Crude garlic extract significantly inhibits replication of grapevine viruses

Efforts to control viral diseases of grapevine include the production of certified material and development of virus-resistant transgenic grapevines. However, effective antiviral agents, once the viruses have infected the plants, are still lacking. This study shows that a crude garlic extract has significant antiviral activity against grapevine viruses. Replication of grapevine leafroll-associated virus 2 (GLRaV-2) was obviously inhibited in grapevine cv. Cabernet Sauvignon calli treated with diluted (1:100) garlic extract. The relative RNA levels of GLRaV-2 and grapevine fleck virus (GFkV) in cv. Summer Black grapevine in in vitro-grown plantlets 10 days after treatment with diluted (1:100) garlic extract were about 22% and 20%, respectively, of that in controls. The viral RNA accumulation of GLRaV-2, GFkV, grapevine virus A (GVA), grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in field-grown grapevine cv. Centennial Seedless plants sprayed with diluted (1:100) garlic extract were about 31–40%, 26–38%, 18–31%, 17–42% and 15–18%, respectively, of that in controls. Moreover, the garlic extract treatment led to a significant decrease in viral RNA accumulation of GLRaV-3, GLRaV-2, GVA, GFkV, GFLV, GRSPaV and grapevine Pinot Gris virus in pot-grown grapevine cv. Shine Muscat plants, and viral disease symptoms in these plants were obviously attenuated. In addition, this extract significantly induced expression of pathogenesis-related protein genes and stimulated activity of antioxidant enzymes in grapevines. Taken together, these results indicate that the crude garlic extract acts as a significant inhibitor against a broad range of grapevine viruses.     

SIRT1 promotes autophagy of pancreatic cancer cells induced by hypoxia via regulating FOXO1/RAB7 signaling pathway

AIM: To investigate the effect of SIRT1 on the autophagy of pancreatic cancer cells under hypoxia condition, and to analyze the underlying mechanism of regulating FOXO1/RAB7 signaling pathway. METHODS: Western blot and immunofluorescence methods were used to determine the expression of SIRT1 in the pancreatic cancer cells. The small interfering RNA targeting SIRT1 and SIRT1 over-expression plasmid were transfected into the pancreatic cancer Panc-1 cells. Confocal microscopy was used to detect the LC3 expression. Western blot was used to analyze the protein levels of LC3, p62 and FOXO1/RAB7 signaling pathway-related molecules. Co-immunoprecipitation was used to detected the protein interaction between SIRT1 and FOXO1. RESULTS: The expression level of SIRT1 in the nucleus of Panc-1 cells was increased under hypoxia condition. Compared with negative control under hypoxia condition, knock-down of SIRT1 expression attenuated the autophagy flux in the pancreatic cancer Panc-1 cells (P<0.05). Over-expression of SIRT1 increased the protein levels of FOXO1 and RAB7. On the contrary, knock-down of SIRT1 expression inhibited the protein levels of FOXO1 and RAB7. The protein interaction between SIRT1 and FOXO1 in the pancreatic cancer cells was observed. CONCLUSION: SIRT1 in pancreatic cancer Panc-1 cells under hypoxia condition is over-expressed in the nucleus. Down-regulation of SIRT1 inhibits autophagy and its mechanism may be related to FOXO1/RAB7 signaling pathway.     

FAM3C activates Akt to promote viability of oral squamous-cell carcinoma cells

AIM: To investigate the expression and roles of family with sequence similarity 3, member C (FAM3C) in oral squamous-cell carcinoma cells. METHODS: The mRNA and protein expression levels of FAM3C in dysplastic oral keratinocyte (DOK) and oral squamous-cell carcinoma WSU-HN6 cells were detected by RT-qPCR and Western blot. The WSU-HN6 cells were treated with siFAM3C or FAM3C antibody. After 24, 48 and 72 h, the viability of WSU-HN6 cells was measured by CCK-8 assay, and the activation of protein kinase B (Akt) was detected by Western blot. Adenovirus was used to mediate over-expression of FAM3C in the DOK cells. The DOK cell viability was measured by CCK-8 assay after adenovirus infection for 24, 48 and 72 h, and the activation of Akt was detected by Western blot. RESULTS: Compared with the DOK cells, the mRNA and protein levels of FAM3C were significantly increased in the WSU-HN6 cells (P<0.05). The viability of WSU-HN6 cells transfected with siFAM3C was significantly inhibited at 48 h and 72 h (P<0.05). siFAM3C treatment inhibited the activation of Akt (P<0.05). FAM3C antibody treatment also suppressed the viability of the WSU-HN6 cells at 48 h and 72 h and the activation of Akt (P<0.05). Over-expression of FAM3C in the DOK cells promoted the cell viability at 48 h and 72 h and activated Akt (P<0.05). CONCLUSION: FAM3C might promote oral squamous-cell carcinoma cell growth by activating Akt.     

Role of ghrelin in cell protection by up-regulating HSP70 through ERK1/2 signaling pathway in PC12 cells

AIM:To study the role of ghrelin in cell protection by up-regulating heat shock protein 70 (HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway in the PC12 cells. METHODS:Sodium nitoprusside (SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group (incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group (ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group (quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group (ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group (added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS:Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased (P<0.05). Compared with SNP-injured group, ghrelin (100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells (P<0.05), and significantly up-regulated the protein expression of HSP70 (P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin (P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression (P<0.05). CONCLUSION:Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.     

Effects of natural feeding stimulants and glutamic acid supplementation on the feed intake,growth performance and digestive enzyme activities of red sea bream (Pagrus major) fed fish meal‐free soy protein concentrate (SPC)‐based diet

Two experiments were conducted for red sea bream (Pagrus major). In experiment 1, the optimum level of glutamic acid and natural feeding stimulants to enhance feed intake were determined and found that glutamic acid level of 0.5% and fish meat hydrolysate (FMH) were effective. In experiment 2, fish were fed with soy protein concentrate (SPC)‐based diet with synthetic feeding stimulants (Basal diet), the Basal diet with FMH (FMH diet), the FMH diet with glutamic acid (FMHG diet) and with fish meal diet (FM diet) as a control until satiation for 8 weeks. Feed intake of FMHG‐fed fish was significantly higher than others (p < 0.05). Specific growth rate and the feed conversion ratio of FMHG were comparable to those of FM‐fed fish (p > 0.05). Relative visceral fat ratio and crude lipid content of any SPC‐based diet‐fed fish tended to be lower than those of FM diet‐fed fish. There were no significant differences in trypsin and lipase activities hepatopancreas among treatments. SPC can be utilized as a sole protein source in a diet for red sea bream. The lower growth performance in SPC‐based diet‐ fed fish was not due to poor digestive enzyme secretion but could be associated with lipid utilization disorder.     

三种融合标签对大熊猫轮状病毒结构蛋白VP6-VP7可溶性表达的影响

大熊猫轮状病毒(giant panda rotavirus,GPRV)是引起幼龄大熊猫腹泻的主要病原,对圈养大熊猫产生了较大的危害。轮状病毒结构蛋白VP6是一种载体蛋白,可介导黏膜免疫反应。VP7是轮状病毒结构蛋白中主要的中和抗原。因此,VP6-VP7的融合表达作为候选抗原对该病的防治具有重要的意义。传统大肠埃希菌原核表达存在表达量低、可溶性差以及纯度低等弊端。本研究使用醛缩酶(EDA)、谷胱甘肽S-转移酶(GST)、麦芽糖结合蛋白(MBP)3种融合标签,以实现获得表达量高和纯度高的GPRV-VP6-VP7重组表达蛋白。将扩增的VP6、VP7基因片段利用同源重组酶构建到含3种融合标签的表达载体pET21b上,将重组质粒转化至大肠埃希菌Rosetta(DE3)感受态细胞中进行低温诱导表达。用Ni-柱亲和层析法纯化目的蛋白,SDS-PAGE和Image J分析蛋白表达量和可溶性,Western blot分析得到表达的重组表达蛋白正确且具有蛋白活性。实验结果证明,EDA标签能显著促进VP6-VP7蛋白的原核可溶性表达,提高VP6-VP7蛋白表达量。     

基于多反应监测质谱技术的水稻叶片蛋白绝对定量方法

【目的】建立水稻叶片蛋白的多反应监测(MRM)质谱绝对定量方法。【方法】水稻叶片蛋白经含1.0%十二烷基硫酸钠(SDS)的磷酸盐(PBS)缓冲液提取,丙酮沉淀除杂纯化、胰蛋白酶消化,酶解液经液相色谱分离,MRM质谱监测,外标法定量。【结果】向提取缓冲液中加入1.0%SDS可增强水稻叶片中16种靶蛋白和总蛋白质的提取效果;不同有机试剂处理,总蛋白质沉淀量存在显著差异(P<0.05),沉淀能力从强到弱依次为乙腈>丙酮>异丙醇>甲醇>乙醇;对于16种目标蛋白,总体以丙酮沉淀效果最好,其次是异丙醇和乙腈,甲醇和乙醇效果较差。该方法线性范围均达到3个数量级,定量限为0.1~2.5 nmol/L,灌浆期16种水稻叶片蛋白质含量为6.0~2818.1μg/g,相对标准偏差均小于14%。【结论】SDS可显著提高水稻叶片蛋白提取效果,采用丙酮或乙腈可获得较好的蛋白沉淀效果,但不同蛋白质略有差异。结合MRM质谱监测技术可实现水稻叶片蛋白的绝对定量,方法线性范围宽、敏度高、重复性好。     

色氨酰-tRNA合成酶基因WRS1调控水稻种子发育

【目的】氨酰-tRNA合成酶（aminoacyl-tRNA synthetases, aaRSs）与遗传信息传递密切相关,已发现植物中aaRSs家族蛋白在维持翻译功能之余,还参与配子发生与胚发育、质体的早期发育以及免疫信号的感知与病害防御等生物学过程。本研究利用水稻胚乳发育缺陷突变体,分析水稻色氨酰-tRNA合成酶（WRS1）在胚乳发育中的作用,证明WRS1基因编码一个影响水稻胚乳发育的关键因子。【方法】本研究通过甲烷磺酸乙酯（ethyl methane sulfonate, EMS）诱变籼稻（Oryza sativa subsp. indica）品种N22,筛选到一个稳定遗传的水稻粉质胚乳突变体（wrs1）,图位克隆获得目标基因。对wrs1成熟种子进行形态学观察以及淀粉相关理化性质测定,利用细胞学切片分析wrs1发育中胚乳的结构,利用实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）和GUS活性染色分析基因表达模式,通过qRT-PCR比较野生型与突变体花后12 d胚乳中淀粉合成相关基因表达情况,免疫印迹检测野生型与突变体成熟种子中淀粉合成酶蛋白积累情况,使用全自动氨基酸分析仪测定游离氨基酸含量。【结果】 wrs1突变体幼苗表现出明显的发育滞后且逐渐蔫萎死亡,从杂合突变体（WRS1wrs1）中分离到的粉质籽粒呈现明显的腹部皱缩,粒厚、千粒重下降,同时总淀粉含量下降,糊化淀粉的峰值黏度和崩解值均低于野生型。wrs1突变体发育胚乳中复合淀粉颗粒变小,排列疏松。WRS1定位于第12染色体长臂约183 kb的区间内,测序发现编码色氨酰-tRNA合成酶（tryptophanyl-tRNA synthetase, WRS）基因的第6外显子上发生单碱基替换,导致一个保守位置上的甲硫氨酸被替换。wrs1突变体中大部分淀粉合成相关基因表达量下调,且野生型与突变体间基因表达的变化与相应蛋白积累的差异存在不一致的趋势。wrs1突变体籽粒中蛋白质积累降低,而游离氨基酸含量显著升高。【结论】 WRS1编码色氨酰-tRNA合成酶,该基因突变后通过影响氨基酸稳态和蛋白质合成,造成淀粉合成相关基因异常表达从而影响淀粉的合成与积累,导致种子发育缺陷。