OBJECTIVETo study the phylogeny relationship and molecular identification of 10 species from Huperzia (Huperziaceae) based on matK gene sequences data.

METHODTotal DNA of nine species from Huperzia was extracted; matK gene sequence was amplified by PCR. PCR product was directly sequenced after purification.

RESULTThe chloroplast matK gene nucleotide sequences from 9 species of Huperzia species were sequenced. The matK gene nucleotide sequences length was 1 589 bp. Analysis with Huperzia lucidula matK gene nucleotide sequences (download from GenBank) and taking Lycopodiella cernua as outgroup, Maximum Parsimony, Neighbor-Joining analyses and genetic distances were conducted using MEGA 3.1 software. 35 variable sites and 35 parsimony informative sites have been found. Pairwise genetic distances among 10 species of Huperzia was 1.59% - 0.25%.

CONCLUSIONThe results were consistent with the taxonomy in morphological of Huperzia. But H. longipetiolata and H. serrata were resolved into in different clade. There are 19 different sites of matK gene sequences between H. longipetiolata and H. serrata, the genetic distances is 0.121%. It is suggested H. longipetiolata should be as an independent species.

DNA, Chloroplast ; genetics ; DNA, Plant ; chemistry ; genetics ; Endoribonucleases ; genetics ; Huperzia ; classification ; enzymology ; genetics ; Molecular Sequence Data ; Nucleotidyltransferases ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Plants, Medicinal ; classification ; enzymology ; genetics ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Species Specificity

OBJECTIVEThe study the patterns of rDNA ITS sequence variation of 9 medicinal species of genus Bupleurum in China to find the DNA molecular markers for identification of these crude drugs.

METHODThe ITS regions of these species were amplified by PCR and sequenced, and their sequences were analyzed by DNAssist Version 2.2.

RESULTSHomologous alignment indicated that the consanguinity of the ITS sequences between genus Bupleurum and outgroups was lower than 75%, and that within genus Bupleurum was higher than 88%. For the same species, the consanguinity was higher than 99%.

CONCLUSIONThe ITS sequences may serve as reliable molecular markers for identification of Radix Bupleuri.

Bupleurum ; classification ; genetics ; DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Molecular Sequence Data ; Phylogeny ; Plant Roots ; genetics ; Sequence Analysis, DNA ; Species Specificity

Objective : To investigate the clinical efficacy and degree of impact of placebo in the patients with primary insomnia. Methods ¡¤ Databases including PubMed, Cochrane, and Embase were searched. The Consolidated Standards of Reporting Trials (CONSORT) was used as a reference standard for enrollment and evaluation. The objective and subjective sleep assessment indicators were collected before and after pla-cebo treatment. Meta-analysis was performed using RevMan 5.3 and Comprehensive Meta-Analysis V2 softwares. Results ¡¤ A total of 17 arti-cles (18 studies) were included in the systematic review, and the total sample size was 1 980. These indicators were analyzed by age subgroups (adults and elderly adults). The objective indicators showed that the number of awaking was only statistically different in elderly adults group (P=0.043), while the latency period of sleep, wakefulness time after sleep onset and total sleep time were statistically different in both groups (P<0.05). The absolute value of total effect range was 0.153-0.414. Subjective indicators showed that the subjective total sleep time, subjective wakefulness time after sleep onset, and subjective number of awaking were statistically different in both groups (P<0.05), while subjective la-tency period of sleep was only statistically different in adults group (P=0.000). The absolute value of total effect range was 0.289-0.474. Con-clusion ¡¤ Placebo in the patients with primary insomnia can improve objective and subjective sleep assessment indicators, but the effect is small.

OBJECTIVETo analyze the nuclear ribosome DNA (nrDNA) internal transcribed spacer (ITS) sequences of 4 Leonurus species, and the possibility of using them for molecular authentication of the crude drugs from the genus.

METHODSThe nrDNA ITS sequence (including ITS1, 5.8S rDNA, ITS2, and partial 18S rDNA and 26S rDNA) of L. japonicus and its 3 adulterant species were amplified and sequenced, and CLUSTRAL X and MEGA software was employed for analysis.

RESULTSThe variation of ITS1 and ITS2 between L. japonicus and its adulterant species ranged between 7.2% and 18.8% and between 14.2% and 27%, respectively. The phylogenic tree derived from the dendrograms based on the ITS sequence data contained some discrepancy from the traditional classification.

CONCLUSIONThe nrDNA ITS sequences can be used potentially as efficient markers for identification of L. japonicus and its adulterants, and further study is needed for studying the phylogeny of Leonurus.

DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Leonurus ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Sequence Analysis, DNA ; Species Specificity

OBJECTIVETo observe the biocompatibility of new biomaterials porous calcium phosphate (CPC) and ectopic bone formation of CPC with bone marrow stromal cells (BMSCs).

METHODSThe BMSCs were cultured from Beagle dog and combined with the porous CPC with the best concentration after transfect green fluorescent protein (GFP). The adhesion and growth of BMSCs on CPC were observed under inversion, fluorescence and scanning electron microscopy. The ectopic bone formation were observed at the 8th week after CPC and BMSCs were implanted subcutaneously into nude mice.

RESULTSWhen BMSCs with CPC were cultured at the 1st day, cells were climbing out from CPC with normal morphology. At the 7th day cells can be seen protruding pseudopods, secretion of matrix. Bone formation could be seen histomorphologically at the 8th week.

CONCLUSIONPorous CPC has good biocompatibility and is an ideal scaffold material for bone tissue engineering.

Animals ; Biocompatible Materials ; Bone Cements ; Bone and Bones ; Calcium Phosphates ; Dental Cementum ; Dogs ; Mesenchymal Stromal Cells ; Mice ; Mice, Nude ; Tissue Engineering

OBJECTIVETo demonstrate molecular characterization of a newly isolated enterovirus.

METHODSVirus were isolated from patient with unknown-causing disease and tested by reverse transcription-polymerase chain reaction (RT-PCR) and 5'3'RACE (rapid amplification of cDNA ends, RACE), in an attempt to obtain the sequence of this newly isolated enterovirus.

RESULTSSequence analysis showed that this newly isolated enterovirus shared 83%-94% nucleotide identity and 91%-100% amino acid identity with enterovirus 89. Phylogenetic analysis indicated that it was probably a new subtype of enterovirus 89.

CONCLUSIONThis newly isolated enterovirus in the stool specimen from patient has the same serotype with enterovirus 89, but it was probably a new subtype of enterovirus 89.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Enterovirus ; classification ; genetics ; isolation & purification ; Feces ; virology ; Genome, Viral ; genetics ; Humans ; RNA, Viral ; genetics ; Sequence Analysis, DNA

Objective To evaluate the value of transesophageal echocardiography (TEE) in transthoracic minimally invasive device closure of ventricular septal defect(VSD).Methods A total of 164 cases of VSD were recruited as candidates to receive transthoracic minimally invasive device closure between January 2007 and October 2010,including 138 perimembranous VSDs,3 muscular VSDs and 23 supracristal VSDs.Among these groups,85 male patients were included.Four-champer view,five-champer view,left ventricular long-axis view,short-axis view and right ventricular inflow view were detected to evaluate the availability of device closure.Results A total of 152 cases(92.7%)were successfully closed with a device.All the patients were followed up more than 3 months arranged with a standard protocol.No complete atrioventricular block (CAVB) or associated valvular complications were observed.Three of the five cases with traced residual shunt after device closure closed spontaneously.Conclusion TEE plays an important role in transthoracic minimally invasive device closure of ventricular septal defect,which has been proved by the good follow-up results without CAVB and associated valvular complications.

Objective To demonstrate molecular characterization of a newly isolated enterovirus.Methods Virus were isolated from patient with unknown-causing disease and tested by reverse transcription-polymerase chain reaction (RT-PCR) and 5' ,3'RACE (rapid amplification of eDNA ends, RACE), in an attempt to obtain the sequence of this newly isolated enterovirus. Results Sequence analysis showed that this newly isolated enterovirus shared 83 %-94% nucleotide identity and 91%-100% amino acid identity with enterovirus 89.Phylogenetic analysis indicated that it was probably a new subtype of enterovirus 89. Conclusion This newly isolated enterovirus in the stool specimen from patient has the same serotype with enterovirus 89, but it was probably a new subtype of enterovirus 89.

Objective To assess if the modulating effect of platelet-derived growth factor(PDGF)BB on p21~(WAF1) was mediated by upregulating transforming growth factor(TGF)-β_1 expression in vascular smooth muscle cells(VSMC).Methods TGF-β_1 mRNA and protein expressions were measured by reverse transeription-PCR and ELISA,the protein expressions of p21~(WAF1) and the downstream TGF-βsignalling including TGF-β type I receptor(ALK-5 in VSMC),Smurf2,pSmad2/3,Smad4,Smad7 were detected by Western blot.Results PDGF-BB significantly upregulated the expressions of TGF-β_1 at mRNA(0.79-fold)and protein(1.98-fold)levels in VSMC,significantly inhibited the expression of p21WAF1(-67±12)%,and enhanced the expressions of ALK-5,pSmad2/3,Smad4,Smurf2 protein by 1.21-fold.0.95-fold,0.69.fold and 2.55-fold respectively,inhibited Smad7 expression(-65±9)%,these alterations were partially restored by anti-TGF-β_1 neutralizing antibody.Conclusions These findings suggested that PDGF-BB inhibited p21~(WAF1) expression in VSMC partially through upregulating TGF-β_1 expression via PDGFBB and TGF-β signalling pathways.

Aim To explore the signaling pathway of matrine derivative ZS10 in inhibiting proliferation and inducing apoptosis of BEL-7402 cells. Methods ZS10 was synthesized by organic synthesis. The inhibitory effect of ZS10 on the proliferation of BEL-7402 cells was analyzed by MTT method at the time of 24 h, 48 h and 72 h, respectively, and IC50 was calculated. DAPI staining was used to observe the state of BEL-7402 cells. Clone formation method was used to observe the colony formation of BEL-7402 cells, flow cytometry was used to observe the cell cycle arrest and apoptosis of BEL7402 cells, and Western blot was used to detect the expression level of PI3K/AKT pathway and related proteins. Results MTT results showed that the IC50 was(6.62±1.11)μmol·L-1; DAPI staining showed that the cell state changed significantly with the increase of drug concentration, and the results of colony formation showed that ZS10 significantly inhibited the colony formation of BEL-7402 cells. The results of flow cytometry showed that ZS10 induced S phase arrest and cycle apoptosis of BEL-7402 cells. Western blot showed that ZS10 at the concentration of 08 μ mol·L-1 could regulate the PI3K/AKT pathway and its related proteins in a dose-dependent manner. Compared with the control group, the expression of PI3K, AKT, P-AKT and anti-apoptotic protein Bcl-2 significantly decreased, the expression of pro-apoptotic protein Bax significantly increased, the expression of Cyclin D1 and CDK2 significantly decreased, and the expression of EGFR and N-cadherin, Vimentin significantly decreased in the treatment group. The expression of E-cadherin increased. Conclusions Matrine derivative ZS10 can inhibit the growth and proliferation of hepatocellular carcinoma cell line BEL-7402.