The specific fragment of Pneumococcal surface protein A(PspA)and Pneumococcal Surface Adhesin A(PsaA)gene was amplified by PCR from Streptococcus pneumonia 5 and Streptococcus pneumonia 19.The amplified fragnent of PspA and PsaA gene was ligated into pET-27b(+)vector and transformed into BL 21 E.coli for expression and obtain the expressive production of PspA and PsaA.Induced by IPTG,the expression level was as high as 75 % of the total disolube protein.The result showed that the recombinant plasmid could express a specific 75 kDa and 37 kDa fusion protein in E.coli BL 21,which showed the good immunogenicity and a broadly cross reactivity with the other serotypes.

OBJECTIVETo investigate the effect of siRNA silencing the role of C-Jun N-terminal Kinase (JNK) gene in excessive endoplasmic reticulum stress on lung ischemia/reperfusion injury.

METHODSMouse model of pulmonary ischemia reperfusion injury (PIRI) in situ was established with unilateral lung in vivo. Seventy experimental mice were randomly allocated into seven groups (n = 10): Sham group (Sham group), ischemia reperfusion group (I/R), PBS+ Lipofectamine2000TM transfection reagent group (I/R + PBS+ Lipo group), negative control group (I/R+ SCR group), JNK-siRNA group (I/R + siRNA(JNK1), siRNA(JNK2), siRNA(JNK3)). Mice were euthanized after experimental time out, and left lung tissue was extracted. Wet/dry lung weight ratio (W/D) and total lung water content (TLW) were tested. Light microscope, alveolar damage quantitative evaluation index (IQA) and electron microscope were observed. The expression levels of JNK and glucose regulatex protein(GRP78) were detected by RT-PCR and Western blot. Apoptosis of lung tissue was determined by TUNEL.

RESULTSCompared with Sham group, all indicators above of I/R + PBS + Lipo group and I/R + SCR group were significantly increased (P < 0.01), and compared with I/R group, those indicators of the three groups all had no notable difference; those indicators were not statistically different between I/R + PBS + Lipo group and I/R + SCR group, and compared to the three groups, the above indicators in JNK-siRNA group were lower (P < 0.05, P < 0.01) except that the expression levels of GRP78 was not statistically different.

CONCLUSIONI/R induces excessive ERS in lung tissue, in which JNK pathway participates in apoptosis, leading to lung tissue injury.

Animals ; Apoptosis ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; metabolism ; JNK Mitogen-Activated Protein Kinases ; genetics ; Lung ; physiopathology ; Lung Injury ; genetics ; MAP Kinase Signaling System ; Mice ; RNA, Small Interfering ; Reperfusion Injury ; genetics

The expression of specific genes in sex chromosomes is the basis of sex-specific membrane protein in mammalian spermatozoa. The gene expression products are shared among spermatozoa through intercellular bridges, however, the phenomena of male transmission-ratio distortion and sex ratio distortion proved that differential proteins exist between X and Y spermatozoa. In addition, the existence of sex-specific proteins was confirmed by the separation experiment of X/Y chromosome bearing spermatozoa and the detection result of sex specific proteins. At the same time, it was also confirmed that the difference of the sex-specific protein is weak . The advance of separation techniques as well as the integration and optimization among these techniques has made it possible to separate sex-specific membrane proteins in mammalian spermatozoa.

Objective: To study the involvement of MAPK MEK/ERK signaling transduction pathway in the apoptosis process of SW620 tumor cell line and the inhibition effect of resveratrol. Methods: SW620 cell lines were divided into 5 groups, namely, control group, PD98059 group, low-dose resveratrol group, mid-dose resveratrol group and high-dose resveratrol group. The inhibition rate of cell proliferation was detected by MTT method. The expression of apoptotic molecules and MEK/ERK signaling pathway related proteins were assayed by real-time PCR and Western blotting. Results: Compared with control group, the proliferation of cells treated with resveratrol was significantly inhibited. In the case of apoptotic molecules, the expression of Bax, Caspase 3 and Caspase 9 was increased significantly while the expression of anti-apoptotic molecule Bcl2 was decreased significantly in resveratrol groups with a dose-dependent manner. In the case of molecules in MEK/ERK signaling pathway, the expression of Ras, Raf, MEK and ERK1/2 was decreased significantly in resveratrol groups with a dose-dependent manner. Conclusions: PD98059 and resveratrol can effectively inhibit the proliferation of SW620 through inhibiting the MEK/ERK signaling pathway.

Ligustrazine, one of the major effective components of the Chinese traditional medicinal herb Ligusticum Chuanxiong Hort, has been reported plenty of biological activities, such as protect cardiovascular and cerebrovascular, neuroprotection and anti-tumor, et al. Because of its remarkable effects, studies on structural modification of ligustrazine have attracted much attention. Ligustrazine synthetic derivatives reported in recent decades are mainly derived from four primary intermediates (TMP-COOH, TMP-OH, TMP-NH2, HO-TMP-OH). To explore the neuroprotection activitiy of ligustrazine intermediates, six ligustrazine intermediates (2, 5, 8, 11, 12, 13) were synthesized and their protective effects against CoCl2-induced neurotoxicity in differentiated PC12 cells were studied. The target compounds were prepared via different chemical methods, including oxidation, substitution, esterification and amidation without changing the structure nucleus of ligustrazine. Compared with TMP (EC50 = 56.03 micromol x L(-1)), four compounds (2, 5, 12 and 13) exhibited higher activity (EC50 < 50 micromol x L(-1)) respectively, of which, compound 2 displayed the highest protective effect against the damaged PC12 cells (EC50 = 32.86 micromol x L(-1)), but target compounds 8 and 11 appeared lower activity (EC50 > 70 micromol x L(-1)). By structure-activity relationships analysis, the introduction of carboxyl, amino to the side chain of ligustrazine and appropriately increase the proportion of ligustrazine may contribute to enhance its neuroprotective activity, which provides a reference for the design, synthesis and activity screening of relevant series of ligustrazine derivatives in the future.
Animals ; Cell Differentiation ; drug effects ; Chemistry Techniques, Synthetic ; Cobalt ; toxicity ; Drugs, Chinese Herbal ; chemistry ; Neuroprotective Agents ; chemical synthesis ; chemistry ; pharmacology ; Neurotoxins ; toxicity ; PC12 Cells ; Pyrazines ; chemical synthesis ; chemistry ; pharmacology ; Rats

BACKGROUNDAlveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis (E. multilocularis) and is a rare but life-threatening disease. This disease commonly is characterized by an infiltrative, tumor-like growth of the E. multilocularis metacestode in the liver of human. Liver transplantation is an effective therapy for end-stage of hepatic AE, but the characteristics of host immunity associated with E. multilocularis infection with organ transplantation are poorly defined. We hereby aimed to study the immunological status and allograft heart survival in inbred rats with E. multilocularis infection.

METHODSRat models of AE were established by injecting the E. multilocularis suspension made from E. multilocularis infected tissues into the abdomen of Lewis (LEW) rats. Three months later, in the experimental group, allograft heart transplantation was performed from Brown-Norway (BN) rats to the E. multilocularis infected LEW rats. In the control group, we transplanted hearts from BN rats to healthy LEW rats. The influence of the disturbed immune system in E. multilocularis infected rats on the heart transplantation was assessed, including observation of allograft heart survival time, histopathological examination of grafts and immunohistochemical examination of infiltrating cells (CD4(+) T cells, CD8(+) T cells and eosinophile granulocytes), measurement of interleukin (IL)-4 and interferon (IFN)-γ in the serum by enzyme-linked immunosorbent assay (ELISA) and analysis of CD4(+)CD25(+) regulatory T cells in peripheral blood by fluorescence activated cell sorting (FACS) flow cytometric analysis.

RESULTSThe survival time of recipients in the experimental group was prolonged compared with those in the control group. The numbers of graft infiltrating CD8(+) T cells were decreased whereas the graft infiltrating eosinophil granulocytes (CD15(+)) were increased in grafts in the experimental group (P < 0.05). Furthermore, the proportion of CD4(+)CD25(+) regulatory T cells in the peripheral blood was 10.8% on average in the experimental group, which was significantly higher than that in the control group (6.1%). In addition, the level of serum IL-4 in E. multilocularis infected rats was higher than that in the control group rats, whereas the level of serum IFN-γ in experimental group was lower than that in the control group when graft rejection occurred (P < 0.05).

CONCLUSIONSThis study suggests that E. multilocularis infection could prolong the allograft survival time through the polarization of Th1/Th2-type cells and induction of CD4(+)CD25(+) regulatory T cells. This strategy may provide a new idea for establishing transplantation tolerance.

Animals ; Echinococcosis ; blood ; immunology ; Echinococcus multilocularis ; immunology ; pathogenicity ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Gerbillinae ; Heart Transplantation ; Immunohistochemistry ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Male ; Rats

In this study, daphnetin and its major metabolites in the intestinal wall of rats were identified by liquid chromatography and quatrupole-time of flight mass spectrometry. Perfusion fluid of duodenum, jejunum, ileum and colon were collected separately for 2 hours from the rat intestine following perfusion with daphnetin. The metabolites of daphnetin in the perfusion fluid of different intestine segments were analyzed by the liquid chromatography and quatrupole-time of flight mass spectrometry. It is shown that the parent drug daphnetin and four metabolites were found in the perfusion fluid of duodenum, jejunum and ileum. However, no metabolites were found in the colon. Among the four metabolites, two daphnetin sulfates (m/z 257) were first discovered as the phase II metabolites of daphnetin in rats, which revealed a new way of daphnetin metabolism in rats.
Animals ; Chromatography, High Pressure Liquid ; Colon ; metabolism ; Duodenum ; metabolism ; Ileum ; metabolism ; Intestines ; metabolism ; Jejunum ; metabolism ; Male ; Perfusion ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spectrometry, Mass, Electrospray Ionization ; Umbelliferones ; metabolism ; pharmacokinetics

BACKGROUNDTo investigate the mutation of HBV polymerase gene in chronic hepatitis B patients treated with lamivudine.

METHODSThe restriction-fragment-length-polymorphism (RFLP) assay for HBV DNA sequence determination at the codon 528 and 552 in the HBV polymerase gene associated with lamivudine resistance in vitro. HBV DNA samples extracted from sera of 240 patients were subjected to PCR amplification with primer pairs F2/R2 (552), F3/R2 (528). Each PCR product was digested with Nde I or Nla III.

RESULTSSerum HBV DNA mutation was found in 51/240 patients (38/51M552V, 26/38L528M, 13/51M552I) after therapy for 52 weeks. DNA sequence analysis was performed on samples of 3 patients, and the results were consistent with those of RFLP assay.

CONCLUSIONThe RFLP assay was able to detect the mutation of HBV DNA at codon 552 and 528 which are the principal site of HBV DNA resistant to lamivudine. The specific PCR method for HBV DNA mutation is rapid, simple and specific.

Drug Resistance, Viral ; Gene Products, pol ; genetics ; Hepatitis B virus ; enzymology ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; therapeutic use ; Mutation ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length ; Reverse Transcriptase Inhibitors ; therapeutic use

OBJECTIVETo develop an approach to the determination of saponins in Radix Cynanchi Atrati, and to optimize the parameters for purified the preparation of total saponins by macroporous resin column chromatography.

METHODUsing cynanversicoside A as a reference, the determination of saponins was performed; according to the elution rate and the purity of the products, the preparation performance of total saponins by macroporous resin was investigated, and its parameters were optimized.

RESULTThe saponins in Radix Cynanchi Atrati were successfully determined at 518 nm by vanillin-perchloric acid as spray reagent. The macroporous resin HP-20 showed static absorption ratio of 59. 3 mg x g(-1); the 70% ethanol extraction of Radix Cynanchi Atrati was eluted from column of macroporous resin HP-20 by water and 30% ethanol, and the saponins were concentrated in 90% ethanol solution. The content of saponin part eluted from HP-20 column was 77.62%.

CONCLUSIONThe proposed approach allows convenient and efficient preparation and purification of saponin in Radix Cynanchi Atrati.

Absorption ; Benzaldehydes ; chemistry ; Calibration ; Cynanchum ; chemistry ; Ethanol ; chemistry ; Perchlorates ; chemistry ; Porosity ; Reproducibility of Results ; Resins, Plant ; chemistry ; Saponins ; chemistry ; isolation & purification ; Sensitivity and Specificity