神经介素B及其受体NMBR参与抗A型流感病毒H1N1亚型感染的信号通路

NMB/NMBR通过调节A型流感病毒（IAV/H1N1/PR8）感染诱导的细胞因子表达而参与抗IAV的先天性免疫反应。为探究其发挥抗IAV/H1N1感染的信号通路，本文用PR8和WSN毒株分别感染MLE-12细胞和小鼠，用NF-κB抑制剂BAY11-7028单独或联合NMB处理MLE-12细胞，小鼠后腿肌内注射NMB和NMBRA，采用RT-PCR和qRT-PCR分析NMB、NMBR、IL-6、IFN-α和NP基因表达变化，采用Western blot分析NMB、NMBR、P65/p-P65、IκBα和NP蛋白表达的变化。结果显示，BAY11-7028可促使PR8和WSN感染的MLE-12细胞中NMB、NMBR、IL-6和IFN-α基因表达水平均下降和NP基因表达水平上升，并降低NMB、NMBR和p-P65蛋白表达水平和提升IκBα和NP蛋白表达水平。然而，NMB联合BAY 11-7028诱导PR8或WSN感染后的细胞中IL-6和NP表达出现极显著下降和IFN-α显著上升。此外，NMB抑制PR8和WSN感染的小鼠肺组织内p-P65和NP蛋白表达水平和促进IκBα蛋白表达水平；NMBRA联合NMB抵消NMB对PR8或WSN感染后的这些蛋白表达水平的调节作用。综上表明，NMB/NMBR通过调节PR8和WSN感染的MLE-12细胞和小鼠体内的NF-κB信号通路上P65蛋白磷酸化和IκBα的表达，进而影响下游细胞因子IL-6和IFN-α基因的表达，从而发挥抗IAV/H1N1感染的先天性免疫应答反应。     

PPARγ mediates effects of diosgenin on proliferation and apoptosis in human glioblastoma U87MG cells

AIM:To investigate the effect of diosgenin (Dio) on the proliferation, apoptosis and expression of peroxisome proliferator-activated receptor γ (PPARγ) in human glioblastoma U87MG cells and its possible mechanism. METHODS:Human astrocytes (HA) and U87MG cells were cultured in vitro and treated with Dio (0, 10, 20, 30, 40 and 50 μmol/L) and GW9662 (5 μmol/L) for 48 h, and then the cell viability was detected by CCK-8 assay. Cell colony formation assay was used to assess the proliferation potential. Flow cytometry was used to analyze the cell cycle distribution and apoptosis. The mRNA expression level of PPARγ was measured by RT-PCR. Western blot was used to determine the protein levels of PPARγ, cyclin D1, cyclin E1, Bcl-2 and Bax. RESULTS:Dio had no significant influence on the viabi-lity of HA (P>0.05). However, Dio remarkably reduced the viability of U87MG cells in a dose-dependent manner (P<0.05) with IC₅₀ of 24.31 μmol/L. Meanwhile, Dio remarkably diminished colony formation ability (P<0.05), induced G₀/G₁ phase arrest of the cell cycle and apoptosis (P<0.05), up-regulated the expression of PPARγ at mRNA and protein levels, increased the protein level of Bax (P<0.05), and down-regulated the protein levels of cyclin D1, cyclin E1 and Bcl-2 (P<0.05) in a dose-dependent manner. However, these effects induced by Dio were inhibited by GW9662 (P<0.05), a specific inhibitor of PPARγ. CONCLUSION:Dio may inhibit proliferation and induce apoptosis in human glioblastoma U87MG cells most likely via up-regulating the expression of PPARγ, and then down-regulating the protein levels of cyclin D1, cyclin E1 and Bcl-2, and up-regulating the protein level of Bax.     

蛙皮素样肽家族及其受体氨基酸序列信息学比较分析

【目的】研究反刍动物蛙皮素样肽家族多肽及其受体的保守性。为不同动物,特别是反刍动物这2种蛙皮素样多肽及其受体蛋白抗原设计和活性多肽筛选等研究提供参考。【方法】采用生物信息分析学的方法,通过UniProt数据库比较牛的蛙皮素样肽家族胃泌素释放肽(Gastrin-releasing peptide, GRP)和神经介素B(Neuromedin B, NMB)2种多肽及其特异性受体GRP-R和NMB-R与其他动物氨基酸序列的差异性。【结果】13种动物成熟GRP多肽C-端的8个氨基酸序列完全相同,牛GRP氨基酸序列与绵羊、猪和豚鼠最高,分别为88.7%、77.8%和77.8%,与其他动物相似性低于74.1%。10种动物成熟NMB多肽C-端10个氨基酸序列完全相同。牛GRP-R与狗、马和猪等相似性最高,分别为96.1%、94.3%和94.0%,牛NMB-R与猪、人和马相似性最高,分别为92.3%、91.5%和91.0%;牛GRP-R与NMB-R相似性仅为62.2%。【结论】GRP和NMB的C-端氨基酸序列在不同动物之间均具有高度的保守性,而N-端为变化区域,且GRP-R和NMB-R氨基酸序列在动物之间保守性较高。     

SCFAs对奶牛瘤胃上皮细胞Ca2+信号通路相关基因表达的影响

为探究短链脂肪酸(SCFAs)对奶牛瘤胃上皮细胞(BRECs)Ca~(2+)信号通路相关基因表达的影响,试验分为3个处理组,分别为野生型BRECs组,含20mmol/L SCFAs BRECs组,含20mmol/L SCFAs且通过CRISPR/Cas 9系统敲除GPR41基因的BRECs组,每个处理组3个重复,每组细胞均培养24h后,收集细胞提取总RNA,通过qRT-PCR对Ca~(2+)信号通路相关基因的mRNA表达量和细胞内Ca~(2+)浓度进行测定。结果表明,与野生型BRECs相比,添加20mmol/L SCFAs可极显著增加PLCB2的mRNA表达量(P0.01),显著增加IP3R1的mRNA表达量(P0.05),对PLCE1、PLCL1、PKCB和PKCG的mRNA表达量无显著差异(P0.05),可增加细胞内Ca~(2+)浓度但无显著差异(P0.05);敲除SCFAs的受体GPR41后,添加20 mmol/L SCFAs可显著降低IP3R1的mRNA表达量(P0.05),极显著上调PLCE1和PLCB2的mRNA表达量(P0.01),但对PLCL1、PKCB和PKCG的mRNA表达量无显著影响(P0.05),细胞内Ca~(2+)浓度有降低趋势但无显著差异(P0.05)。综上,SCFAs可以通过激活其受体GPR41来调控BRECs内Ca~(2+)信号通路中相关基因的表达和细胞内Ca~(2+)的释放。     

蛋鸡育成期和产蛋高峰期生殖激素水平及激素受体基因表达规律研究

【目的】探究蛋鸡育成期和产蛋高峰期相关生殖激素含量及其受体基因mRNA表达量在24 h内的变化规律与差异,分析血液生殖激素含量变化与产蛋排卵行为的关系。【方法】以育成期和产蛋高峰期的罗曼蛋鸡为研究对象,监测其在产蛋高峰期的产蛋时间点,运用酶联免疫吸附法(ELISA)测定2个时期蛋鸡血液中前列腺素(PGs)、雌二醇(E2)、促卵泡素(FSH)、黄体生成素(LH)含量在24 h内的动态变化,实时荧光定量PCR技术检测相应时间点的前列腺素F受体(PTGFR)、雌激素受体(ERα)、促卵泡素受体(FSHR)和黄体生成素受体(LHR)基因mRNA在输卵管子宫部的日表达水平。【结果】蛋鸡产蛋时间集中在早上08:00-10:00。产蛋高峰期时,一天中06:00、10:00、14:00、18:00的PGs含量显著高于育成期(P0.05),E2含量在06:00和18:00极显著高于育成期(P0.01),FSH含量在18:00显著高于育成期(P0.05),LH含量在14:00、22:00和02:00显著高于育成期(P0.05)。育成期除LH外,其余3种激素含量在一天中波动不大。产蛋高峰期PGs含量在产蛋前显著高于产蛋后,PTGFR mRNA表达量在产蛋前后4 h均显著(P0.05)升高;FSH和E2含量在产蛋后(18:00)极显著(P0.01)上升。子宫组织的激素受体基因表达量变化与相应血液激素含量变化一致。【结论】蛋鸡产蛋时间集中于上午,产蛋高峰期各生殖激素含量总体高于育成期,蛋鸡育成期和产蛋高峰期的血液生殖激素水平变化规律与输卵管子宫部激素受体基因mRNA表达量的变化规律基本一致。PGs参与蛋鸡产蛋高峰期的产蛋和排卵过程,E2和FSH可能在蛋壳形成过程中发挥调控作用。     

Functional polymorphism among members of abscisic acid receptor family(ZmPYL) in maize

Pyrabactin resistance 1-like proteins(PYLs) are direct receptors of abscisic acid(ABA). For the redundant and polymorphic functions, some members of the PYL family interact with components of other signaling pathways. Here, 253 positive colonies from a maize cDNA library were screened as interacting proteins with the members of ZmPYL family. After sequencing and function annotation, 17 of 28 interaction combinations were verified by yeast two-hybrid(Y2 H). The germination potential, taproot length and proline content of a quartet mutant of Arabidopsis PYL genes were significantly deceased comparing to the wild type(WT) under alkaline stress(pH 8.5) and 100 μmol L–1 methyl jasmonate(MeJA) induction. The malondialdehyde(MDA) content was significantly increased. After germinating in darkness, the characteristics of dark morphogenesis of the quartet mutant seedlings were more obvious than those of the WT. The differential expression of the related genes of photomorphogenesis in the mutant was much more than that in the WT. Three light and two JA responsive cis-affecting elements were identified during the promoter sequences of the AtPYL1 and AtPYL2 genes. These results suggested that functional polymorphism has evolved among the members of ZmPYL family. In response to developmental and environmental stimuli, they not only function as direct ABA receptors but also interact with components of other signaling pathways mediated JA, brassinosteroid(BR), auxin, etc., and even directly regulate downstream stress-related proteins. These signaling pathways can interact at various crosstalk points and different levels of gene expression within a sophisticated network.     

New angiotensin-converting enzyme inhibitory peptide from Coix prolamin and its influence on the gene expression of renin-angiotensin system in vein endothelial cells

Coix seed, which is a traditional Chinese medicine, has been used to treat hypertension for thousands of years. It has been shown that Coix prolamin peptides display high levels of angiotensin I converting enzyme (ACE) inhibitory activity. Hence, we purified the ACE inhibitory peptides from Coix prolamin hydrolysates and evaluated the influence of the most potent peptide on the renin-angiotensin system (RAS) genes expression in human umbilical vein endothelial cells (HUVECs). In this study, Coix prolamin peptides were sequentially separated by ultrafiltration, ion exchange chromatography, gel filtration chromatography and RP-HPLC, while the peptide structure was analyzed by mass spectrometry. Next, in silico proteolysis, pharmacophore and molecular docking were further applied to screen and optimize the structure of peptides. Finally, a novel ACE inhibitory peptide VDMF was obtained, in which its influence on the gene expression of RAS signaling pathway in AngⅡ-injury HUVECs was evaluated by quantitative real-time PCR. VDMF significantly down-regulated ACE, AngII type 1 receptor (AT1R) and ACE2 mRNA expression in comparation with model group, while up-regulating Mas gene expression. Hence, we obtained a novel antihypertensive candidate that was derived from the Coix peptides, which could involve a multi-modulation mechanism that regulates blood pressure.     

Role of IP3R in LH-EGFR-induced mouse oocyte meiotic resumption

AIM: To explore the effect of inositol 1, 4, 5-trisposphate receptor (IP₃R) in luteinizing hormone-epidermal growth factor receptor (LH-EGFR)-induced oocyte meiotic resumption. METHODS: Models of mouse cumulus-oocyte complexs (COCs) culture and follicle culture in vitro were generated to study the effects of 2-aminoethyl diphenyl borate (2-APB) and heparin (IP₃R specific inhibitors) on LH/EGF-induced oocyte meiotic resumption and EGF-induced cumulus cell expansion. Real-time PCR was used to detect the mRNA expression of cumulus expansion-related factors. The changes of the intracellular calcium level were monitored using Fluo 3-AM, and the cGMP level was measured by ELISA. RESULTS: The inhibitors of IP₃R, 2-APB and heparin, dramatically reversed EGF-induced oocyte maturation (P<0.05) and decreased cGMP levels in COCs (P<0.05). In addition, 2-APB and heparin reversed EGF-induced cumulus expansion, and significantly inhibited EGF-induced cumulus expansion-related factor expression (P<0.05). The activation of IP₃R increased intracellular calcium level, and the study found that 2-APB and heparin dramatically reversed EGF-induced elevation of calcium level in cumulus cells (P<0.05). Follicular culture in vitro showed that 2-APB and heparin significantly reversed the LH-induced oocyte maturation (P<0.05). CONCLUSION: LH-EGFR signaling pathway increases calcium level in cumulus cells through IP₃R, resulting in meiotic resumption.     

昆虫嗅觉相关蛋白的研究进展

昆虫对自然环境中复杂化学信号的识别多依赖于其灵敏的嗅觉系统,选择寄主、觅食、寻找配偶等行为的发生都以嗅觉识别为基础,而完成嗅觉识别还需要多种嗅觉相关蛋白的参与。嗅觉相关蛋白主要包括6种,即气味结合蛋白、化学感受蛋白、气味受体、感觉神经元膜蛋白、离子型受体和气味降解酶。不同种类和性别的昆虫中,嗅觉蛋白的种类、数量和分布各不相同。由于嗅觉蛋白在昆虫识别外界气味分子中的重要作用,国内外近年来对其展开了广泛、深入的研究。本文从几种嗅觉相关蛋白的生化特性、分子结构、生理功能、分布表达部位和研究概况等角度,较详细地综述了近年来国内外昆虫嗅觉相关蛋白的研究进展。     

斑节对虾Toll9受体基因免疫功能的研究与表达分析

Toll受体蛋白(Toll receptors)是一类重要的模式识别受体,在无脊椎动物先天性免疫系统中发挥重要作用。文章对斑节对虾(Penaeus monodon)新型Toll9受体基因(Pm Toll9)进行了研究:以人源胚胎肾细胞(HEK293T)成功构建体外细胞免疫模型,通过免疫印迹方法证实Pm Toll9重组真核蛋白可在HEK293T中成功表达。双荧光素酶报告系统检测发现在200 ng转染浓度处Pm Toll9对NF-κB报告基因的激活效果显著。q RT-PCR数据证明PmToll9成功激活HEK293T细胞Toll-like receptor(TLR)信号通路,促进通路下游髓样分化因子(My D88)、肿瘤坏死因子(TNF-α)和白介素(IL-10)的上调表达,同时酶联免疫吸附测定结果证明Pm Toll9可促进TNF-α蛋白表达水平显著上调。斑节对虾体内细菌刺激实验结果显示无乳链球菌(Streptococcus agalactiae)可激活Pm Toll9在肝胰腺、肠、淋巴和鳃中的表达,哈维氏弧菌(Vibrio harveyi)可显著抑制Pm Toll9在肝胰腺中的表达。提示无乳链球菌可通过Pm Toll9激活Toll信号通路,引起机体免疫防御反应,而哈维氏弧菌对此过程具有一定的抑制作用。