胡萝卜14-3-3基因家族的鉴定与分析

以胡萝卜为试材,采用分子生物学和生物信息学方法对胡萝卜14-3-3基因家族各成员进行鉴定,研究分析其理化特性、基因结构、保守结构域、复制事件、系统进化及顺式作用元件,以期为进一步研究胡萝卜14-3-3蛋白的功能提供参考依据。结果表明:胡萝卜中共鉴定到11个14-3-3基因,不均匀分布在6条染色体上,含有3~7个外显子,其中1对基因发生片段复制事件;编码的蛋白序列长度为236~264 aa,分子量为26.64~30.14 kD,等电点为4.65~5.33,具有7个Motif,全部为酸性非分泌型蛋白,定位在细胞核。系统进化分析显示,胡萝卜14-3-3蛋白分为ε类(3个)与非ε(8个)类2个亚族。此外,顺式作用元件分析结果表明,胡萝卜14-3-3基因含有大量激素与逆境胁迫相关元件。

烟粉虱MED隐种14-3-3基因克隆及时空表达谱分析

【目的】真核生物中,14-3-3蛋白是一类可与多种蛋白互作的调节蛋白,能够参与信号转导、免疫反应、生长发育和胁迫响应等。本研究旨在克隆烟粉虱Bemisia tabaci MED隐种的14-3-3基因的全长cDNA序列,了解该基因编码的蛋白特征和该基因的时空表达模式。【方法】使用RT-PCR技术克隆烟粉虱MED隐种的14-3-3基因全长cDNA序列,通过生物信息学软件和在线网站分析14-3-3基因的生物学特性;使用RT-qPCR测定14-3-3基因在烟粉虱MED隐种不同发育阶段(卵、1-4龄若虫和成虫)、雌雄成虫以及雌成虫头、胸和腹中的表达量。【结果】克隆并鉴定了烟粉虱MED隐种14-3-3基因的两个亚型:Bt14-3-3 epsilon(GenBank登录号:XM_019046102.1)和Bt14-3-3 zeta(GenBank登录号:XM_019057395.1),开放阅读框(ORFs)分别长771和744 bp,分别编码256和247个氨基酸,编码的蛋白是无跨膜螺旋区和信号肽的亲水性蛋白,其二级结构主要由α旋螺旋组成。系统发育树分析表明,Bt14-3-3 epsilon与褐飞虱Nilaparvata lugens、温带臭虫Cimex lectularius和茶翅蝽Halyomorpha halys的14-3-3 epsilon聚为一支,同源性较高;Bt14-3-3 zeta与褐飞虱的14-3-3 zeta的亲缘关系更近。RT-qPCR结果表明,Bt14-3-3 epsilon和Bt14-3-3 zeta在烟粉虱MED隐种的卵、雌成虫和雌成虫腹部中的表达量较高。【结论】明确了烟粉虱MED隐种14-3-3基因的两个亚型的全长序列、编码蛋白特征及时空表达特点,为后续研究14-3-3蛋白的分子功能奠定了基础。

血清14-3-3β蛋白联合呼出气一氧化氮及常规通气肺功能参数对儿童支气管哮喘的诊断效能

目的探讨血清14-3-3β蛋白联合呼出气一氧化氮(fractional exhaled nitric oxide,FeNO)及常规通气肺功能参数对儿童支气管哮喘(简称“哮喘”)的诊断效能。方法前瞻性纳入136例初次诊断为哮喘且处于急性发作期的儿童为哮喘组,选择同期85例健康体检儿童为健康对照组,比较两组血清14-3-3β蛋白浓度的差异,分析血清14-3-3β蛋白与临床指标的相关性,评估14-3-3β蛋白联合FeNO及常规通气肺功能参数对儿童哮喘的诊断效能。结果哮喘组血清14-3-3β蛋白浓度高于健康对照组(P<0.001)。血清14-3-3β蛋白与中性粒细胞百分比、血清总免疫球蛋白E呈正相关,与常规通气肺功能参数呈负相关(P<0.05)。联合指标交叉验证显示14-3-3β蛋白+FeNO+用力呼出75%肺活量的呼气流量占预测值百分比预测哮喘的曲线下面积为0.948,灵敏度和特异度分别为88.9%和93.7%,具有较好的诊断效能(P<0.001),模型的外推性最好。结论血清14-3-3β蛋白联合FeNO、用力呼出75%肺活量的呼气流量占预测值百分比可以显著提高儿童哮喘的诊断效能。

GM130通过14-3-3ζ对卵巢癌细胞增殖的影响及机制研究

目的探讨高尔基体基质蛋白130(GM130)通过14-3-3ζ对卵巢癌细胞增殖的影响及可能机制。方法采用免疫组化法检测在20例正常卵巢上皮组织和42例卵巢上皮恶性肿瘤中GM130和14-3-3ζ的表达,分析二者相关性。采用Western blot和RT-PCR法检测卵巢癌SKOV3/A2780细胞株GM130和14-3-3ζ蛋白及其mRNA表达情况,筛选出高表达细胞株。采用免疫荧光染色法检测GM130与14-3-3ζ在卵巢癌细胞中的共表达情况。将设计好的重组表达质粒shRNA-GM130片段稳转进入卵巢癌细胞中,采用Western blot和RT-PCR筛选出降低GM130的最佳片段。细胞分为三组:空白组(WT)、对照组(NC)、低表达组(313),采用CCK法和流式细胞术分别检测各组卵巢癌细胞生殖情况及周期变化情况,采用Western blot法检测各组14-3-3ζ、GRASP65、P115及增殖相关蛋白表达水平。结果GM130和14-3-3ζ在卵巢恶性肿瘤组织中的表达水平高于正常卵巢组织(P<0.001)。在卵巢恶性肿瘤组织中,GM130和14-3-3ζ呈正相关关系(r=0.873,P<0.001)。GM130蛋白及其mRNA在SKOV3细胞中的表达量高于A2780细胞(P<0.05),14-3-3ζ蛋白及其mRNA在SKOV3细胞和A2780细胞中比较差异无统计学意义(P>0.05)。后续实验选用SKOV3细胞。免疫荧光共定位染色显示,GM130与14-3-3ζ主要在胞质表达,前者近核膜部分呈堆叠样表达量较高,后者胞核中表达较少,二者可能存在共定位关系。转染313组片段后,对GM130的抑制效果最好。转染质粒313组细胞增殖能力降低,细胞在G1期阻滞的数量增多,G2期阻滞数量减少,S期阻滞的细胞数量稍减少,GM130、14-3-3ζ、P115、Cyclin A、Cdc25A蛋白相对表达水平降低,P21蛋白相对表达水平升高(P<0.05)。结论抑制GM130可抑制卵巢癌细胞增殖,其机制可能与抑制14-3-3ζ表达,影响卵巢癌细胞周期有关。

Cloning and Sequence Analysis of Ma-14-3-3d Encoding a Homologue 14-3-3 Protein from Banana

[Objective] The aim of the study is to clone and analyze the gene encoding 14-3-3 protein from banana. [Method] Combined with PCR amplification, RACE (rapid amplification of cDNA ends) technique was employed to clone 14-3-3 gene from banana; then the amplified sequence was sequenced and homologically analyzed. [Result] A new cDNA homologous with 14-3-3 protein genes were obtained by RT-PCR and RACE ( rapid amplification of cDNA ends ) approaches. The full length of this cDNA was 866 bp encoding 197 amino acids. Alignment of deduced amino acid sequence with those from other plants revealed that the cDNA shared high homology with 14-3-3 protein genes from other plants, and was designated as Musa acuminata 14-3-3 gene (Ma-14-3-3d). Phylogenetic analysis reveals that Ma-14-3-3d has closer genetic relationship with those from monocotyledon species than those from other species. [Conclusion] Ma-14-3-3d belongs to the same lineage of 14-3-3 from monocotyledon.

菊芋14-3-3基因家族的鉴定及其对非生物胁迫响应的分析

[目的]本文旨在鉴定菊芋14-3-3基因家族成员并分析它们对高温、低温、盐和干旱胁迫响应的表达模式,为研究14-3-3蛋白功能及菊芋育种提供依据。[方法]采用克隆和生物信息学研究基因性质,采用RNA-seq数据分析和RT-qPCR研究基因对非生物胁迫的响应模式。[结果]从菊芋中克隆到14-3-3基因家族的10个成员HtGRF1—HtGRF10,GenBank登录号为OP132618—OP132627。根据进化关系将其分为2个亚家族:HtGRF1—HtGRF7属于非ε组,HtGRF8—HtGRF10属于ε组,通常形成同源或异源二聚体。组织表达分析表明,HtGRF2/3/6/9在芽、根、茎、叶中的表达丰度较高,HtGRF3/5/9在块茎发育过程中前高后低。综合分析根和叶中HtGRF对非生物胁迫响应时发现,HtGRF6表达水平在高温、盐和干旱胁迫下下降;HtGRF2/3/7/8/9表达水平在盐胁迫下下降,而在干旱胁迫下上升;HtGRF1表达水平在低温胁迫下上升;HtGRF4/5表达水平在干旱胁迫下下降;而HtGRF10表达水平变化不显著。[结论]菊芋14-3-3蛋白是一个高度保守的多基因编码家族,在菊芋的生长发育和适应复杂环境中起着重要作用。

14-3-3ɛ蛋白与肿瘤发生发展关系的研究进展

14-3-3ε蛋白也被称为YWHAE蛋白,是属于14-3-3蛋白家族的一种小相对分子质量的蛋白质。不同于蛋白激酶,14-3-3ε蛋白在细胞内并不参与蛋白质磷酸化反应,而是通过形成特殊的二聚体结构并与特定的磷酸化蛋白质结合,调节蛋白质配体的活性和亚细胞定位,从而参与多种细胞生命活动的调节。目前,在多种肿瘤中均已发现14-3-3ε蛋白的异常表达,异常表达的14-3-3ε蛋白对蛋白质配体的调节效应改变,进而影响蛋白质配体的亚细胞定位和酶活性,导致肿瘤细胞发生发展和侵袭转移。14-3-3ε蛋白的二聚体结构是其在肿瘤发生发展过程中发挥生物学功能的重要结构基础,破坏这一二聚体结构可有效靶向攻击肿瘤细胞。现基于国内外对14-3-3ε蛋白的研究成果,针对14-3-3ε蛋白的结构、作用机制及其在胃癌、肝癌、皮肤癌和其他多种肿瘤中发生发展过程中的影响及作用机制进行综述。

铁皮石斛14-3-3基因家族鉴定及表达分析

【目的】14-3-3蛋白,亦称通用调节因子(GRF),由多基因家族编码,在植物生长发育和逆境应答发挥关键的作用。鉴定铁皮石斛(Dendrobium officinale)GRF基因家族,为铁皮石斛GRF基因功能研究及遗传改良提供理论依据。【方法】通过生物信息学的方法鉴定铁皮石斛14-3-3家族成员,分析其理化性质、染色体定位、系统进化发育、基因结构和启动子顺式作用元件等,同时通过荧光定量PCR技术检测它们在不同组织、低温处理及盐胁迫处理后的表达量。【结果】铁皮石斛有17个GRF家族成员,分为ε类和非ε类亚族,不均匀地分布在7条染色体上,且存在7对串联复制基因。同一亚族成员基因结构、保守基序和蛋白质二级结构相类似。DoGRF家族基因的启动子区域存在大量激素和环境胁迫应答相关的调控元件。DoGRF家族基因在铁皮石斛各组织中均有表达,具有组织表达特异性,大多数基因在花器官中表达最高,其次是茎和根。同时,在低温处理、盐胁迫处理下呈现差异化表达,可能受到低温和盐胁迫的调控,特别是DoGRF2在铁皮石斛逆境应答过程中起着关键的作用。【结论】在全基因组水平从铁皮石斛中鉴定出17个DoGRF家族成员,不同基因对非生物胁迫响应不一,并具有器官表达特异性。DoGRF2对低温和盐胁迫呈现正向响应。

14-3-3蛋白及其在植物中的功能研究进展

14-3-3蛋白是由不同基因编码的高度同源的酸性蛋白质家族,在真核生物中广泛存在,且在结构上相对保守。14-3-3蛋白主要是通过识别靶蛋白上的磷酸化位点与靶蛋白发生相互作用,导致靶蛋白的稳定性、亚细胞定位或与其他蛋白之间的相互作用发生显著变化,进而调控靶蛋白的功能。不同物种中含有多种亚型的14-3-3蛋白,这些不同的亚型蛋白通过与其他蛋白的相互作用来影响植物的生长发育等过程。本文概述了14-3-3蛋白在植物中的种类、亚细胞定位以及在组织中的表达情况,重点总结了14-3-3蛋白在植物激素信号转导、生长发育及胁迫响应中的功能,以期为今后系统开展14-3-3蛋白的研究提供理论依据。

急性呼吸窘迫综合征患者血清lncRNA SNHG14、miR-124-3p表达变化及其与预后的关系

目的观察急性呼吸窘迫综合征(ARDS)患者血清长链非编码核糖核酸(lncRNA)核仁核糖核酸宿主基因14(SNHG14)、微小核糖核酸-124-3p(miR-124-3p)表达变化,并探讨两者与患者预后的关系。方法选取ARDS患者100例作为ARDS组,同期体检健康志愿者60例作为对照组。采用实时荧光定量PCR法检测血清lncRNA SNHG14、miR-124-3p相对表达量,Pearson相关分析法分析两者的关系。ARDS患者入院后28 d,根据生存状态评估预后,记录死亡和存活情况,采用多因素Logistic回归分析血清lncRNA SNHG14、miR-124-3p对患者死亡的影响,绘制两者单独及联合预测ARDS患者预后的受试者工作特征(ROC)曲线,分析预测价值。结果ARDS组血清lncRNA SNHG14、miR-124-3p相对表达量分别为2.78±0.45、、0.43±0.08,对照组分别为1.13±0.15,1.03±0.20,两组比较P均<0.05。ARDS患者血清lncRNA SNHG14、miR-124-3p相对表达量呈负相关关系(r=-0.789,P<0.05)。100例ARDS患者28 d内死亡39例、存活61例,死亡的ARDS患者血清lncRNA SNHG14、miR-124-3p相对表达量分别为3.07±0.42、0.91±0.17,存活的ARDS患者分别为2.60±0.37、1.11±0.17,两者比较P均<0.05。多因素Logistic回归分析结果显示,血清lncRNA SNHG14表达升高、miR-124-3p表达降低是ARDS患者死亡的独立危险因素(P均<0.05)。血清lncRNA SNHG14、miR-124-3p单独及联合预测ARDS患者预后的AUC分别为0.790、0.785、0.881(P均<0.05),敏感度分别为87.18%、61.54%、92.31%,特异度分别为59.02%、80.33%、72.13%。结论ARDS患者血清lncRNA SNHG14表达升高、miR-124-3p表达降低,两者均与患者预后有关,联合检测有助于预测预后。

Overexpression of 14-3-3 protein protects pheochromocytoma cells against 1-methyl-4-phenylpyridinium toxicity

Objective To investigate the effects of 14-3-3 protein overexpression on the 1-methyl-4-phenylpyridinium （MPP^＋） induced pheochromocytoma （PC12） cell death and the potential mechanisms. Methods pcDNA3.1（＋）-14-3-3 plasmids, which could be expressed in mammalian cell, were constructed and transfected into PC 12 cells with Lipofectamine 2000. The expression of 14-3-3 protein, Bcl-2 protein, and BAD protein were determined by western blot. 3-（4,5- dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, microplate reader, and flow cytometric analysis were used to measure cell viability, the caspase activity, and apoptotic ratio respectively. Results （1） The expression of 14-3-3 protein increased significantly three weeks after pcDNA3.1（＋）-14-3-3 plasmids transfected into PC 12 cells. （2） MPP^＋ caused a decrease of cell viability in a dose-dependent manner. At 100μmol/L MPP^＋, cell viability reduced approximately 50%. （3） The caspase activity increased along with the MPP^＋ concentrations rising and reached its maximum value （0.34 μmol/mg protein） at 100 μmol/L MPP＊. However caspase activity decreased significantly when the MPP^＋ concentration exceeded 100 μmol/L. （4） Overexpression of 14-3-3 protein decreased the apoptosis ratio of PC 12 cells treated with 100μmol/L MPP^＋ from 26.5% to 8.6%. （5） Bcl-2 protein tended to decrease but BAD protein tended to increase after treatment of PC 12 cells with 100 μmol/L MPP^＋. Overexpression of 14-3-3 protein significantly increased the cellular level of Bcl-2 protein and decreased that of BAD protein. Conclusion Overexpression of 14-3-3 protein may reduce MPP^＋-induced apoptotic cell death in PC12 cells by up-regulating the Bcl-2 expression and down-regulating the BAD expression. These results may provide a promising target for treatment of Parkinson＇s disease.

Involvement of ERK1/2 and p38 MAPK in up-regulation of 14-3-3 protein induced by hydrogen peroxide preconditioning in PC12 cells

Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.

miR-637、miR-223-3p、PARP14在甲状腺癌中的表达及与临床特征的相关性

目的 探究miR-637、miR-223-3p、多聚(腺苷二磷酸核糖)聚合酶14(PARP14)在甲状腺癌中的表达及与临床特征相关性。方法 选取2018年5月至2020年5月衡水市人民医院73例甲状腺癌患者。采用PCR法检测患者癌组织及癌旁组织miR-637、miR-223-3p、PARP14表达;所有患者随访3年,根据生存情况将其分为死亡组及生存组,分析患者癌组织miR-637、miR-223-3p、PARP14表达与预后的关系及对预后的预测价值。结果 甲状腺癌组织miR-637 mRNA表达量低于癌旁组织,miR-223-3p、PARP14 mRNA表达量高于癌旁组织(P<0.05)。miR-637 mRNA在临床分期Ⅲ~Ⅳ期、包膜浸润及淋巴结转移患者中均为低表达(P<0.05),PARP14 mRNA在临床分期Ⅲ~Ⅳ期、肿瘤直径≥2 cm、包膜浸润及淋巴结转移患者中均为高表达(P<0.05),miR-223-3p mRNA在肿瘤直径≥2 cm、包膜浸润及淋巴结转移患者中均为高表达(P<0.05)。死亡组癌组织miR-637mRNA表达量低于生存组,miR-223-3p、PARP14表达mRNA表达量高于生存组(P<0.05)。多因素Cox回归显示,癌组织miR-637 mRNA表达量升高为影响甲状腺癌患者预后的危险因素,miR-223-3p、PARP14 mRNA表达量升高为保护因素(P<0.05)。癌组织miR-637、miR-223-3p、PARP14表达联合检测预测甲状腺癌患者预后的AUC值大于各指标单独检测(P<0.05)。癌组织miR-637低表达患者的累积生存率低于高表达患者(P<0.05);癌组织miR-223-3p、PARP14高表达患者的累积生存率低于低表达患者(P<0.05)。结论 甲状腺患者癌组织miR-637、miR-223-3p、PARP14 mRNA呈异常表达现象,上述指标mRNA表达量与临床分期、肿瘤直径、淋巴结转移等临床特征及预后有关,且miR-637、miR-223-3p、PARP14联合可用于评估患者预后。

外周血sCD14、miR-140-3p及TBIL对冠心病患者冠脉斑块稳定性的评估价值

目的探究外周血可溶性白细胞分化抗原14(sCD14)、微小RNA-140-3p(miR-140-3p)及总胆红素(TBIL)对冠心病患者冠脉斑块稳定性的评估价值。方法选取郸城县人民医院2021年8月至2023年8月收治的80例冠心病患者为研究对象,行血管内超声检查,对患者的斑块性质进行分类,并根据检查结果对患者进行分组:稳定组55例与不稳定组25例。收集并对比两组患者一般资料信息及血清指标水平,将有统计学意义的变量进行分析,探究影响冠心病患者冠脉斑块稳定性的因素。结果稳定组和不稳定组患者的年龄、性别、体质量指数(BMI)、糖尿病、高血压、尿素氮、尿肌酐以及血中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平比较差异无统计学意义(P>0.05);稳定组sCD14水平显著低于不稳定组,差异具有统计学意义(P<0.05),miR-140-3p、TBIL水平显著高于不稳定组,差异均有统计学意义(P<0.05);Logistic回归分析显示,高水平sCD14、低水平miR-140-3p及低水平TBIL均为冠心病患者冠脉斑块不稳定性斑块形成的危险因素,差异均有统计学意义(P<0.05);受试者工作特征(ROC)曲线分析表明,高水平sCD14、低水平miR-140-3p及低水平TBIL均对不稳定型斑块均具有一定诊断价值,其曲线下面积(AUC)分别为0.706、0.939、0.807,差异均有统计学意义(P<0.05)。结论冠心病患者外周血sCD14、miR-140-3p及TBIL表达水平与冠脉不稳定型斑块的形成具有密切关系,对于斑块稳定性具有一定的评估价值。

14-3-3 proteins—an update

14-3-3 is a highly conserved acidic protein family, composed of seven isoforms in mammals. 14-3-3 protein caninteract with over 200 target proteins by phosphoserine-dependent and phosphoserine-independent manners. Little isknown about the consequences of these interactions, and thus are the subjects of ongoing studies. 14-3-3 controls cellcycle, cell growth, differentiation, survival, apoptosis, migration and spreading. Recent studies have revealed newmechanisms and new functions of 14-3-3, giving us more insights on this fascinating and complex family of proteins.Of all the seven isoforms, 14-3-3σ seems to be directly involved in human cancer. 14-3-3σ itself is subject to regulationby p53 upon DNA damage and by epigenetic deregulation. Gene silencing of 14-3-3σ by CpG methylation has beenfound in many human cancer types. This suggests that therapy-targeting 14-3-3σ may be beneficial for future cancertreatment.

Echinococcus Granulosus 14-3-3 Protein:A Potential Vaccine Candidate Against Challenge with Echinococcus Granulosus in Mice

Objective To investigate the protective immunity against Echinococcus granulosus in mice immunized with rEg14-3-3. Methods ICR mice were subcutaneously immunized three times with rEg14-3-3, followed by the challenge with Echinococcus granulosus protoscoleces intraperitoneally and then sacrificed after six months of post-challenge to detect the proliferation of splenocytes by MTT assay, and to measure the secretion of IL-2, IL-4, IL-20, and IFN -y by ELISA. The rate of reduced hydatid cyst and the levels of IgE, igG and IgG subclasses in sera were examined. Results Mice vaccinated with rEg14-3-3 and challenged with protoscoleces revealed significant protective immunity of 84.47%. ELISA analysis indicated that the immunized mice generated specific high levels of IgG and the prevailing isotypes of IgG were IgG1 and IgG2a. Splenocytes from mice immunized with rEg14-3-3 showed a significant proliferation response. The secretion of IFN-V and IL-2 increased significantly in the vaccinated mice whereas there was no significant difference in IL-4 and IL-20 levels between vaccinated and control mice. Conclusion The results indicate that the rEg24-3-3 vaccine could induce a high level of protective immunity as a promising vaccine candidate to prevent cystic echinococcosis.

Accumulation of Carbohydrate and Regulation of 14-3-3 Protein on Sucrose Phosphate Synthase(SPS) Activity in Two Tomato Species

To explore the differences of carbohydrate metabolism in two tomato species and discuss the possible regulation of 14-3-3 proteins on the sucrose phosphate synthase （SPS） activity, we determined the contents of soluble sugar and starch through high performance liquid chromatography （HPLC）. The activities of sugar-metabolizing enzymes were assayed in desalted extract, and the relative expression levels of related genes in sugar metabolism were determined though real-time RT-PCR. The results indicated that glucose and fructose were mainly accumulated during the maturation of the fruit because of the high acid invertase （AI） and neutral invertase （NI） in Micro-Tom （Solanum lycopersicum） fruit, while in Solanum chmielewskii fruit, SPS which went along with the change of sucrose content led to the rapid sucrose increase during the fruit ripening. TFT1 and TFT10, belonging to 14-3-3 protein in tomato, were likely to down-regulated SPS activity during young and intumescence period.

A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins

hPFTAIRE1 （PFTK1）, a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals （NLSs） in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms （β,ε,η,τ） were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif（RHSSPSS） in the hPFTAIRE 1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser^119 gwithAla in the conserved binding motif abolished the specific interaction between the hPFTAIRE 1 and the 14-3 -3 proteins. The mutant S 120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser^119 is crucial for the interaction between hPFTAIREI and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells （SH-SY5Y） when fused to the C-terminus of a green fluorescent protein （GFP）, indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation.

The crystal structure of the non-liganded 14-3-3σ protein: insights into determinants of isoform specific ligand binding and dimerization

Seven different, but highly conserved 14-3-3 proteins are involved in diverse signaling pathways in human cells. It isunclear how the 14-3-3σ isoform, a transcriptional target of p53, exerts its inhibitory effect on the cell cycle in thepresence of other 14-3-3 isoforms, which are constitutively expressed at high levels. In order to identify structuraldifferences between the 14-3-3 isoforms, we solved the crystal structure of the human 14-3-3σ protein at a resolutionof 2.8 ? and compared it to the known structures of 14-3-3ζ and 14-3-3τ. The global architecture of the 14-3-3σ foldis similar to the previously determined structures of 14-3-3ζ and 14-3-3τ: two 14-3-3σ molecules form a cup-shapeddimer. Significant differences between these 14-3-3 isoforms were detected adjacent to the amphipathic groove, whichmediates the binding to phosphorylated consensus motifs in 14-3-3-ligands. Another specificity determining region islocalized between amino-acids 203 to 215. These differences presumably select for the interaction with specific ligands,which may explain the different biological functions of the respective 14-3-3 isoforms. Furthermore, the two 14-3-3σmolecules forming a dimer differ by the spatial position of the ninth helix, which is shifted to the inside of the ligandinteraction surface, thus indicating adaptability of this part of the molecule. In addition, 5 non-conserved residues arelocated at the interface between two 14-3-3σ proteins forming a dimer and represent candidate determinants of homo-and hetero-dimerization specificity. The structural differences among the 14-3-3 isoforms described here presumablycontribute to isoform-specific interactions and functions.

14-3-3 gamma and zeta protein expression in active microglia Immune response mechanisms of Parkinson’s disease

BACKGROUND： The progressive degeneration of dopaminergic neurons in Parkinson＇s disease is associated with an activated glial reaction, combined with an inflammatory process. These responses lead to the production of cytokines, such as interferon- γ, tumor necrosis factor- α （TNF- α ）, and interleukin-1 β. In addition, 14-3-3 protein is a component of Lewy bodies in Parkinson＇s disease. OBJECTIVE： To observe the expression of 14-3-3 γ and ζ protein, as well as TNF-α, in mouse microglia, as well as changes after lipopolysaccharide （LPS） activation. To investigate possible mechanisms of dopaminergic neuronal injury due to activated microglia. To and clarify the immune response mechanisms of Parkinson＇s disease. DESIGN： Randomized controlled observation, cell study.SETTING： Laboratory of Department of Neurology, the Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： The BV-2 immortalized murine microglia cell line was purchased from China Unit cell center. LPS was provided by Sigma Company. Cell cultures were purchased from Gibco. Phospho-（Ser） 14-3-3 binding motif antibody was purchased from Santa Cruz Biotechnologies. FITC was provided by Linfei Biotechnology, Wuhan, China. TNF- α ELISA was provided by Jingmei Biotech Co, Wuhan, China. The flow cytometer was provided by Becton Dickinson, Canada. METHODS： The present experiment was performed at the Laboratory of Department of Neurology, the Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Technology from April to December 2006. The microglial cell line, BV-2, was cultured in vitro and stimulated with LPS for 2, 6, 12, and 24 hours. BV-2 cultures without LPS were used as controls. MAIN OUTCOME MEASURES： Expression of 14-3-3 γ protein was detected by flow cytometry. 14-3-3 ζ percentage expression and the mean fluorescence intensity was detected by immunofluorescence. TNF- α expression was detected by ELISA. RESULTS： 14-3-3 γ protein expression analysis： following LPS-induction in BV-2 cells, the fluorescence intensity of the 14-3-3 γ proteins gradually decreased. The 12 and 24 hours groups exhibited significantly lower expression than the normal control group （P 〈 0.05）. 14-3-3 ζ percentage expression and the mean fluorescence intensity： the percentage of 14-3-3 ζ protein expression gradually decreased with LPS stimulation. The mean fluorescence intensity from the 6, 12, and 24 hours groups was significantly less than the control group （P 〈 0.05）. TNF-α expression： resting BV-2 cells did not express TNF-α. Following 2 hours of LPS stimulation, TNF-α was highly expressed in BV-2 cells, but decreased again by 24 hours. CONCLUSION： Dopaminergic neuronal injury, due to activated microglial cells, might be related to the participation of 14-3-3 proteins and the release of TNF-α.