Critical contribution of immunoproteasomes in the induction of protective immunity against Trypanosoma cruzi in mice vaccinated with a plasmid encoding a CTL epitope fused to green fluorescence protein

Acquired immunity against infection with Trypanosoma cruzi is dependent on CD8(+)T cells. Here, to develop a vaccine strategy taking advantage of activated CD8(+)T cells, we constructed a DNA vaccine, designated pGFP-TSA1, encoding a fusion protein linking GFP to a single CTL epitope of TSA1, a leading candidate for vaccine against T. cruzi. C57BL/6 mice vaccinated with this plasmid showed suppressed parasitemia and prolonged survival. Vaccination with pGFP-TSA1 enhanced epitope-specific cytotoxicity and IFN-gamma secretion by CD8(+)T cells. Furthermore, the depletion of CD8(+)T cells prior to challenge infection with T. cruzi completely abolished this protection, indicating that CD8(+)T cells are the principal effector T cells involved. When mice deficient in the proteasome activator PA28alpha/beta or the immunoproteasome subunits LMP2 and LMP7 were used, the protective immunity against infection was profoundly attenuated. Our findings clearly demonstrate that vaccination with pGFP-TSA1 successfully induces protection dependent on CD8(+)T cell activation, in which immunoproteasomes play a crucial role. It is noteworthy to document that physical binding of the epitope and GFP is required for induction of this protection, since mice vaccinated with pTSA1-IRES-GFP failed to acquire resistance, probably because the epitope and GFP are separately expressed in the antigen-presenting cells.     

Long-Term Prognostic Analysis after Endoscopic Endonasal Surgery for Olfactory Neuroblastoma: A Retrospective Study of 13 Cases

ObjectivesTo summarize the characteristics and long–term outcomes of olfactory neuroblastoma through the analysis of 13 cases in single institution, with the assessment of treatment modality, prognostic factors.MethodA retrospective study of thirteen cases diagnosed as olfactory neuroblastoma and underwent combined treatments during the period 2000–2010. Statistical analysis was performed to search for prognostic factors and compared different treatment modalities.Results13 patients were enrolled in this study, including 8 male and 5 female, ranging from 15 to 69 (median 43) years old. One patient at stage A was only treated with endoscopic endonasal surgery (EES). Seven patients were treated with preoperative radiotherapy and EES, two with EES and postoperative radiotherapy, and the other three with combined radiotherapy and chemotherapy. The range of follow-up time varied from 23 to 116 months (median 65 months). The 5-year overall survival rate was 46.2% (6/13). To date, these thirteen patients have not suffered local recurrences while two patients had lymph node recurrences and one had distant metastasis in the bone marrow. In 13 patients, 61.5% were diagnosed as late T stage (T3/4), 69.2% late Kadish stage (C/D) and 53.8% were high Hyams grade (I/ II), which indicated poor prognosis. Related prognostic factors were the TNM stage (T stage P = 0.028, N stage P = 0.000, M stage P = 0.007), Kadish stage (P = 0.025) and treatment modality (P = 0.015).ConclusionLate stage of TNM and Kadish staging system indicated a poor prognosis. Combined treatment modality, including endoscopic endonasal surgery, achieved a better outcome than non-surgical approach.     

新疆地区小麦白粉病菌寄主范围的研究

用小麦白粉病菌11个生理小种的混合菌种,对新疆地区的小麦近缘植物的7个属22个种的47份材料进行接种,除6份免疫外,其余均接种成功.用其中6个属19个种的29份小麦近缘植物产生的白粉病菌,对小麦回接,参试的29份材料全部回接成功.小麦白粉病菌对小麦近缘植物的寄生像在小麦上一样,有明显的寄生专化性.感病的小麦近缘植物的78.0%对小麦白粉病菌的感病性,随生育期增长而急剧下降.文中并对小麦白粉病中间寄主的作用进行了讨论.     

Phylogenetic analysis of epiphytic marine bacteria on Hole-Rotten diseased sporophytes of <Emphasis Type="Italic">Laminaria japonica</Emphasis>

During an occurrence of Hole-Rotten Disease of Laminaria japonica in a cultivating farm in Ma Shan Shandong province, China, 42 Gram-negative epiphytic marine bacteria were isolated and purified on Zobell 2216E marine agar medium. Morphological and biochemical characteristics of each isolated bacterium were studied, and molecular identification of bacterial strains was conducted with polymerase chain reaction amplification to 16S rRNA gene sequence analysis. Based on nearly full length of 16S rRNA gene sequence analysis, the isolated strains were bacteria that belong to genus Pseudoalteromonas, Vibrio, Halomonas and Bacillus. The percentage of each group was 61.9%, 28.6%, 7.1% and 2.4% respectively. The results of pathogenicity assay showed that 12 strains could cause the disease symptoms in sporophytes of L. japonica. They belonged to the genera Pseudoalteromonas, Vibrio and Halomonas with 58.3%, 33.3%, 8.3% respectively. The results suggest that these bacteria are the dominant marine bacteria on diseased sporophytes of L. japonica and may be the potential pathogenic bacteria associated with Hole-Rotten Disease of L. japonica.     

Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis: Streptokinase with Longer Functional Half-Life

The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent. During the clot-dissolving process, streptokinase is processed to smaller intermediates by plasmin. Two of the major processing sites are Lys59 and Lys386. We engineered two versions of streptokinase with either one of the lysine residues changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient Bacillus subtilis WB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation and showed different degrees of resistance to plasmin depending on the nature of the mutation. Muteins with double mutations had half-lives that were extended 21-fold when assayed in a 1:1 molar ratio with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.     

Bisphenol A exposure at an environmentally relevant dose induces meiotic abnormalities in adult male rats

Whether environmental exposure to bisphenol A (BPA) may induce reproductive disorders is still controversial but certain studies have reported that BPA may cause meiotic abnormalities in C. elegans and female mice. However, little is known about the effect of BPA on meiosis in adult males. To determine whether BPA exposure at an environmentally relevant dose could induce meiotic abnormalities in adult male rats, we exposed 9-week-old male Wistar rats to BPA by gavage at 20 μg/kg body weight (bw)/day for 60 consecutive days. We found that BPA significantly increased the proportion of stage VII seminiferous epithelium and decreased the proportion of stage VIII. Consequently, spermiation was inhibited and spermatogenesis was disrupted. Further investigation revealed that BPA exposure delayed meiosis initiation in the early meiotic stage and induced the accumulation of chromosomal abnormalities and meiotic DNA double-strand breaks (DSBs) in the late meiotic stage. The latter event subsequently activated the phosphatidylinositol 3-kinase-related protein kinase (ATM). Our results suggest that long-term exposure to BPA may lead to continuous meiotic abnormalities and ultimately put mammalian reproductive health at risk.     

DDPH对低氧内皮细胞条件培养液诱导肺动脉平滑肌细胞增殖及α-SM-actin表达的影响

目的探讨1-(2,6-二甲基苯氧基)-2-(3,4-二甲氧基苯乙氨基)丙烷盐酸盐(DDPH)抑制低氧内皮细胞条件培养液(HECCM)诱导肺动脉平滑肌细胞增殖及对α-SM-actin表达的影响.方法利用低氧内皮细胞条件培养液建立猪肺动脉平滑肌细胞(PASMC)的增殖模型;以四甲基偶氮唑盐(MTT)比色法、α平滑肌肌动蛋白(α-SM-actin)为指标,采用免疫细胞化学染色法观察低氧内皮细胞条件培养液对肺动脉平滑肌细胞增殖的影响以及DDPH对低氧内皮细胞条件培养液促肺动脉平滑肌细胞增殖后的逆转效应.结果低氧内皮细胞条件培养液显著促进肺动脉平滑肌细胞增殖,低氧内皮细胞条件培养液促肺动脉平滑肌细胞增殖后,肺动脉平滑肌细胞的表型发生转化,由收缩表型转化为合成表型,肺动脉平滑肌细胞胞浆内的α-SM-actin含量下降;DDPH能显著抑制低氧内皮细胞条件培养液对肺动脉平滑肌细胞的增殖作用,并使肺动脉平滑肌细胞的表型发生逆转,即由合成表型逆转为具有执行正常收缩功能的收缩表型,肺动脉平滑肌细胞胞浆内的α-SM-actin含量回升.结论提示DDPH能显著抑制低氧内皮细胞条件培养液促肺动脉平滑肌细胞的增殖作用,其作用机制可能是通过肺动脉平滑肌细胞的表型发生逆转来实现的.     

Influence of pulsed electromagnetic field with different pulse duty cycles on neurite outgrowth in PC12 rat pheochromocytoma cells

The influence of low frequency (50 Hz repetition rate) pulsed electromagnetic field (EMF) on PC12 cell neurite outgrowth in vitro was investigated in this study. We studied the percentage of neurite bearing cells, average length of neurites, and directivity of neurite outgrowth in PC12 cells cultured for 96 h in the presence of nerve growth factor (NGF). PC12 cells were exposed in one incubator to pulsed EMF at 1.36 mT (peak value) generated by a pair of Helmholtz coils, and the control samples were placed in another identical incubator. We found that the pulse duty cycle had significant effect on neurite outgrowth. Low (10%) pulse on-time significantly inhibited the percentage of neurite bearing cells, but at the same time increased the average length of neurites, while 100% on-time (DC) had exactly the opposite effects. Furthermore, we found that neurites were prone to extend along the direction of pulsed EMF with 10% pulse on-time. Our studies show that neurite outgrowth in PC12 cells is sensitive to the pulse duty and this sensitivity was associated with NGF concentration.     

miRConnect: identifying effector genes of miRNAs and miRNA families in cancer cells

micro(mi)RNAs are small non-coding RNAs that negatively regulate expression of most mRNAs. They are powerful regulators of various differentiation stages, and the expression of genes that either negatively or positively correlate with expressed miRNAs is expected to hold information on the biological state of the cell and, hence, of the function of the expressed miRNAs. We have compared the large amount of available gene array data on the steady state system of the NCI60 cell lines to two different data sets containing information on the expression of 583 individual miRNAs. In addition, we have generated custom data sets containing expression information of 54 miRNA families sharing the same seed match. We have developed a novel strategy for correlating miRNAs with individual genes based on a summed Pearson Correlation Coefficient (sPCC) that mimics an in silico titration experiment. By focusing on the genes that correlate with the expression of miRNAs without necessarily being direct targets of miRNAs, we have clustered miRNAs into different functional groups. This has resulted in the identification of three novel miRNAs that are linked to the epithelial-to-mesenchymal transition (EMT) in addition to the known EMT regulators of the miR-200 miRNA family. In addition, an analysis of gene signatures associated with EMT, c-MYC activity, and ribosomal protein gene expression allowed us to assign different activities to each of the functional clusters of miRNAs. All correlation data are available via a web interface that allows investigators to identify genes whose expression correlates with the expression of single miRNAs or entire miRNA families. miRConnect.org will aid in identifying pathways regulated by miRNAs without requiring specific knowledge of miRNA targets.     

Dysregulation of Claudin family genes in colorectal cancer in a Chinese population

Claudins play an important role in tumor metastasis and in invasiveness of colorectal cancer (CRC). We have evaluated the relationship between CRC and expression of the claudin genes in Chinese patients with CRC. We measured CLDN1 and CLDN7 mRNA using quantitative PCR, and protein levels with immunohistochemistry in cancer tissues and adjacent normal tissue. Cancer tissues had significantly higher levels of CLDN1, and significantly lower levels of CLDN3, CLDN4, and CLDN7 than did normal tissue. CLDN3, CLDN4, and CLDN7 expression levels were higher in CRC of the protruded type than in CRC of the infiltrative type. Expression of CLDN7 correlated with lymph node metastasis. Stage N0 cancer tissues had higher levels of CLDN7 than did stages N1 and N2, suggesting that CLDN7 expression was closely related to the extent of lymph node metastasis. CLDN1 protein was upregulated, but CLDN7 protein was downregulated in cancer tissues when compared with expression in adjacent normal tissues. In conclusion, CLDN3, CLDN4, and CLDN7 were significantly downregulated, whereas CLDN1 was significantly upregulated in CRC. The altered expression of claudin genes may play a role in the initiation and development of CRC.