3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。     

3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）北大核心CSCD

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。     

Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

The apoptosis of lens epithehal cells has been proposed as the common basis of cataract formation, with oxidative stress as the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolide against oxidative stress-induced apoptosis of human lens epithelial （HLE） cells and the possible molecular mechanisms involved. HLE cells （SRA01-04） were incubated with 50 μM H2O2 in the absence or presence of different doses of parthenolide （10, 20 and 50 μM）. To study apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expression of caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 and caspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H202 for 18 h, a high fraction of riLE cells underwent apoptosis, while in the presence ofparthenolide of different concentrations, dose-dependent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H202 in HLE cells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation ofcaspase-3 and caspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLE cells from oxidative stress-induced apoptosis through inhibition of the activation ofcaspase-3 and caspase-9, suggesting a potential protective effect against cataract formation.     

Antiviral Effect of Interferon-Induced Guanylate Binding Protein-1 against Coxsackie Virus and Hepatitis B Virus B3 in Vitro

Guanylate binding protein-1(GBP-1)is an interferon-induced protein.To observe its antiviral effect against Hepatitis B virus(HBV)and Coxsackie virus B3(CVB3),we constructed an eukaryotic expression vector of human GBP-1(hGBP-1).Full-length encoding sequence of hGBP-1 was amplified by long chain RT-PCR and inserted into a pCR2.1 vector,then subcloned into a pCDNA3.1(-)vector.Recombinant hGBP-1 plasmids and pHBV1.3 carrying 1.3-fold genome of HBV were contransfected into HepG2 cells,and inhibition effect of hGBP-1 against HBV replication was observed.Hela cells transfected with recombinant hGBP-1 plasmids were challenged with CVB3,and viral yield in cultures were detected.The results indicated that recombinant eukaryotic expression plasmid of hGBP-1 was constructed successfully and the hGBP-1 gene carried in this plasmid could be efficiently expressed in HepG2 cells and Hela cells.hGBP-1 inhibit CVB3 but not HBV replication in vitro.These results demonstrate that hGBP-1 mediates an antiviral effect against CVB3 but not HBV and perhaps plays an important role in the interferon-mediated antiviral response against CVB3.     

Phenotypic Characterization of Porcine IFNc-Producing Lymphocytes in Porcine Reproductive and Respiratory Syndrome Virus Vaccinated and Challenged Pigs

Porcine reproductive and respiratory syndrome(PRRS) continues to be one of the most important swine diseases worldwide. Interferon-γ(IFNγ)-mediated type Ⅰ cell-mediated immune response plays an important role in protection from,and clearance of, PRRS virus(PRRSV). Several lymphocyte subsets including T-helper, CTLs, Th/memory cells, and cd T lymphocytes were previously reported to produce IFNc during PRRSV infection. However, the proportion and phenotypic characterization of these IFNγ-secreting lymphocytes have not been explored. In this study, IFNc producted by different lymphocyte subsets was assessed by multi-color flow cytometry after vaccination with PRRSV modified live vaccine(PRRSV-MLV) and challenge with homogeneous or heterogeneous PRRSV. The results showed that T-helper cells were the major IFNγ-secreting cell population after PRRSV-MLV vaccination and PRRSV challenge. Additionally, the proportion of IFNγ producing Th/memory cells and γδ T cells increased after PRRSV challenge. This difference was accounted for an enhanced ability to produce IFNγ in Th/memory cells and an enlarged quantity of γδ T cells. The results presented here could contribute to our understanding of the roles of IFNγ in protective immunity against PRRSV infection and may be useful for assessment of cell-mediated immunity in vaccine tests.     

Effects of Nitrogen Source and Bacterial Elicitor on Isoflavone Accumulation in Root Cultures of Albizzia kalkora （Roxb.） Prain

Changes in cellular isoflavone （daidzein and genistein） contents were monitored in root cultures of Albizzia kalkora （Roxb.） Prain after feeding different ratios of NH4^＋/NO3^- and treatment with a biotic elicitor （three strains of Rhizobium sp.）. The NH4^＋/NO3^- ratio appears to be positively correlated with daidzein content in the roots and shows a negative correlation with genistein. Among the three different strains of Rhizobium used, the strain ATCC 15834 caused a 35% increase in daidzein production by infection. In the case of genistein, maximum production （94%） was obtained when cultures were treated on Day 6 by the strains ATCC 15834 and KCTC 1541. The biosynthetic pathway of the two isoflavones apparently reacts differently to the same culture conditions and the same strains of Rhizobium. Therefore, the present data suggest that the production of daidzein and genistein could be modulated by changing the NH4^＋/NO3^- ratio and the application of Rhizobium.     

Water Channels Are Involved in Stomatal Oscillations Encoded by Parameter-Specific Cytosolic Calcium Oscillations

Earlier studies have shown that various stimuli can induce specific cytosolic calcium （[Ca^2＋]cyt） oscillations in guard cells and various oscillations in stomatal apertures. Exactly how [Ca^2＋]cyt oscillation signaling functions in stomatal oscillation is not known. In the present study, the epidermis of broad bean （Vicia faba L.） was used and a rapid ion-exchange treatment with two shifting buffers differing in K^＋ and Ca^2＋ concentrations was applied. The treatment for fivetransients at a 10-min transient period induced clear and regular stomatal oscillation. However, for other transient numbers and periods, the treatments induced some Irregular oscillations or even no obvious oscillations in stomatal aperture. The results indicate that stomatal oscillation Is encoded by parameter-specific [Ca^2＋]cyt oscillation： the parameters of [Ca^2＋]cyt oscillation affected the occurrence rate and the parameters of stomatal oscillation. The water channel inhibitor HgCl2 completely Inhibited stomatal oscillation and the inhibitory effect could be partially reversed by β-mercaptoethanol （an agent capable of reversing water channel inhibition by HgCl2）. Other Inhibitory treatments against Ion transport （i.e. the application of LaCIs, EGTA, or tetraethylammonlum chloride （TEACI）） weakly impaired stomatal oscillation when the compounds were added after rapid ion-exchange treatment. If these compounds were added before rapid-ion exchange treatment, the inhibitory effect was much more apparent （except In the case of TEACI）. The results of the present study suggest that water channels are involved In stomatal oscillation as a downstream element of [Ca^2＋]cyt oscillation signaling.     

Selective proliferation of human γδ T cells in vitro

The effect of monoethylphosphate (MEP,commercial available or synthesized) together with IL-2 on the selective proliferation of human γδT cells in vitro from peripheral blood mononuclear cells (PBMC) of healthy donors and of cancer patients was investigated.The γδT cells were stimulated by MEP to proliferate in a dose-dependent manner.The effect of synthesized MEP was 10 times greater than that of commercial MEP.When the PBMCs of healthy donors were cultured for 25 d in the medium containing different concentrations of MEP,the total cell number increased about 1000-3000 fold;and the ratio of γδT cells reached to 70-80%.The selective expansion of γδT cells depended on the synergic action of MEP and IL-2.The bulk cultured γδT cells exhibited obvious cytotoxic activities against allogenic tumor cell lines (SQ-5,K562 and Daudi) and autologous tumor cells.The culture system described here not only offers a simple method for obtaining a large number of γδT cells which may become a new effector in the adoptive immunotherapy,but also provides a useful model for the further studies of the structure and function of γδT cells in vitro.     

Biological character of human adipose-derived adult stem cells and influence of donor age on cell replication in culture

To investigate the biological character of human adipose-derived adult stem cells (hADAS cells) when cultured in vitro and the relationship between hADAS cell’s replication activity and the donor’s age factor, and to assess the stem cells as a new source for tissue engineering. hADAS cells are isolated from human adipose tissue of different age groups (from adolescents to olds: <20 years old, 21―40 years old, 41―60 years old and >61 years old groups). The protein markers (CD29, CD34, CD44, CD45, CD49d, HLA-DR, CD106) of hADAS cells were detected by flow cytometry (FCM) to identify the stem cell, and the cell cycle was examined for P20 hADAS cells to evaluate the safety of the subculture in vitro. The generative activity of hADAS cells in different age groups was also examined by MTT method. The formula “ log2T D = t logN t ? logN 0” was used to get the time doubling (TD) of the cells. The results showed that the cells kept heredity stabilization by chromosome analysis for at least 20 passages. The TD of these cells increased progressively by ageing, and the TD of the <20 years old group was lower than that of the >61 years old group (statistical analysis of variance (ANOVA), P=0.002, P<0.05). These find- ings suggested that a higher level of hADAS cells replication activity was found in the younger dona- tors, and they represent novel and valuable seed cells for studies of tissue engineering.     

Biological character of human adipose-derived adult stem cells and influence of donor age on cell replication in culture

To investigate the biological character of human adipose-derived adult stem cells (hADAS cells) when cultured in vitro and the relationship between hADAS cell's replication activity and the donor's age factor, and to assess the stem cells as a new source for tissue engineering, hADAS cells are isolated from human adipose tissue of different age groups (from adolescents to olds ＜20 years old, 21-40years old, 41-60 years old and ＞61 years old groups). The protein markers (CD29, CD34, CD44, CD45,CD49d, HLA-DR, CD106) of hADAS cells were detected by flow cytometry (FCM) to identify the stem cell,and the cell cycle was examined for P20 hADAS cells to evaluate the safety of the subculture in vitro.The generative activity of hADAS cells in different age groups was also examined by MTT method. The formula "TD = t log2/logNt - logN0 "was used to get the time doubling (TD) of the cells. The results showed that the cells kept heredity stabilization by chromosome analysis for at least 20 passages. The TD of these cells increased progressively by ageing, and the TD of the ＜20 years old group was lower than that of the ＞61 years old group (statistical analysis of variance (ANOVA), P=-0.002, P＜0.05). These findings suggested that a higher level of hADAS cells replication activity was found in the younger donators, and they represent novel and valuable seed cells for studies of tissue engineering.     

Bcl-2 down modulation in WEHI-3B/CTRES cells resistant to Cholera Toxin （CT）-induced apoptosis

The very different effects of Cholera Toxin （CT） on cell growth and proliferation may depend on the type of ganglioside receptors in cell membranes and different signal transduction mechanisms triggered, but other functions related to the drug resistance mechanisms can not be excluded. The effect of CT treatment on the ＂in vitro＂ clonogenicity, the Population Doubling Time （PDT）, apoptosis, PKA activation and Bax and Bcl-2 expression was evaluated in WEHI-3B cell line and its CT-resistant subclone （WEHI-3B/CTRES）. In WEHI-3B parental cells the dramatic accumulation of cAMP induced by CT correlated well with PKA activation, increased PDT value, inhibition of clonogenicity and apoptosis. H-89 treatment inhibited PKA activation by CT but did not protect the cells from apoptosis and growth inhibition. In WEHI-3B/CTRES no significant CT-dependent accumulation of cAMP occurred with any increase of PKA activity and PDT. In CT resistant cells （WEHI-3B/CTRES）, Bcl-2 expression was down regulated by both CT or drug treatment （eg, ciprofloxacin, CPX） although these cells were protected from CT-dependent apoptosis but not from drug-induced apoptosis. Differently from other cell models described, down regulation of Bcl-2 is proved to be independent on cAMP accumulation and PKA activation. Our observations support the implication of cAMP dependent kinase （PKA） in the inhibition of WEHI-3B cells growth and suggest that, in WEHI-3B/CTRES, Bcl-2 expression could be modulated by CT in the absence of cAMP accumulation. Also in consideration of many contradictory data reported in literature, our cell models （of one sensitive parental cell strain and two clones with different uncrossed specific resistance to CT and CPX） provides a new and interesting tool for better investigating the relationship between the CT signal transduction mechanisms and Bcl-2 expression and function.     

Hepatitis B surface antigen expression in NT-1 cells of tobacco using different expression cassettes

Nicotiana tabacum 1 (NT-1) cells were transformed with four different expression cassettes of hepatitis B surface antigen (HBsAg). The transformed nature of the cells was confirmed by polymerase chain reaction (PCR). The expression levels were assayed by enzyme linked immunosorbent assay (ELISA). The expressivities varied among the different cassettes and the maximum expression of 16.6 ng g⁻¹(f.m.) of cells was noted in pEFEHER transformed cells. Salicylic acid (100 μM) treatment resulted in 1.8 fold increase of expression in pEFEHBS transformed cells. The effect of different concentrations of kanamycin and geneticin was studied on the growth of transformed cells and HBsAg expression. The cell growth was optimum at lower concentrations of the antibiotics, and the maximum expression was noted at 200 mg dm⁻³ of kanamycin.     

Association of swine influenza HIN1 pandemic virus （SIV- HINlp） with porcine respiratory disease complex in sows from commercial pig farms in Colombia

Porcine respiratory disease complex （PRDC） is a serious health problem that mainly affects growing and finishing pigs. PRDC is caused by a combination of viral and bacterial agents, such as porcine reproductive and respiratory syndrome virus （PRRSV）, swine influenza virus （SIV）, Mycoplasma hyopneumoniae （Myh）, Actinobacillus pleuropneumoniae （APP）, Pasteurella multocida and Porcine circovirus 2 （PCV2）. To characterize the specific role of swine influenza virus in PRDC presentation in Colombia, 11 farms from three major production regions in Colombia were examined in this study. Nasal swabs, bronchial lavage and lung tissue samples were obtained from animals displaying symptoms compatible with SIV. Isolation of SIV was performed in 9-day embryonated chicken eggs or Madin-Darby Canine Kidney （MDCK） cells. Positive isolates, identified via the hemagglutination inhibition test, were further analyzed using PCR. Overall, 7 of the 11 farms were positive for SIV. Notably, sequencing of the gene encoding the hemagglutinin （HA） protein led to grouping of strains into circulating viruses identified during the human outbreak of 2009, classified as pandemic H1N1-2009. Serum samples from 198 gilts and multiparous sows between 2008 and 2009 were obtained to determine antibody presence of APP, Myh, PCV2 and PRRSV in both SIV-H1Nlp-negative and -positive farms, but higher levels were recorded for SIV- HI Nlp-positive farms. Odds ratio （OR） and P values revealed statistically significant differences （p〈0.05） in PRDC presentation in gilts and multiparous sows of farms positive for SIV-HINlp. Our findings indicate that positive farms have increased risk of PRDC presentation, in particular, PCV2, APP and Myh.     

Effect of 5-azacytidine on the protein expression of porcine bone marrow mesenchymal stem cells in vitro

Bone marrow-derived mesenchymal stem cells （MSCs） are pluripotent stem cells that show a vital potential in the clinical application for cell transplantation. In the present paper, proteomic techniques were used to approach the protein profiles associated with porcine bone marrow MSCs and investigate the regulation of MSC proteins on the effect of 5-azacytidine （5-aza）. Over 1,700 protein species were separated from MSCs according to gel analysis. Compared with the expression profiling of control MSCs, there were 11 protein spots up-regulated and 26 downregulated in the protein pattern of 5-aza-treated cells. A total of 21 proteins were successfully identified by MALDI-TOF-MS analysis, among which some interesting proteins, such as alpha B-crystallin, annexin A2, and stathmin 1, had been reported to involve in cell proliferation and differentiation through different signaling pathways. Our data should be useful for the future study of MSC differentiation and apoptosis.     

Expression and intercellular trafficking of the VP22 protein of CVI988/Rispens vaccine strain of Marek’s disease virus

The viral protein 22 (VP22) in the tegument of Marek’s disease virus serotype 1 (MDV-1) plays an im-portant role in cell-to-cell spread and viral propagation. Antiserum against the carboxyl terminus of VP22 was prepared by immunizing mice with recombinant VP22 expressed in E. coli, and used to in-vestigate its expression in chicken embryo fibroblast (CEF) cells infected with different MDV-1 strains. At an infection dose of PFU=50, intercellular trafficking of the VP22 into the nuclei of the surrounding receipt cells was detected as early as 3 hours post infection. By 6 hours after infection (before viral plague formation), the protein was detected in the whole nuclei of the recipient cells with no difference among MDV-1 strains CVI988/Rispens, GA and RB1B. Intra-nuclear accumulation of the VP22 protein was further increased when the viral plagues started to form. These results indicate that, albeit the ex-istence of the 201TKSERT206 deletion, the VP22 of the CVI988/Rispens vaccine strain has also intercel-lular-trafficking function, which might serve as a potential alternative delivering protein instead of virulent strains VP22.     

Induction of Epstein-Barr virus lytic replication by recombinant adenoviruses expressing the zebra gene with EBV specific promoters

The latent Epstein-Barr virus (EBV) is found in the cells of many tumors. For example, EBV is detectable in almost all cases, and in almost all tumor cells, of non-keratinizing nasopharyngeal carcinoma.Activating the latent virus, which will result in its lytic replication and the death of tumor cells, is a potential approach for the treatment of EBV-associated cancers. In this study, three recombinant adenoviruses were constructed to express the Zebra gene, an EBV gene responsible for switching from the latent state to lytic replication. EBV-specific promoters were used in order to limit Zebra expression in EBV-positive cells, and reduce the potential side effects. The EBV promoters used were Cp, Zp and a dual promoter combining both promoters, CpZp. The Zebra protein was detected in HEK293 cells as well as the EBV-positive D98-HR1 cells infected with recombinant viruses. An EBV lytic replication early antigen, EA-D, was also detected in infected D98-HR1, implying the initiation of lytic replication. In the cell viability assay, Zebra-expressing adenoviruses had little effect on EBV-negative HeLa cells, while significantly reducing the cell viability and proliferation of D98-HR1 cells. The results indicate that EBV virus promoters can be used in adenovirus vectors to express the Zebra gene and induce EBV lytic replication in D98-HR1 cells.     

Photosynthetic Regulation of the Cyanobacterium Synechocystis sp. PCC 6803 Thioredoxin System and Functional Analysis of TrxB （Trx x） and TrxQ （Trx y） Thioredoxins

The expression of the genes encoding the ferredoxin-thioredoxin system including the ferredoxin-thioredoxin reductase （FTR） genes ftrC and ftrV and the four different thioredoxin genes trxA （m-type; sir0623）, trxB （x-type; sir1139）, trxC （sll1057） and trxQ （y-type; sir0233） of the cyanobacterium Synechocystis sp. PCC 6803 has been studied according to changes in the photosynthetic conditions. Experiments of light-dark transition indicate that the expression of all these genes except trxQ decreases in the dark in the absence of glucose in the growth medium. The use of two electron transport inhibitors, 3-（3,4-dichlorophenyl）-1,1-dimethylurea （DCMU） and 2,5-dibromo-3-methyl-6-isopropyl-p- benzoquinone （DBMIB）, reveals a differential effect on thioredoxin genes expression being trxC and trxQ almost unaffected, whereas trxA, trxB, and the ftr genes are down-regulated. In the presence of glucose, DCMU does not affect gene expression but DBMIB still does. Analysis of the single TrxB or TrxQ and the double TrxB TrxQ Synechocystis mutant strains reveal different functions for each of these thioredoxins under different growth conditions. Finally, a Synechocystis strain was generated containing a mutated version of TrxB （TrxBC34S）, which was used to identify the potential in-vivo targets of this thioredoxin by a proteomic analysis.