Protective effects of histone deacetylase inhibitor on stress-induced myocardial injury in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the protective effects of histone deacetylase inhibitor on stress-induced myocardial injury.</p><p><b>METHODS</b>Healthy male Wistar rats were randomly divided into 3 groups( n = 6), and the stress-induced myocardial injury model was established with chronic restraint stress method. The protective effects of histone deacetylase inhibitor on stress-induced myocardial injury were observed with Trichostatin A (TSA) intervention. Histone acetylation levels in myocardium of rats were detected by Western blot method, spectrophotometry method was used to dynamically determine the activity of rat serum lactate dehydrogenase (LDH), serum creatine kinase isoenzyme-MB (CK-MB) and Caspase 3, and nagar Olsen staining were used to observe the early myocardial damage.</p><p><b>RESULTS</b>Restraint stress could significantly reduce the level of histone acetylation of myocardium in rats, and TSA intervention could inhibit the stress-induced reduction of myocardial levels of histone acetylation. Restraint stress could cause the significant increase of serum LDH activity ( P < 0.05), serum CK-MB activity ( P < 0.05), and the Caspase 3 activity of myocardial tissue (P < 0.05), and early myocardial damage also occurred during restraint stress. ISA intervention could significantly reduce the serum LDH activity (P < 0.05), the serum CK-MB activity (P < 0.05), the activity of myocardial tissue caspase 3 induced by restraint stress (P < 0.05), and the stress-induced myocardial injury was also attenuated by TSA intervention.</p><p><b>CONCLUSION</b>The histone deacetylase inhibitor TSA can protect stress-induced myocardial injury.</p>

The construction and application of a novel apparatus for detecting oxygen consumption of mice under normobaric hypoxia / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To establish a method for real-time recording the oxygen consumption of mice under normobaric hypoxia.</p><p><b>METHODS</b>The experimental apparatus was made up of animal container, filling water control system, electronic balance, hose, a computer with weight recording software, etc. The working principle was that the oxygen consumed by animal was replaced by water filling which was controlled by the pneumatic and hydraulic actuator. The water was weighted by an electronic balance and the weight signal was recorded into excel file at the same time. The accuracy and precision of the apparatus were detected by a 10 ml syringe. The oxygen consumption characteristics of 6 acute repetitive hypoxia mice and 6 normal mice were observed.</p><p><b>RESULTS</b>The P value for the paired t test was 1 and the CV value was 4%. The survival time and total oxygen consumption of acute repetitive hypoxia mice were both significantly increased compared to normal mice (P < 0.05), which were (58.8 +/- 6.8) min and (46.0 +/- 8.7) min respectively for the survival time and (85.1 +/- 8.5) ml and (73.6 +/- 5.4) ml respectively for total oxygen consumption.</p><p><b>CONCLUSION</b>The hypoxia tolerance of the acute repetitive hypoxia mice can significantly improved by taking more oxygen in the animal cabin. The accuracy and precision of the apparatus are high and it can be used for the determination of oxygen consumption in hypoxia research.</p>

Establishment of mammalian cell lines for constitutive expression of influenza virus matrix protein 2 / 病毒学报

To establish a mammalian cell line for stable expression of the matrix protein 2 (M2) of influenza virus type A. M2 gene was amplified by PCR from the influenza virus strain A/PR/8/34. The PCR product was cloned into eukaryotic expression vector pcDNA5/FRT. After identification with restriction enzyme digestion, the plasmid was co-transfected with plasmid pOG44 which expressed Flp in Flp-In-CHO cells. The target gene was integrated into chromosome of CHO cells by homologous recombination in vivo. Recombinant CHO-M2 cell lines were selected for hygromycin B resistance. A total of 15 recombinant cell strains with high expression of M2 protein were screened by hygromycin, and the expression of M2 protein was determined by IFA and Western blot. After subculturing for 10 passages, the presence of M2 gene in the CHO-M2 cells was confirmed by PCR, and the expression of M2 protein were proved by IFA and Western blot. We successfully constructed a mammalian cell line which stably expressed M2 protein of influenza virus type A. The cell line will be useful for studies on function of M2 protein and provide tools for novel influenza virus vaccine development.

Construction of recombinant baculovirus co-expressing M1 and HA of influenza A virus / 病毒学报

The M1 and HA genes of H1N1 influenza virus were amplified and then cloned into the pFastBac dual donor plasmid. The recombinant pFastBac Dual-M1-HA was identified by restriction enzyme digestion. After the pFastBacdual-M1-HA was transformed into the baculovirus shuttle plasmid (bacmid) in DH10Bac competent cells, the colonies were identified by antibiotics and blue-white selection. The rBac-mid-M1-HA was verified by PCR and transfected into S f9 cells to produce recombinant baculovirus (rBac-M1-HA). Gene insertion of rBac-M1-HA was verified and the expression of M1 and HA genes was analyzed by IFA and Western-blot, demonstrating M1 and HA were co-expressed successfully. This study provides the foundation for researching the formation mechanism of influenza VLP and developing new influenza vaccines.

Construction of vectors expressing M2 and NA genes of H5N1 influenza virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct vectors expressing M2 and NA genes of H5N1 influenza virus.</p><p><b>METHODS</b>Based on the human H5N1 avian influenza virus (A/Anhui/1/2005) isolated in china, M2 and NA genes were amplified by PCR. M2 or NA gene was subcloned into pStar vector to construct recombinant pStar-M2/, pStar-/M2, pStar-NA/and pStar-NA/. Furthermore, both of the M2 and NA genes were subcloned into pStar to construct two genes co-expressing recombinant pStar-M2/NA and pStar-NA/M2. Expression of the genes were detected by IFA after transfection of 293 cells with the recombinant plasmids.</p><p><b>RESULTS</b>Recombinant plasmids were constructed and identified by restriction endonuclease digestion. Expression of the genes cloned into the recombinant plasmids was confirmed by IFA.</p><p><b>CONCLUSION</b>Recombinant plasmids expressing M2 and/or NA genes of H5N1 influenza virus were constructed, which provided basis for development of influenza DNA vaccine.</p>

Subcloning of M1 gene fragment of H5N1 influenza virus and its expression in Escherichia coli / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To generate the Escherichia col vector expressing human H5N1 influenza virus M1 protein. To provide useful tools for detection of human H5N1 influenza virus and study on biological function of M1 protein.</p><p><b>METHODS</b>M1 gene fragment was amplified by PCR using the influenza virus gene segment 7 as template, and was subcloned into pQE80-L vector. The recombinant plasmid pQE80-L/M1 was transformed into Escherichia coil BL21 (DE3) strain. The expression of M1 was induced by isopropy-beta3-D-thiogalactopyranoside. We purified the recombinant M1 protein with polyhistidine tag with Ni2+ affinity chromatography. Mouse were immunized with the purified M1 protein for preparing antibodies against M1.</p><p><b>RESULTS</b>The recombinant Ml protein was recognized by antiserum against H5N1 subtype influenza virus, elicit specific antibody in immunized animals.</p><p><b>CONCLUSION</b>These confirmed that we successfully constructed the Escherichia coli vector expressing human H5N1 influenza virus M1 protein.</p>

Construction, expression and immunogenicity analysis of a fusion protein containing M2e of influenza A virus fused to a modified Pseudomonas aeruginosa exotoxin A / 病毒学报

M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.

Epidemiologic and clinical characteristics of 59 persons suspecting of being infected by the HIV virus despite having repeated negative laboratory findings / 中华流行病学杂志

Objective To describe the epidemiologic characteristics and clinical manifestations of 59 persons recruited via an intemet chat group who complained of AIDS-like symptoms, so as to formulate effective intervention strategies and measures. Methods Case was defined as onset of any three of the following self-reported AIDS-like symptoms in a member of relevant "intemet chat groups": persistent low grade fever, rash, swollen lymph node, fatigue, diarrhea, weight loss and low CD4+T count. We administered an internet-based questionnaire, and invited 59 of the 88 casepersons for voluntary physical examination and laboratory testing. Results The 59 case-persons came from 22 provinces; 54 (91.5 %)were men; the median age was 34 (range: 22-53)years; 84.7% of them had high-risk sexual behaviors before the onset of self-reported symptoms. The median time interval from exposure to onset was 15 d (range: 1-365 d). Blood specimens for all the 59 case-persons were tested negative for HIV and syphilis antibodies. There was also no evidence of Xenotropic murine leukemia virus-related virus infection. One case-person was tested positive for hepatitis C virus antibody. The average CD4'T lymphocyte count was 707/μl. Of the 59 case-persons,57 (96.6%) sought medical care from multiple providers; 40 were diagnosed to have no physical disorders. Conclusion None of the 59 case-persons had any evidence of infection with HIV or any other infectious agents that could explain their self-reported symptoms.

Construction of recombinant adenovirus co-expressing M1 and HA genes of influenza virus type A / 病毒学报

Based on the human H5N1 influenza virus strain A/Anhui/1/2005, recombinant adenovirus co-expressing M1 and HA genes of H5N1 influenza virus was constructed using an internal ribosome entry site (IRES) sequence to link the two genes. The M1 and HA genes of H5N1 influenza virus were amplified by PCR and subcloned into pStar vector separately. Then the M1-IRES-HA fragment was amplified and subcloned into pShuttle-CMV vector, the shuttle plasmid was then linearized and transformed into BJ5183 bacteria which contained backbone vector pAd-Easy. The recombinant vector pAd-Easy was packaged in 293 cells to get recombinant adenovirus Ad-M1/HA. CPE was observed after 293 cells were transfected by Ad-M1/HA. The co-expression of M1 and HA genes was confirmed by Western-blot and IFA (immunofluorescence assay). The IRES containing recombinant adenovirus allowed functional co-expression of M1 and HA genes and provided the foundation for developing new influenza vaccines with adenoviral vector.

Study on preparation and identification of monoclonal antibodies immunized with H5N1 influenza virus M1 protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies specific for M1 protein of H5N1 subtype human influenza virus, this work may provide new tool in rapid diagnosis and study of type A influenza virus.</p><p><b>METHODS</b>BALB/c mice were immunized with purified recombinant H5N1 (A/Anhui/1/2005)/M1 protein expressed in E. coli. Spleen cells of the immunized mice were fused with sp2/0 cells to produce hybridoma cell lines. ELISA was performed to identify the monoclonal antibody against M1 protein of H5N1. Immunofluorescence assay (IFA) were applied to identify the specificity of these antibodies.</p><p><b>RESULTS</b>Three hybridoma cell lines steadily secreting anti-H5N1/M1 McAb were obtained, and their cross reactivity was confirmed by cross-reaction test and IFA.</p><p><b>CONCLUSION</b>Monoclonal antibodies immunized with H5N1 subtype influenza virus M1 protein are cross-reactive, which can be used to detect different subtype of influenze virus type A.</p>

Effects of human HSF1 on transcriptional regulation of prohibitin promoter / 中国应用生理学杂志

<p><b>AIM</b>To assay the transcriptional regulation effect of hHSF1 on prohibitin gene promoter.</p><p><b>METHODS</b>The total length of hHSF1 exon was amplified by PCR method and cloned into pcDNA3. 1(+) vector. pcDNA3. 1(+)-hHSF1 and pGI3 prohibitin were co-transfected into HEK293 cells. The luciferase activity was detected by Dual-Luciferase Reporter Assay System to evaluate the transcriptional regulation effect of hHSF1 on prohibitin gene promoter.</p><p><b>RESULTS</b>The pcDNA3.1(+)-hHSF1 vector was constructed successfully. The assay of luciferase activity showed that the transcription of pGL3-prohibitin was dramatically upregulated by hHSF1.</p><p><b>CONCLUSION</b>hHSF1 can transcriptionally regulate prohibitin expression.</p>

Construction and immunogenicity study of DNA vaccine expressing human H5N1 influenza virus hemagglutinin / 病毒学报

Based on the first isolated human H5N1 influenza virus strain A/Anhui/1/2005 in China, HA and HA1 genes were amplified and cloned into the eukaryotic expression vector pStar. The recombinant plasmids pStar-HA and pStar-HA1 were transfected into COS7 cells. Western blot and IFA showed that the two recombinant DNA plasmids were successfully expressed in eukaryotic cells. BALB/c mice were immunized with the plasmids DNA by intramuscular injection. Anti-HA specific antibody in peripheral blood of immunized mice was tested by ELISA. The results showed that the recombinant plasmids successfully induced the anti-HA humoral immune response, and there was no significant difference between HA and HA1 as immunogen. This work provides basis for future development of novel avian flu vaccine.

Expression of sTNFR-IgGFc fusion gene in endothelial cell and its application in gene therapy for rheumatoid arthritis / 生物工程学报

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine, acting as a regulator of inflammation and immunity. TNFalpha plays a critical role in the pathogenesis of rheumatoid arthritis. Blocking of TNFa activity suppressed inflammatory tissue damage. In present study, the chimeric gene of soluble TNF receptor and IgG Fc fragment (sTNFR-IgG FC) was cloned into the mammalian cell expression vector pStar. When the plamid pStar/sTNFR-IgGFc-GFP was transfected into endothelial cells, a considerable expression of the sTNFR-IgG Fc fusion protein was detected. Moreover, the product in 100microL expression supernatant could completely antagonize the cytolytic effect of 1ng TNFa on L929 cells, even at 1/64 dilution. Then the plasmid was delivered into CIA-induced rheumatoid arthritis mice by tail vein injection. The expression of sTNFR-IgG Fc was detected in liver by RT-PCR. Animals in treatment group showed reduced symptoms of arthritis and more active. This treatment induced decrease of synovial incrassation and prevented the cartilage destruction of the mice RA model. These results show that tail vein injection is an effective way for gene therapy and sTNFR-IgGFc expression plasmid is potential for the treatment of rheumatoid arthritis.

Research on the physical components stimulated by rotating grating using functional magnetic resonance imaging / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: Using functional magnetic resonance imaging, to inspect v sual cortex physical reactions stimulated by rotating grating, and to its components. METHODS: On 1.5T MR scanner, GRE-EPI imaging sequence was carried on 9 h discover ealthy volunteers, the visual cortex response data were processed after delinearation by SPM99.RESULTS: Different components of rotating grating excited different areas of the visual cortex. Dramatic response in the central part of the occipital lobe, which was related to white light stimuli, located at primary visual cortex. Response area in bilateral Broadaman 19 areas was related to vision-motion function. And weak response area in the central part of the occipital lobe was related to shape perception.CONCLUSION: Rotating grating conclude plenty of visual information, and it excites different areas of the optic center as a stimuli. fMRI is a valuable equipment to study the physiology of visual cortex.

Screening for new binding proteins which interact with BM2 of influenza B virus with yeast two-hybrid system / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the role of BM2 protein in the life cycle of influenza B virus.</p><p><b>METHODS</b>The authors screened human kidney MATCHMAKER cDNA library for new binding partners of BM2 of influenza B virus by using the yeast two hybrid system with truncated BM2 (26-109 aa) as the bait.</p><p><b>RESULTS</b>Six positive plasmids encoding N-acetylneuraminate pyruvate lyase, angiopoietin 3, zinc finger protein 251, ribosomal protein S20, protein arginine N-methyltransferase 1 variant 1 (PRMT) and transcription factor-like 1 (TCFL1) were obtained.</p><p><b>CONCLUSION</b>The results suggest that BM2 may play an important role in the life cycle of influenza B virus.</p>

Progress of EBNA1/oriP-based plasmid applied in gene therapy / 生物工程学报

The nonviral gene delivery systems are usually not very effective in transferring gene into target cells, and the intensity and duration of the gene expression is very poor. The EBNA1/oriP maintain EBNA1/oriP-based plasmids as episome, contribute to nuclear transport of the plasmid and transcriptional up-regulation of target gene. The EBNA1/oriP based plasmid enhances the transfection rate as well as magnitude and longevity of gene expression. This article reviews recent preclinical gene therapy studies with the EBV plasmid vectors conducted against various diseases. For gene therapy against malignancies, the EBNA1/ oriP based plasmid encoding the HSV1-TK suicide gene was combined with a cationic polymer to transfer into HCC cell line. The expression level of TK gene was 100- to 1000-fold higher than the conventional plasmid. The sensitivity of HCC to ganciclovir (GCV) elevated several hundred-fold. The EBNA1/oriP based plasmid equipped with tumor specific promoter, such as CEA promoter, enabled targeted killing of CEA-positive tumor cell. Transfection of EBNA1/oriP based plasmid carrying IL-12 and IL-18 gene either locally, or systemically, induced therapeufic antitumor immune responses including augmentation of the cytotoxic T lymphocyte and natural killer activities and growth retardation of tumors. For gene therapy of congenital diseases and chronic diseases, the EBNA1/oriP based plasmid encoding the adenosine deaminase gene was transfered into human hematopoietic progenitor cells. The ADA activity was elevated 1.5-to 2-fold. Intracardiomuscrlar transfer of the EBNA1/oriP based plasmid encoding the beta-AR gene may be useful for the treatment of severe heart failure. Human tumor necrosis factoralpha (hTNFalpha) is one of the most important inflammatory cytokines. It has been implicated in many autoimmune and inflammatory diseases. sTNFR can efficiently neutralize the bioactivities of hTNFalpha. In primary study we cloned the chimeric protein sTNFR II-IgG Fc and expect to use it in the gene therapy of the inflammatory disease relative to TNF. In summary, The EBNA1/oriP based plasmid shows advantage in gene therapy of cancer, congenital and inflammatory diseases. Moreover, the EBNA1/oriP element may greatly contribute to the engineering of a human artificial chromosome, the ultimate device for controllable gene therapy.

Cytokine aptamers as therapeutic and diagnostic agents / 生物工程学报

Aptamers are oligonucleotides derived from an in vitro evolution process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers specially binding to targets could recognize and inhibit the function of targets. Using this method, many powerful antagonists of cytokines have been found. In order for these antagonists to work in animal models of disease and in humans, it is necessary to modify the aptamers. First of all, 2'-F, 2'-NH2 and 2'-CH3O modifications of nucleoside triphosphates could prolong half-lives in blood. Aptamers can be kept in the circulation from hours to days by conjugating them to higher molecular weight vehicles. After modified, conjugated aptamers are injected into animals, they inhibit physiological functions known to be associated with their target cytokines. Exhibiting binding characteristics comparable to or even better than monoclonal antibodies, these ligands can be used as detection probes, highly efficient inhibitors of protein function or specific competitors in high-throughput screening (HTS) assays. Recently several aptamers of cytokines have been characterized. Some of them have been used as diagnostic agent for the detection of target cytokines. The first aptamer that has proceeded to phase II clinical studies is NX-1838, an injectable angiogenesis inhibitor that can be potentially used to treat macular degeneration-induced blindness. Aptamers will be versatile tools that can greatly enhance the efficiency of modern diagnose and therapy development.

Novel methods to detect cytokines by enzyme-linked oligonucleotide assay / 生物工程学报

The development of the systematic evolution of ligands by exponential enrichment (SELEX) process has made it possible to isolate oligonucleotide sequences with the capacity of recognizing virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers", are useful as a class of molecules that rival antibodies in diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. To meet the shortcomings of antibodies, aptamers have the following advantages. Aptamer does not depend on animals, cells, or even in vivo conditions and produced by chemical synthesis with extreme accuracy and reproducibility. Once denatured, functional aptamers could be regenerated easily within minutes. They are stable to long-term storage and can be transported at ambient temperature. We describe here an enzyme -linked oligonucleotide assay that use a SELEX-derived RNA aptamer to detect hTNFalpha. In order to protect from nuclease attack, the RNA aptamer was modified by replacement of 2'-NH2 for 2'-OH at all ribo-purines. In a sandwich micro-plate assay, hTNFalpha monoclonal antibody was coated on the surface of the plate, biotin-labeled RNA aptamer was used as a detect molecle. HTNFalpha was diluted by pooled human serum as standard, and streptavidin-horseradish peroxidase-substrate system was added for detection. Accuracy, precision, sensitivity, specificity of ELONA method were analyzed. The levels of hTNF-alpha in normal human serum samples were assayed by the ELONA and the ELISA processes. The resultes demonstrate that a sandwich assay using a SELEX-derived RNA aptamer has parameters for accuracy, precision, sensitivity, specificity well within the limits expected of a typical enzyme-linked assay. There is no significant difference between the results of ELONA and ELISA. The minimum detection level was 100 pg/mL. This method will be useful for detection of almost all the cytokines and other protein molecules.

Construction, expression and bioactivity characterization of targeting toxin DT-VEGF / 生物工程学报

Tumor rapid growth depends on neovascularization. Vascular endothelial growth factor, the main mediator during the occurrence and formation of vascularization, has specific receptors whose expression rate shows difference of orders of magnitude between tumor and the normal tissue, so it can be used to transport toxin molecules to the proliferative tumor endothelial and kill cancer cells. In our experiment, we constructed fusion protein DT-VEGF by linking diphtheria toxin's forward 389 amino acids's gene and VEGF165 via a linker. DT-VEGF is expressed in E. coli and purified. Our experiment proves in can kill vascular endothelial cells specifically, and the inhibition of neovascularization of chicken chorionic membrane is also confirmed.