐ence of different drug concentrations on the proliferation of H .pylori were detected .The effects of rhLF on the mRNA and protein expressions of CagA and Ure in H .pylori were detected by RT‐PCR and Western blot ,respectively .The animal study :Balb/c mice were adopted and assigned randomly into four groups ,including the standard triple+rhLF(group A) ,rhLF(group B) ,standard tri‐ple(group C) and normal saline(group D) .The histopathological HE staining was used to observe the gastric inflammation and ELISA was used to detect the IL‐8 level of gastric tissue in each group .Results MIC was 0 .5 mg/mL ,moreover rhLF inhibited the bacterial growth and proliferation with a concentration‐dependent manner .rhLF could reduce the expression of H .pylori major viru‐lence factor CagA ,mRNA and protein of Ure .Comparing the group A with the group B ,C and D ,the gastric mucosal inflammation score and the IL‐8 levels of gastric tissue homogenates had statistically significant differences(P<0 .05) .Conclusion rhLF inhibits the growth and proliferation of H .pylori ,moreover inhibit the expression of major virulence factor CagA in H .pylori ,mRNA and protein of Ure in different degrees ,weakens its pathogenicity ,meanwhile reduces the IL‐8 level in mice gastric mucosa ,and allevi‐ates H .pylori related gastric mucosal inflammatory response .

Preparation and crystallization of Polygonum cuspidatum benzalacetone synthase / 生物工程学报

The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation.

Apoptosis effect of Siraitia grosvenorii extracts on lung cancer cells A549 and its mechanisms / 中国药理学通报

Aim To study the apoptosis effect of Sir-aitia grosvenorii extract on human lung cancer cells A549 and its mechanisms.Methods MTT assay was applied to determine A549 cell proliferation.Hoechst 33258 staining was applied to investigate morphological changes in A549 cells.To find out the cause of cell growth inhibition,several experiments on cell cycle distribution and apoptosis were performed by flow cy-tometry analysis.The expression of p21 and Bcl-2 was determined by Western blot.Results Flow cytometry analysis showed that treatment with mogrol arrested A549 cells in the G0 /G1 phase and induced apoptosis. After treatment with Siraitia grosvenorii extract,West-ern blot experiment showed cell cycle regulator p21 was up-regulaed,while the apoptosis inhibitor Bcl-2 was down-regulated.Conclusion Treatment with Siraitia grosvenorii extract arrests the A549 cells at G0 /G1 phase and induces apoptosis that may contribute to the anti-proliferation activity of mogrol through the regula-tion of p21 and Bcl-2 expression.

A stereological analysis of ginsenoside Rg1 in inhibiting hepatic fibrosis / 重庆医学

Objective To investigate the effect of Panax notoginsenosides monomers ginsenoside Rg 1 in inhibiting hepatic fibro-sis .Methods The rat model of hepatic fibrosis was established by using 50% Ccl4 ,total 35 d .The different doses of Rg1was ad-ministered by hypodermical injection .At the end of the treatment ,the pathological changes of hepatic tissue were observed by light and transmission electron microscope .The stereological method was adopted to measure the volume density (Vvm) ,area density (Svm) ,specific surface(Qm) and surface number density (Nam) of liver cell mitochondria in various groups .Results The stereo-logical data of liver cell mitochondria showed that the statistical differences existed among various groups .Vvm in the Panax Notog-insenosides ,low dose Rg1 and isotonic saline groups were significantly increased compared with the normal control group with sta-tistical difference(P0 .05);Vvm in the high ,middle and low dose Rg1 ,Panax No-toginsenosides and colchicine groups showed the decreasing trend compared with the isotonic saline group without statistical differ-ence(P>0 .05) .Svm in the low dose Rg1 ,Panax Notoginsenosides ,colchicine and isotonic saline groups were significantly increased compared with the normal group with statistical difference (P0 .05) .Conclu-sion Rg1 has antifibrosis effects of Panax notoginsenosides ,even exceeds Panax notoginsenosides in some aspects ,and the above-mentioned effect is positively correlated with dose .Rg1 is an ideal drug for preventing and treating liver fibrosis .

Comparison of stilbene synthase from different plant sources for resveratrol biosynthesis / 生物工程学报

Resveratrol is a natural phytoalexin with special pharmacological and health functions. Stilbene synthase (STS) is a key and rate-limiting enzyme in the biosynthesis of resveratrol that is present only in a limited number of plants. The content of resveratrol from Polygonum cuspidatum is more than 1000 times higher than grapes and peanuts. We speculate that the catalytic ability of different STS may be one of the reasons causing differences in the content of resveratrol. To verify the above speculation, Vitis vinifera stilbene synthase gene (VvSTS) was amplified according to overlap PCR protocol with genomic DNA as template. VvSTS and PcSTS (PcPKS5) were analyzed through heterologous expression in Escherichia coli. The expression products were purified with Ni-NTA sepharose affinity chromatography and desalted through PD-10 column. The molecular weight of the two fusion proteins was about 43 kDa. Enzyme reaction and product analysis showed that the two products were resveratrol. The enzyme kinetic analysis showed that the catalyze efficiency (Kcat/Km) of PcPKS5 was 2.4 times of the VvSTS. Our findings confirms that STS from certain plants has much higher catalytic capability.

Crystal structures of plant uridine diphosphate-dependent glycosyltransferases / 生物工程学报

Glycosyltransferases (GTs) catalyze the transfer of a sugar residue of an activated sugar donor to an acceptor molecule. Many families 1 GTs utilize an uridine diphosphate (UDP) activated sugar as donor in the glycosylation reaction, and most of these belong to a group of GTs referred to as the UGTs. The relationship between the degree of amino acid sequence identity and substrate specificity of the plant UGTs is highly complicated, and the prediction of substrate specificity based on phylogenetic analyses need to be improved by more biochemical characterization. This review summarizes the three dimensional structures of plant UGTs published in the Protein Data Bank (PDB), including the detailed substrate interactions with the sugar and receptor binding pockets and mutational analyses of some critical amino acids. It will be helpful for biochemical characterization the substrate specificity of the individual UGT, and lay the foundation for the enzymatic and genetic manipulation of plant UGTs in the future.

Plant-specific type III polyketide synthases superfamily: crystal structures and functions / 生物工程学报

Plant type III polyketide synthase (PKS) generates backbones of a variety of plant secondary metabolites with diverse functions, and has long been models to elucidate the relationship between the three-dimensional structure and function. More than 80 type IIII PKS crystal structures with different functions have been reported in Protein Data Bank, including the crystal structures of the well-studied Chalcone Synthase of plant type III PKS, as well as the 6 other kinds of PKSs in the family, which are critical for understanding the structural basis for diverse starter molecule selectivity, polyketide chain length and the cyclization reaction. Structure-based analysis and site-directed mutagenesis are foundation for the investigation of enzyme engineering, genetic and metabolic engineering. This review summarized 7 plant-specific type III PKS in the aspects of their crystal structures and functions.

Salidroside biosynthesis pathway: the initial reaction and glycosylation of tyrosol / 生物工程学报

Salidroside, the 8-O-beta-D-glucoside of tyrosol, is a novel adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor. Due to the scarcity of R. sachalinensis and its low yield of salidroside, there is great interest in enhancing the production of salidroside by biotechnological process. Glucosylation of tyrosol is thought to be the final step in salidroside biosynthesis. In our related works, three UGT clones were isolated from the roots and the cultured cells. Our intention was to combine the catalytic specificity of these UGTs in vitro in order to change the level of salidroside in vivo by over-expression of the above UGTs. However, as the aglycone substrate of salidroside, the biosynthetic pathway of tyrosol and its regulation are less well understood. The results of related studies revealed that there are two different possibilities for the tyrosol biosynthetic pathway. One possibility is that tyrosol is produced from a p-coumaric acid precursor, which is derived mainly from phenylalanine. The second possibility is that the precursor of tyrosol might be tyramine, which is synthesized from tyrosine. Our previous work demonstrated that over-expression of the endogenous phenylalanine ammonia-lyase gene (PALrs1) and accumulation of p-coumaric acid did not facilitate tyrosol biosynthesis. In contrast, the data presented in our recent work provide in vitro and in vivo evidence that the tyrosine decarboxylase (RsTyrDC) is most likely to have an important function in the initial reaction of the salidroside biosynthesis pathway in R. Sachalinensis.

Plant-specific type III polyketide synthase superfamily: gene structure, function and metabolites / 生物工程学报

Plant-specific type III polyketide synthase (PKS) produces a variety of plant secondary metabolites with notable structural diversity and biological activity. So far 14 plant-specific type III PKS have been identified according to their enzymatic products, and the corresponding genes have been cloned and characterized. The differences among the various PKS are mainly in their substrate specificities, the number of their condensation reactions, and the type of ring closure of their products. However, numerous studies have revealed the common features among the plant-specific type III PKS, which include sequence homology, similar gene structure, conserved amino acid residues in the reaction center, enzymatic characteristics and reaction mechanism. We briefly reviewed 14 plant-specific type III PKS to better understand genetic and metabolic engineering of plant-specific type III PKS.

Abscisic acid inhibits proliferation of human hepatocarcinoma cells / 基础医学与临床

Objective To investigate the mechanism of Depression proliferation in human hepatocarcinoma cells by Abscisic acid.Methods To detect protein expression o of P53,Ki-67 and Cyclin D1 by immunocyte chemistry; detect mRNA expression of P53 and telomerase by RT-PCR. Results The protein expression level of mtP53, Cyclin D1, Ki-67 and the mRNA expression level of mtP53 and hTERT all decreased in cells treated by ABA,HMBA and ABA+ HMBA(P