To investigate the genetic diversity among wild Dipsacus asperoides in China, 66 germplasmic resources of D. asperoides were analyzed by Start Codon Targeted Polymorphism (SCoT) molecular markers. Genetic distance was calculated by TREECONW software and the systematic diagram of genetic relationship was clustered by UPGMA method. The results showed that the totals of 181 bands were detected using 20 primers , among which 109 were polymorphic bands. The average percentage of polymorphic bands was 60.13%. Genetic distance changed from 0.030 6 to 0.181 4. The clustering results showed that there was no significant correlation between the classification of the wild D. asperoides and their geographical origin. The relatively high genetic diversity of D. asperoides provides the basis for breeding new varieties.
China ; DNA Primers ; genetics ; DNA, Plant ; genetics ; Dipsacaceae ; chemistry ; classification ; genetics ; Genetic Variation ; Phylogeny ; Polymorphism, Genetic

To reveal the genetic diversity and genetic structure in Artemisia annua varieties (strains) populations, we detected the genetic polymorphism within and among eight varieties (strains) populations (192 individuals) by the approach of Start Codon Targeted Polymorphism (SCoT). The associated genetic parameters were calculated by POPGENE1.31 and the relationship was constructed based on UPGMA method. The results showed that, using 20 screened primers, a total of 145 bands were produced, of which 122 were polymorphic loci. At species level, there was a high level of genetic diversity among eight varieties (strains) populations (PPB = 84.1% ,H = 0.217 3 and H(sp) = 0.341 9). However, at the variety (strains) population level, genetic diversity was lower, the average of genetic parameters was PPB = 41.9%, H = 0.121 5, H(pop) = 0.186 8. The Nei's genetic differentiation coefficient was 0.441 0, indicate that most of the genetic variation in this species existed within the variety populations. The gene flow (N(m) = 0.633 9) was less among populations, indicating that the degree of genetic differentiation was higher. Genetic similarity coefficient were changed from 0.755 1 to 0.985 7. By clustering analysis, eight varieties (strains) were clustered into two major categories and it was also showed the same or similar genetic background varieties (strains) have a tendency to gather in the same group. Results suggest that, in variety breeding, breeders should strengthen the exchange of bred germplasm and increase mutual penetration of excellent genes, which would broaden the genetic base of A. annua.
Artemisia annua ; classification ; genetics ; Codon, Initiator ; genetics ; Genetic Markers ; genetics ; Genetic Structures ; Genetic Variation ; Genetics, Population ; methods ; Phylogeny ; Polymorphism, Genetic ; Species Specificity

Background People recently realized that it is important for artificial vascular biodegradable graft to bionically mimic the functions of the native vessel.In order to overcome the high risk of thrombosis and keep the patency in the clinical small-diameter vascular graft (SDVG) transplantation,a double-layer bionic scaffold,which can offer anticoagulation and mechanical strength simultaneously,was designed and fabricated via electrospinning technique.Methods Heparin-conjugated polycaprolactone (hPCL) and polyurethane (PU)-collagen type Ⅰ composite was used as the inner and outer layers,respectively.The porosity and the burst pressure of SDVG were evaluated.Its biocompatibility was demonstrated by the 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide (MTT) test in vitro and subcutaneous implants in vivo respectively.The grafts of diameter 2.5 mm and length 4.0 cm were implanted to replace the femoral artery in Beagle dog model.Then,angiography was performed in the Beagle dogs to investigate the patency and aneurysm of grafts at 2,4,and 8 weeks post-transplantation.After angiography,the patent grafts were explanted for histological analysis.Results The double-layer bionic SDVG meet the clinical mechanical demand.Its good biocompatibility was proven by cytotoxicity experiment (the cell's relative growth rates (RGR) of PU-collagen outer layer were 102.8％,109.2％ and 103.5％,while the RGR of hPCL inner layer were 99.0％,100.0％ and 98.0％,on days 1,3,and 5,respectively) and the subdermal implants experiment in the Beagle dog.Arteriography showed that all the implanted SDVGs were patent without any aneurismal dilatation or obvious anastomotic stenosis at the 2nd,4th,and 8th week after the operation,except one SDVG that failed at the 2nd week.Histological analysis and SEM showed that the inner layer was covered by new endothelial-like cells.Conclusion The double-layer bionic SDVG is a promising candidate as a replacement of native small-diameter vascular graft.

Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpene synthesis pathway, catalyzed two units of acetyl-CoA to acetoacetyl-CoA. In order to study the tanshinone biosynthesis in Salvia miltiorrhiza, a novel AACT gene, SmAACT, was cloned using cDNA microarray and RACE strategy. The full length cDNA of SmAACT is 1 623 bp (accession No. EF635969), which contained a 1 200 bp open reading frame (ORF) encoding a 399 amino acid protein. Nine introns were found in the genomic sequence. SmAACT was upregulated by YE and Ag+ elicitors both with cDNA microarray and quantitative RT-PCR analyses along with the accumulation of tanshinones. Sequence homology comparison and phylogenetic analysis all suggested that SmAACT belonged to the class of acetyl-CoA C-acetyltransferase. The transcription level of SmAACT was relatively higher in root than that in stem and leaf tissues. SNP analysis revealed that SmAACT was highly variable in the region of 6 to 9 introns with 33 SNPs in the 600 bp region, there are 5 SNPs in the cDNA region while they are all synonymous cSNPs. Some special genotypes were found in Salvia miltiorrhiza from different areas. SmAACT will be an useful gene for further analyze the mechanism of gene regulation among the tanshinones biosynthesis.
Acetyl-CoA C-Acetyltransferase ; genetics ; metabolism ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Gene Expression Regulation, Plant ; Genotype ; Introns ; Oligonucleotide Array Sequence Analysis ; Open Reading Frames ; Phylogeny ; Plant Leaves ; enzymology ; genetics ; Plant Roots ; enzymology ; genetics ; Plant Stems ; enzymology ; genetics ; Plants, Medicinal ; classification ; enzymology ; genetics ; Polymorphism, Single Nucleotide ; Reverse Transcriptase Polymerase Chain Reaction ; Salvia miltiorrhiza ; classification ; enzymology ; genetics

OBJECTIVETo study the metallothionein genes of Salvia miltiorrhiza through bioinformatics and characterization of tissue expression in regenerated shoots.

METHODMetallothionein genes were obtained by cDNA microarray analyze. BLAST was used for align, ORF finder software was used to find open reading frame, Prosite database was used to analyze the protein. Semi-quantitative RT-PCR method was used to detect the gene expression level.

RESULTTwo metallothionein genes were obtained which were contained a deduced amino acid sequence of 80 and 79 residues, named as SmMT-2a and SmMT-2b, they had a homology of 71.25%. Semiquantitative RT-PCR indicated that metallothionein genes were expressed in all tissues such as root, stem and leaf in regenerated shoots, while the expression level was higher in leaf than in root and stem.

CONCLUSIONIt was the first time that metallothionein genes were obtained from S. miltiorrhiza. It provides a good basis for further functional study of S. miltiorrhiza.

Amino Acid Sequence ; Base Sequence ; DNA, Complementary ; genetics ; Gene Expression Regulation, Plant ; Genes, Plant ; Genomics ; Metallothionein ; genetics ; metabolism ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Open Reading Frames ; Plants, Medicinal ; genetics ; metabolism ; Salvia miltiorrhiza ; genetics ; metabolism ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid

This paper firstly introduced the acquired full length cDNA of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase from hairy roots of Salvia miltiorrhiza (Abbr: SmCMK, GenBank number: EF534309). Results of KEGG analysis showed that SmCMK was belong to the upstream of nonemevalonate pathway, the only one kinase of the pathway. The full-length cDNA was deduced as encoding 4-(cytidine 5'-diphospho)-2-C-methylerythritol kinase (designated as SmCMK), and the sequence had a 1493 bp including 5' UTR 71 bp and 3' UTR 232 bp, an open reading frame (ORF) encoding a protein of 396 amino acid residues. The deduced protein had isoelectric point (pI) of 6.78 and a calculated molecular weight about 43 kDa, similar to cloned diterpene of CMK from other species of plants such as Mentha piperita and Lycopersicon esculentum reported previously. Real time PCR results indicated that elicitors of MJ stimulated the increase of mRNA expression of SmCMK. At the same time, results of high performance liquid chromatography (HPLC), used to examine the accumulation of diterpenoid tanshinones in hairy roots, showed that the contents of diterpenoid tanshinones in hairy root of Salvia miltiorrhiza were increased dramatically after treated with methyl jasmonate (MJ). This result showed a positive correlation between the levels of mRNA expression and tanshinones accumulation in Salvia miltiorrhiza stimulated by MJ. It proved primarily that the increased expression level of mRNA of SmCMK helps to enhance tanshinones' accumulation, which will be the basis for further study on the mechanism of gene regulation of secondary metabolism of tanshinones.
Acetates ; pharmacology ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Conserved Sequence ; Cyclopentanes ; pharmacology ; DNA, Complementary ; genetics ; Diterpenes, Abietane ; Gene Expression Regulation, Plant ; Genes, Plant ; Molecular Sequence Data ; Open Reading Frames ; Oxylipins ; pharmacology ; Phenanthrenes ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; enzymology ; Plants, Medicinal ; enzymology ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Salvia miltiorrhiza ; enzymology ; genetics ; metabolism ; Sequence Homology, Amino Acid

The aim of the study is to explore the effects of luteolin preconditioning on hepatic ischemia/reperfusion injury in rats and its mechanism, and investigate the effects of the change of heme oxygenase-1 (HO-1) activity on hepatic ischemia/reperfusion injury. Sprague-Dawley rats were divided into 5 groups randomly: control, model, luteolin, luteolin + zinc protoporphyrin (ZnPP, an inhibitor of HO-1) and hemin groups (n = 8 for each group). The rats in control, model and hemin groups received a standard chow daily. The rats in luteolin and luteolin + ZnPP groups received a chow supplemented with luteolin (200 mg/kg) daily. After 4 weeks, ZnPP (25 μmol/kg) and hemin (20 μmol/kg) were injected hypodermically 6 h before ischemia/reperfusion in luteolin + ZnPP and hemin groups, respectively. Portal vein and hepatic artery supplying the middle and left hepatic lobe were clamped with an atraumatic vascular clip for induction of partial hepatic ischemia in all rats except control group. After the 60 min of hepatic ischemia, a 60-minute reperfusion period was initiated by removal of the arterial clip. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected in serum, and the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in serum and liver were measured with assay kit. The expression of HO-1 protein and activity of HO-1 were examined in liver. The results showed that the luteolin and hemin pretreatment led to significant decreased levels of AST and ALT in serum, increased activity of SOD and decreased content of MDA in serum and liver compared with model group (P < 0.01). In addition, the expression of HO-1 protein and activity of HO-1 were elevated in luteolin and hemin groups (P < 0.01). ZnPP markedly increased the levels of AST and ALT in serum, and decreased the activities of SOD and HO-1, elevated MDA content in liver when compared with those in luteolin group (P < 0.01). Cytoplasmic vacuolation and swelling of hepatocytes were revealed in the model group after ischemia/reperfusion. Treatments with luteolin and hemin markedly relieved the liver structural changes. These results suggest that HO-1 protects rat liver from ischemia/reperfusion injury, and luteolin reduces the content of MDA and increases the activity of SOD and the expression of HO-1, which indicate that luteolin can elevate the antioxidation in rat liver, and thus protects rat liver from ischemia/reperfusion injury.
Animals ; Female ; Heme Oxygenase (Decyclizing) ; metabolism ; Ischemic Preconditioning ; methods ; Liver ; blood supply ; Luteolin ; therapeutic use ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; Superoxide Dismutase ; metabolism

OBJECTIVETo estimate the effect of the lymph duct ligation on systemic inflammatory factors and endotoxins during intestinal ischemia-reperfusion (I/R).

METHODSMale SD rats underwent occlusion of superior mesenteric artery for 60 min followed by reperfusion for 120 min plus lymph duct ligation or not. Forty rats were randomly divided into 4 groups: group A (blank); group B (sham); group C (intestinal I/R); group D (intestinal I/R plus lymph duct ligation). Mesenteric lymph nodes were harvested for standard bacteriologic cultures. The endotoxin, D-lactate, diamine oxidase (DAO), and cytokines in serum were detected.

RESULTSThe rates of bacterial translocation to mesenteric lymph nodes were 40% in group C and 20% in group D. No positive lymph node cultures were encountered in any of group A and B. The serum cytokines (except for sICAM-1) , D-lactate, DAO and endotoxin levels were lower in group D than those in group C (P<0.05), but both were higher than those in group A and B (P<0.05).

CONCLUSIONDuring intestinal I/R injury, blockage the lymph flow from gut into bloodstream decreases the levels of cytokines, and significantly attenuates the increase in intestinal permeability.

Animals ; Disease Models, Animal ; Inflammation ; Intestinal Diseases ; metabolism ; microbiology ; pathology ; Intestines ; blood supply ; pathology ; Ligation ; Lymph Nodes ; pathology ; Lymphatic System ; surgery ; Male ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; microbiology ; pathology

Objective To study the clinical efficacy of anus-preserving rectectomy by using telescopic anastomosis of colon and rectal mucosa for the middle and lower rectal cancer. Methods A retrospective analysis was carried out in 402 cases with middle and lower rectal cancer undergoing telescopic anastomosis for anus-preserving procedure, including 241 males and 161 females, age ranging from 21 to 99 years, averaging at 55. 7 years. The distal margins of the tumors were within 6 - 9 cm to anal verge. According to TNM staging, there were 123 cases in Stage Ⅰ , 244 cases in Stage Ⅱ , 31 cases in Stage Ⅲ,and 4 cases in Stage Ⅳ. Results 345(345/402, 85. 8% ) cases were followed up, the median time of the follow-up was 6. 1 years. Postoperative complications included 17(4.2%) cases of stomal leakage, 11(2.7% ) cases of stomal stenosis. All patients recovered normal defecating function 12-24 weeks post operation. Local recurrence rate was 6. 3% (22/345). Hepatic and lung metastasis was 13. 6% (47/345) and 2. 6% (9/345)respectively. The five year survival rate was 68. 7% (112/163). Conclusions Anuspreserving rectectomy by using telescopic anastomosis is safe and effective procedure to treat middle and lower rectal cancer, with the preservation of anal function and without the increasing rate of local recurrence.

Objective To study clinical therapeutic effects of anus-preserving operation with resecting anal intersphincter to treat ultra-low rectal cancer through abdominal cavity. Methods We retrospectively analyzed 52 cases of ultra-low rectal cancer, with the inferior border of the cancers within 2 cm to anocutaneous line or 5 cm to the edge of anus treated by anus-preserving operation with resecting archos internal sphincter muscles through abdominal cavity and anus. There were 29 males, and 23 females, with age 28 to 76 years old, averaging 56. 3 years old. The inferior border of the cancer were within 4 cm to the edge of anus in 18 cases, including 6 cases of adenoma cancerization, and 5 cm to the anus in 34 cases. Pathologic diagnosis was well-differentiated adenocarcinoma in 21 cases, moderately differentiated in 29 cases, low differentiated in 2 cases, there were 6 cases with adenoma cancerization. 28 cases were Dukes A stage, and 24 B stage. Results The follow-up rate was 88. 4% (46/52), and the median time was 5.9 years. 2 case developed stoma leak (3.8%), and 3 developed stoma stenosis(5.7% ) after operation. The anus could roughly control defecation in 6 ～ 12 mouths after operation. The local recurrence rate was 5.7%, and the 5-year-survival rate was 72.7%. Conclusion By anus-preserving operation with resecting archos internal sphincter muscles, defecation controlling was well reserved by anus, and the 5-year-survival rate was not cut down. This operation is one of the safe and effective operations of anus-preserving procedure.