The genetic analysis of herpesviruses has been a constant challenge, due to the large, complex genomes of herpesviruses and mutagenesis of viral genes by conventional recombination methods in cell culture. Recently, a completely new approach for full-length infectious clones of herpesviruses based on bacterial artificial chromosomes (BACs) has been developed. This technique allows the maintenance, propagation and genetic modification of the viral genome as a BAC plasmid in E.coli, thus making the procedures fast, safe and effective in prokaryotic cells. This technique also makes it possible for the reconstitution of viral progeny or mutants by transfection of the BAC plasmid into eukaryotic cells, thereby facilitating the analysis of viral gene functions in the context of genome. In this presentation, Epstein-Barr virus was used as an example to describe the principle, establishment of the technique and mutation introduction into the BAC plasmid, and to discuss the perspective in the use of BAC-cloned herpesviruses.

OBJECTIVETo explore the protective effect and mechanism of total flavones of Bidens pilosa L. (TFB) on IgA1 induced injury of venous endothelial cells in Henoch-Schönlein purpura (HSP) children patients. METHODS Human umbilical venous endothelial cells (HUVECs) were taken as subject. They were intervened by normal IgA1 and HSP children patients' serum IgA1, and added with different concentrations TFB at the same time. Then they were divided into the blank control group, the normal control group, the HSP IgA1 group, and HSP IgA1 plus TFB (1.0, 0.5, 0.25 mg/mL) groups. Levels of TNF-α and IL-8 in supernate were detected by ELISA. The NO level was detected by nitrate reductase method. mRNA and protein expressions of NF-κB and ICAM-1 in HUVECs were detected by fluorescent quantitative PCR and Western blot respectively.

RESULTSCompared with the normal control group and the blank control group, levels of IL-8, TNF-α, and NO all significantly increased in the HSP group (P < 0.05). Compared with the HSP group, levels of IL-8, TNF-α, and NO significantly decreased after intervention of TFB (1.0 and 0.5 mg/mL; P < 0.05, P < 0.01). Results of fluorescent quantitative PCR and Western blot showed, as compared with the blank control group and the normal control group, mRNA and protein expressions of NF-κB and ICAM-1 in HSP children patients' serum IgA1 induced venous endothelial cells significantly increased with statistical difference (P < 0.05, P < 0.01). Compared with the HSP group, mRNA and protein expressions of NF-KB and ICAM-1 were obviously down-regulated after intervention of TFB (1.0, 0.5, 0.25 mg/mL), with statistical difference (P < 0.05, P < 0.01).

CONCLUSIONTFB could protect vascular damage by inhibiting in vivo high expression of NF-κB, reducing the production of IL-8, TNF-α, and NO in vascular endothelial cells of HSP children patients.

Bidens ; chemistry ; Child ; Flavones ; pharmacology ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Immunoglobulin A ; blood ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-8 ; metabolism ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Purpura, Schoenlein-Henoch ; blood ; RNA, Messenger ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Objective Using the third high sensitive cardiac troponin I assay to establish the reference intervals of serum cardiac marker high sensitivity cardiac troponin I ( hs-cTnI ) in apparently healthy subjects of Beijing area, and to explore the relationship between cTnI and other parameters.Methods Using random selection method, a total of 440 serum samples were collected from apparently healthy subjects of Beijing area.Among them, 217 cases were male, average age was (50.0 ±14.3) years old;223 cases were female, average age was ( 49.0 ±14.0 ) years old.All of these serum samples were detected hs-cTnI using i2000 Abott biochemical analyzer and ARCHITECT STAT Abbott assay kit.And the 99th percentile upper reference limit was used as the reference interval.Non-parametric tests were used to compare the different groups, the U Mann-Whitney test was used between two groups, the Kruskal-Wallis test was used in many groups, and the correlation analysis was used Spearman method.Results The 99th percentiles of hs-cTnI in 440 apparently healthy subjects is 9.66 ng/L.While the male group is 9.66 ng/L, and the female group is 8.25 ng/L.The serum hs-cTnI level of male is higher than female ( 9.95 ng/L vs 8.25 ng/L, P<0.05 ).According to the spearman correlation analysis, the serum hs-cTnI level appears positive correlation with age, brain natriuretic peptide (BNP), alanine aminotransferase (ALT),aspartate transaminase ( AST) ,gamma-glutamyl transferase ( GGT) ,total bilirubin ( TBIL) ,serum creatinine ( SCR) , hemoglobin (HGB)(P<0.05,R>0),and appears negative correlation with platelet (PLT)(P<0.05,R<0).Conclusion The serum hs-cTnI level apparently differents with gender, so it is necessary to establish reference intervals among different gender group of Beijing area.

Animals ; Calcitonin Gene-Related Peptide ; pharmacology ; Ischemic Postconditioning ; Male ; Myocardial Reperfusion Injury ; prevention & control ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley

Caused by Helminthosporium carposaprum, tomato brown lea f spot was a serious disease in green house in Henan Province. The condition for promoting sporulation of fungi were tested in this paper. The results showed th at the number of sporulation were different on the different medium,the fungi c ould sporulate a lot on the PDA+tomato leaf and Czapek medium, but V8、PSA and t omato juice restrained sporulation.The best carbon source and nitrogen source f or the fungi promoting sporulation were fructose and ammonium chloride respectiv ely,mannitol and Peptone ammonium sulfate restrained sporulation. Light and ult raviolet radiation were in favor of sporulation , ultraviolet radiation irradiat ing for 60～80min promoted sporulation. The fungi were promoted sporulation on the condition of lower or higher temperature and alkalescence,which 15℃o r 30℃,pH 8～9.

Cleistocalycis Operculati Cortex is the dry bark of Cleistocalyx operculatus. It is the raw material of Compound Hibiscuse which is external sterilization antipruritic drugs. The quality standard of Cleistocalycis Operculati Cortex in Guangdong Province "standard for the traditional Chinese medicine" (second volumes) only contains TLC identification. It is unable to effectively monitor and control the quality of Cleistocalycis Operculati Cortex. A reversed-phase HPLC method was established for the determination of 3, 3'-O-dimethylellagic acid from Cleistocalycis Operculati Cortex and the content was calculated by external standard method for the first time. Under the selected chromatographic conditions, the target components between peaks to achieve effective separation. 3,3'-O- dimethylellagic acid standard solution at the concentration of 1.00 - 25.0 mg x L(-1) showed a good linear relationship. The standard curve was Y = 77.33X + 7.904, r = 0.999 5. The average recovery was 101.0%, RSD was 1.3%. The HPLC method for the determination of 3,3'-O-dimethylellagic acid in Cleistocalycis Operculati Cortex is accurate and reliable. It can provide a strong technical support for monitoring the quality of Cleistocalycis Operculati Cortex.
Chromatography, High Pressure Liquid ; methods ; Chromatography, Reverse-Phase ; methods ; Drugs, Chinese Herbal ; analysis ; Plant Bark ; chemistry ; Syzygium ; chemistry

OBJECTIVETo observe the effect of Polygonatum sibiricum on Yin deficiency model rats induced by long-term overload swimming.

METHODExcept for the normal group, all of the remaining rats performed the long-term overload swimming for eight weeks, with five days every week and once every day, to establish the Yin deficiency model. The daily swimming time increased from 10 min to 180 min at the end of the 7th week, with the water depth of 60 cm and the water temperature at 30 degrees C. After the success of the modeling, the rats were orally administered with different doses of aqueous extracts from P. sibiricum (2.5, 10 g x kg(-1)) for eight weeks. After the final administration, their blood were collected from orbits to measure immunoglobulin A, G and M (IgA, IgG, IgM), interleukin 2 and 6 (IL-2, IL-6) and cAMP, cGMP contents in plasma General behavioral indicators (weight, facial temperature, pain threshold and holding power) of rats were observed during the drug administration.

RESULTCompared with the model control group, aqueous extracts from P. sibiricum was given for eight weeks to significantly increase the rat weight and holding power of Yin deficiency model rats, decrease the facial temperature and the sensitivity of pain threshold, and increase IgA, IgG, IgM and IL-6 content and IgG content in serum, but without statistical difference. Aqueous extracts from P. sibiricum (10 g x kg(-1)) could also increase IL-2 content in serum, and decrease cAMP content and cAMP/cGMP ratio.

CONCLUSIONP. sibiricum could improve the general behavioral indicators (weight, holding power, pain threshold and facial temperature), immunologic functions (IgA, IgG, IgM) and cyclic nucleotide (cAMP, cAMP/cGMP), so as to ameliorate such Yin deficiency symptoms as dysphoria in chestpalms-soles, weight loss, soreness and weakness of waist and knees, immunologic dysfunction and cyclic nucleotide system disorders.

Animals ; Body Weight ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Male ; Polygonatum ; chemistry ; Rats ; Rats, Sprague-Dawley ; Swimming ; Yin Deficiency ; drug therapy ; physiopathology

Objective To develop a five fluorescence-labeled multiplex amplification system for 15 loci and study genetic polymorphism in Xinjiang Uygur population. Methods The STR loci were screened. The alleles were named according to the number of repeats by sequencing. The sensitivity, species specificity, identity and stability of the five fluorescence-labeled multiplex amplification system for the 15 loci were all tested. Then, the genetic polymorphism was analyzed in Xinjiang Uygur population and compared with other ethnic groups including Xizang Tibetan, Xiuyan Manchu, and Guangzhou Han pop-ulation. Results The 15 loci multiplex amplification system was established. The sensitivity was 0.3 ng with good species specificity, identity and stability. The distributions of genotype for 13 STR loci in Uygur population were in accordance with Hardy-Weinberg equilibrium with no genetic linkage between these loci. Most loci showed statistically significant among different populations. Conclusion The estab-lished system has application value in forensic evidence. The 13 STR loci in Uygur population have high polymorphisms to be the supplements to the existing loci.

Endostatin is a potent, endogenous inhibitor of angiogenesis, which corresponds to the C-terminal fragment of collagen ⅩⅧ. The human endostatin gene was amplified from a human fetal liver cDNA library by means of PCR and was cloned into pPIC9K vector. Soluble endostatin was superexpressed in Pichia pastoris (50.5mg/L) . The recombinant protein was purified by cation exchange chromatography with the final yield of more than 95%. Western blotting analysis showed positive immunoreactivity to the expressed product. Recombinant endostatin was able to suppress the angiogenesis on the CAMs. Also, the endostatin inhibited specifically of the migration of HMECs stimulated by bFGF, with EC50 being about 0.4 ?g/ml.

Objective To investigate the significance of the expression of serum LDH and β2-MG in predicting survival of NHL patients and there correlations with clinical parameters. Methods The serum levels of LDH and β2-MG of 429 patients with NHL were measured by rate method and immunoturbidimetry.Besides,the clinical reference of the patients such as gender,age,B symptoms,clinical stages,extranodal sites and Karnofsky evaluation were summarized and grouped.All patients' survival status were achieved via phone and letter. All statistical analyses were performed using the SPSS program for Windows (version 16.0).Results The levels of LDH and β2-MG in age,B symptom,the advanced stage,extranodal sites≥2,Karnofsky evaluation were obviously higher than their counterpart and the difference was significant (P＜0.001). The median of LDH and β2-MG lever in age group was 229 U/L,190 U/L, 2.4 mg/L,1.7 mg/L. In B symptom was 260 U/L,192 U/L,2.3 mg/L, 1.7 mg/L. In clinical stage was 252 U/L, 175 U/L, 2.5 mg/L, 1.6 mg/L. In extranodal sites group was 278 U/L,195 U/L,2.4 mg/L,1.8 mg/L.In Karnofsky evaluation group was 250 mg/L,195 mg/L,2.2 mg/L,1.8 mg/L.LDH level of invasive lymphoma was significantly higher than that of indolent ones (median 211U/L,175 U/L,P=0.002),but there were no difference in the 32-MG level (P=0.937). There were no statistical significance about LDH and β2-MG levels in gender and cell type (P＞0.05).Overall survival rates were different between the abnormal LDH group and the normal LDH group (χ2=119.029, P＜0.001).Overall survival rates were different between the elevated β2-MG group and the normal β2-MG group (χ2 =104.733,P＜0.001).Overall survival rates of the abnormal LDH/β2-MG group was significantly lower than that of normal group (x2=192.326,P＜0.001).Multivariate analysis in Cox regression showed that LDH,β2-MG,age,clinical stages, extranodal sites, Karnmofsky evaluation and aggressive were independent prognostic factors.Conclusion The level of serum LDH and β2-MG can be taken as an auxiliary clinical index to evaluate the prognosis of the NHL patients.