Objective To construct the recombinant plasmid pYES6-S and express and purify spike protein of severe acute respiratory syndrome(SANS)coronavirus in Saccharomyces cerevisiae. Methods DNA fragments of SANS coronavirus were obtained by reverse transeription.Four over- lapped fragments of spike protein genes were amplified by polymerase chain reaction(PCR)and ligated into an integral spike protein gene by restriction enzyme digestion.The spike protein gene recombined with pYES6 and cloned into E.coll.The recombinant plasmid pYES6-S was induced and expressed in Saccharomyces cerevisiae(INVScl)by galactose.Results The recombinant plasmid pYES6-S was confirmed that inserted fragment was right in length,direction and base matching by restriction enzyme digestion and sequencing.The purified protein encoded by the whole spike protein gene was about Mr 110?10~3 identified by electrophoresis.Conclusion The whole spike protein gene of SARS coronavirus is cloned into E.coli and the protein is expressed in Saccharomyces cerevisiae successful ly.which can be helpful in SARS vaccine research.

To study the chemical constituents from the the deep-sea fungus Alternaria sp. F49. Seven compounds were isolated from the EtOAc extract by using silica gel, Sephadex LH-20, ODS and HPLC methods. Based on the spectroscopic analysis, their structures were identified as (8R)-5-O-methyl-orcinotriol (1), orcinotriol (2), α-acetylorcinol (3), 3'-hydroxyalternariol 5-O-methyl ether (4), altenusiol (5), altenusin (6), and 5'-methoxy-6-methyl-biphenyl-3,4,3'-triol (7). (8R)-5-O-Methyl-orcinotriol (1) is a new phenolic compound which has never been reported in the literature. Compounds 4-7 showed strong DPPH free radical scavenging activity; whereas compounds 1-7 showed strong ABTS free radical scavenging activity.

This study was aimed to explore the single nucleotide polymorphism (SNPs) of breakpoint cluster region of bcr gene in Chinese people and the relationship between SNPs and chronic myelogenous leukemia (CML). A 3.12 kb region spanning from exon 13 to exon 15 in the bcr region were screened by DNA pooling and denaturing high performance liquid chromatography (dHPLC), and the results were verified by sequencing. The results indicated that 6 novel SNP sites and 2 bases different from reference sequence were confirmed in the region studied, and the frequency of 6 novel SNP sites in studied population was obtained, one SNP of which was found in exon 13 and caused a nonsynonymous mutation. The gene frequencies of novel SNPs had no significant difference between CML and control people. It is concluded that sequence polymorphisms in the major breakpoint cluster region of bcr gene are found, most of which are SNPs, No relationship can be confirmed between SNPs and CML disease.
Base Sequence ; Chromosome Breakage ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Molecular Sequence Data ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins c-bcr ; genetics

OBJECTIVE: To investigate the polymorphism and forensic efficiency values of STR loci D12S391/D18S865 METHODS: Population studies on D12S391 and D18S865 were carried out in a sample of 454 unrelated Wenzhou Han individuals using PCR followed by PAGE and silver stain detection. RESULTS: 11/7 alleles and 50/21 genotypes of D12S391/D18S865 were respectively observed among the 454 unrelated individuals. The genotype frequency matched the Hardy-Weinberg equilibrium, The heterozygotes (H) in D12S391/D18S865 were 0.7974/0.7379, Power of Exclusion were 0.9566/0.9077, polymorphism information content (PIC) were 0.8260/0.7279 respectively. CONCLUSION D12S391 and D18S865 were high polymorphic loci and the frequencies were in accordance with Hardy-Weinberg equilibrium (P>0.5), so the two loci can be used in forensic medicine and in other genetic research areas.
Alleles ; China/ethnology* ; DNA/isolation & purification* ; Electrophoresis, Polyacrylamide Gel ; Forensic Medicine ; Gene Frequency ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Tandem Repeat Sequences

OBJECTIVE: To study genetic polymorphism of D7S2846, D19S400 and D18S535 in Han population in Wenzhou. METHODS: DNA was extracted with chelex-100 method from EDTA-blood samples of 194 unrelated individuals in Wenzhou and amplified with PCR technique. The PCR products were analyzed by PAG vertical electrophoresis and silver-stain. RESULTS: 6 alleles and 15 genotypes of D7S2846, 10 alleles and 36 genotypes of D19S400, 8 alleles and 26 genotypes of D18S535 was observed. The heterozygosities of the three loci are 0.644, 0.724 and 0.772; The power of discriminating are 0.854, 0.940 and 0.938. CONCLUSION The heterozygosities of the three loci are high and the frequencies was in accordance with Hardy-Weinberg equilibrium (P>0.05), so the three loci can be used in forensic medicine and in other genetic researches.
Alleles ; China/ethnology* ; DNA/isolation & purification* ; Gene Frequency ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Tandem Repeat Sequences

Objective To investigate the effects of advanced glycation end-products(AGEPs)on the function of normal keratinocytes in vitro so as to explore the role of AGEPs in impaired wound healing. Methods Normal rat keratinocytes were incubated with different concentrations of AGEPs.After 48 hours of culturing,the cell proliferation rates were measured by MTT colorimetric determination.The cell cycle distributions and apoptosis were analyzed with flow cytometry,and the migration was investigated by 24-well fluorimetric cell migration assay kit by exposing to 100?g/ml AGEPs.Nuclear extracts from these cells were examined for binding of nucleotides containing NF-?B consensus by immunocytochemistry and EMSA in vitro.Results The proliferations of normal keratinocytes were significantly arrested and many cells were induced to early apoptosis compared with control ones(P＜0.05)by exposing to AGEPs for 48 hours. Meanwhile AGEPs also irritated keratinocytes migration compared with control ones(P＜0.05).Inhibiting the activation of NF-?B could partly recover the proliferation of keratinocytes,reverse apoptosis and attenu- ate migration.Conclusion AGEPs are correlated with the migration,proliferation and apoptosis of kera- tinocytes by NF-?B.

The purposes of the present study were to investigate the inhibitory effect of quercetin (QUE) preconditioning on endoplasmic reticulum stress (ERS) inducer tunicamycin (TM)-induced apoptosis in RAW264.7 macrophages and the underlying molecular mechanisms. RAW264.7 cells were pretreated with different concentrations (20, 40, and 80 μmol/L) of QUE for 30 min and then treated with TM (5 mg/L) for 12 h. Cell viability and apoptosis were determined using MTT assay and Annexin V-FITC apoptosis detection kit, respectively. The nuclear translocation of activating transcription factor 6 (ATF6) in cells was detected by immunofluorescence analysis and Western blot. Protein and mRNA expressions of C/EBP homologous protein (CHOP) and Bcl-2 were examined by Western blot and real-time PCR, respectively. The results showed that TM reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of TM were significantly inhibited by QUE pretreatment at the concentrations of 40 and 80 μmol/L. Interestingly, we found that QUE also significantly suppressed the TM-induced translocation of ATF6, an ERS sensor, from the cytoplasm to the nucleus. In addition, exposure of RAW264.7 macrophages to TM resulted in a significant increase of the expression of CHOP, a transcription factor regulated by ATF6 under conditions of ERS, as well as a decrease of Bcl-2 at transcript and protein levels. QUE blocked these effects in a dose-dependent manner. These data indicate that QUE can protect RAW264.7 cells from TM-induced apoptosis and that the mechanism at least partially involves its ability to inhibit the ATF6-CHOP signaling pathway.
Activating Transcription Factor 6 ; metabolism ; Animals ; Apoptosis ; Cell Survival ; Endoplasmic Reticulum Stress ; Macrophages ; cytology ; drug effects ; Mice ; Quercetin ; pharmacology ; Transcription Factor CHOP ; metabolism ; Tunicamycin ; pharmacology

OBJECTIVETo investigate the change pattern of substance P (SP) in the portal vein during the rectoanal inhibitory reflex (RAIR), and its physiologic significance; the influence of external splanchnic nerve of rectum and anal to the RAIR.

METHODSThe rats were divided into seven groups, among them there were six groups, which were first divided into two big groups according to whether the external splanchnic nerve to the rectum and anal were cut off, one is no cut-off external splanchnic nerve group, the other is cut-off external splanchnic nerve group. Each group were further divided, according to the distance of the balloon-sac on Foley's tube in the rectum away from anal verge, into 2, 4, 6 centimeter groups; A control group with Foley's tube put into the rectum, but the balloon-sac on Foley's tube did not pumped up with water. Measure and compare the value and change of SP in the portal vein during the RAIR.

RESULTSThe comparison of SP in portal vein, among the 2, 4 centimeter groups with cut-off external splanchnic nerve, all groups with intact external splanchnic nerve supply and control group, had no statistic difference (P>0.05). The comparison between the 6 centimeter group with intact external splanchnic nerve group and the 2, 4 centimeter groups with cut-off external splanchnic nerve, P<0.01, the statistic difference was significant. The comparison between 6 centimeter group of intact and cut-off external splanchnic nerve, P<0.01, the difference was significant.

CONCLUSIONThe reason for the stimulation on upper rectum dose not induce the RAIR is related with this stimulation result in the release of SP, the exciting mediator to internal sphincter. The external splanchnic nerve supply of rectum and anal canal have influence on the change of SP of the portal vein during RAIR.

Anal Canal ; physiology ; Animals ; Female ; Male ; Portal Vein ; physiology ; Rats ; Rats, Sprague-Dawley ; Rectum ; physiology ; Substance P ; metabolism

OBJECTIVETo evaluate the accuracy of optical coherence tomography (OCT) in evaluating neointimal proliferation in canine coronary artery following stenting.

METHODSIn 15 domestic dogs, a single bare-metal stent was implanted in the anterior descending or the circumflex branch of the left coronary artery. Ninety days after stenting, the dogs underwent coronary angiography and OCT, followed by quantitative histological assessment of neointimal proliferation in the target arterial segments. The parameters of OCT and the histological findings were analyzed comparatively.

RESULTSA total of 15 OCT-histology matched frames acquired at the point with the most severe stenosis in every stent, and 60 pathological sections from all the stents were analyzed. The difference of the stent area assessed by OCT was comparable to that defined histologically (5.01∓0.79 mm(2) vs 4.99∓0.81 mm(2), P>0.05). Neointimal thickness and area were smaller with OCT assessment than with histological assessment (0.19∓0.08 mm vs 0.22∓0.10 mm, and 1.52∓0.49 mm(2) vs 1.85∓0.78 mm(2), respectively, P<0.05). The lumen area was larger by OCT assessment than by histological assessment (3.50∓0.66 mm(2) vs 3.15 ∓ 0.43 mm(2), P<0.05). Close correlations were found between OCT and histological evaluations of the neointimal thickness (R(2)=0.5280.767), neointimal area (R(2)=0.5280.537) and stent area (R(2)=0.528), but the correlation was poor for lumen area (R(2)=0.5280.307). All the stents showed full endothelialization without thrombus or aneurysm in the stents.

CONCLUSIONOCT allows precise and reproducible assessment of neointimal proliferation in the coronary artery following stenting, but for measurement of the lumen area, OCT shows a poor correlation to histological evaluation.

Angioplasty, Balloon ; adverse effects ; instrumentation ; Animals ; Coronary Angiography ; Coronary Vessels ; diagnostic imaging ; pathology ; Dogs ; Male ; Models, Animal ; Neointima ; pathology ; Stents ; adverse effects ; Tomography, Optical Coherence ; Tunica Intima ; pathology

To investigate the relationship between the single nucleotide polymorphism (SNPs) of the bcr and abl gene and chronic myelogeous leukemia (CML), the 9 sequence-tagged sites (STS) in bcr and abl gene were screened by DNA pooling and denaturing high performance liquid chromatography (dHPLC), and the results were varified by sequencing. The results showed that the polymorphism sites were detected in 4 out of the 9 STS fragments and there were 3 bases different from the reference sequence found in 3 fragments. In conclusion, the novel SNP in U07000 fragment shows significantly different frequencies between CML and controled people.
Chromatography, High Pressure Liquid ; methods ; Fusion Proteins, bcr-abl ; genetics ; Genes, abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins c-bcr ; genetics ; Sequence Analysis, DNA ; Sequence Tagged Sites