Cellular Expression and Subcellular Localization of Wwox Protein During Testicular Development and Spermatogenesis in Rats

A well-known putative tumor suppressor WW domain–containing oxidoreductase (Wwox) is highly expressed in hormonally regulated tissues and is considered important for the normal development and function of reproductive organs. In this study, we investigated the cellular and subcellular localization of Wwox in normal testes during postnatal days 0–70 using Western blotting and immunohistochemistry. Wwox is expressed in testes at all ages. Immunohistochemistry showed that fetal-type and adult-type Leydig cells, immature and mature Sertoli cells, and germ cells (from gonocytes to step 17 spermatids) expressed Wwox except peritubular myoid cells, step 18–19 spermatids, and mature sperm. Wwox localized diffusely in the cytoplasm with focal intense signals in all testicular cells. These signals gradually condensed in germ cells with their differentiation and colocalized with giantin for cis-Golgi marker and partially with golgin-97 for trans-Golgi marker. Biochemically, Wwox was detected in isolated Golgi-enriched fractions. But Wwox was undetectable in the nucleus. This subcellular localization pattern of Wwox was also confirmed in single-cell suspension. These findings indicate that Wwox is functional in most cell types of testis and might locate into Golgi apparatus via interaction with Golgi proteins. These unique localizations might be related to the function of Wwox in testicular development and spermatogenesis:     

GAS41蛋白的表达、抗体制备及其亚细胞定位

GAS41是新发现的与神经胶质瘤密切相关的基因。为了对该基因进行深入的功能研究,成功地将GAS41基因构建于原核表达载体pQE—N3,转化BL21（DE3）获得融合表达产物。对含融合蛋白的包含体进行溶解和复性,通过免疫新西兰大白兔获得兔抗GAS41多克隆抗体。采用Western印迹技术,用该抗体检测GAS41基因的原核和真核表达产物,证明该抗体有较好的针对GAS41蛋白的专一性,可用于对GAS41的结构和功能研究。同时,用该抗体通过荧光免疫细胞进行定位分析发现,GAS41蛋白均匀分布于COS7细胞的细胞核中,提示GAS41可能是一个重要的转录因子。     

Subcellular Location and Expression of Tobacco Necrosis Necrovirus p7a Protein

A putative movement protein (p7a) of tobacco necrosis Necrovirus , strain D (TNV-D), produced in Escherichia coli using an expression vector, was used to raise an antiserum. Immunoblot analysis using this antiserum showed that the p7a protein was detectable only in the combined cell wall and cell membrane fraction prepared from TNV-D infected Phaseolus vulgaris leaves. The p7a protein was detectable 1 day after inoculation and reached a maximum 3 days later, before declining, whereas coat protein was not detectable until 3 days after inoculation and continued to increase in concentration for a further 2 days before declining. Differences in the detectable amounts of both proteins may reflect their differential stability in extracts from necrotic tissue and/or the transient expression of the putative movement protein early in the replication cycle of TNV-D.     

苹果MdKS、MdKOA1基因克隆与表达分析

内根-贝壳杉烯合成酶基因(KS)、内根-贝壳杉烯酸氧化酶基因(KOA)是赤霉素合成途径的关键基因。通过touchdown PCR从短枝富士茎尖组织中克隆得到KS和KOA基因的开放阅读框(ORF),分别命名为MdKS(GenBank登录号KF437681)和MdKOA1(GenBank登录号为KF437682)。MdKS和MdKOA1基因的ORF分别为2211 bp、1509 bp。氨基酸同源性分析表明,MdKS与其他物种的氨基酸序列具有45.2%~98.8%的同源性;而MdKOA1与其他物种的氨基酸序列同源性为42.8%~99.4%。亚细胞定位显示,MdKS蛋白定位于细胞核、细胞质和细胞质膜;而MdKOA1蛋白定位于细胞质和细胞质膜。荧光定量表明,MdKS与MdKOA1基因在SH40、SH28嫁接品种短枝富士不同组织中的表达模式基本一致。MdKS和MdKOA1在半矮化类型SH28嫁接短枝富士的茎尖、幼果与枝条中的表达量高于矮化类型SH40的嫁接品种。     

Subcellular Location and Neuronal Release of Diazepam Binding Inhibitor

Diazepam binding inhibitor (DBI), a peptide located in CNS neurons, blocks the binding of benzodiazepines and beta-carbolines to the allosteric modulatory sites of gamma-aminobutyric acid (GABAA) receptors. Subcellular fractionation studies of rat brain indicate that DBI is compartmentalized. DBI-like immunoreactivity is highly enriched in synaptosomes obtained by differential centrifugation in isotonic sucrose followed by a Percoll gradient. In synaptosomal lysate, DBI-like immunoreactivity is primarily associated with synaptic vesicles partially purified by differential centrifugation and continuous sucrose gradient. Depolarization induced by high K+ levels (50 mM) or veratridine (50 microM) released DBI stored in neurons of superfused slices of hypothalamus, hippocampus, striatum, and cerebral cortex. The high K+ level-induced release is Ca2+ dependent, and the release induced by veratridine is blocked by 1.7 microM tetrodotoxin. Depolarization released GABA and Met5-enkephalin-Arg6-Phe7 together with DBI. DBI is also released by veratridine depolarization, in a tetrodotoxin-sensitive fashion, from primary cultures of cerebral cortical neurons, but not from cortical astrocytes. Depolarization fails to release DBI from slices of liver and other peripheral organs. These data support the view that DBI may be released as a putative neuromodulatory substance from rat brain neurons.     

Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves

The subcellular location of NADP⁺-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP⁺-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP⁺-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP⁺-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K_m values of 11 micromolar (NADP⁺), 35 micromolar (isocitrate), 78 micromolar (Mn²⁺), 0.3 millimolar (Mg²⁺) and showed optimum activity at pH 8 to 8.5 with Mn²⁺ and 8.8 to 9.2 with Mg²⁺. The NADP⁺-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP⁺-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.     

人微丝相关蛋白hHBRK1突变体的亚细胞定位

 hHBrk1是本研究室利用抑制性消减杂交手段,从人支气管上皮细胞恶性转化株BERP3 5中克隆到的差异高表达基因.hHBRK1蛋白家族序列在动、植物界高度保守,含有一个7重复 (heptad repeat, HR) 结构域.利用绿色荧光蛋白(GFP)报告系统,发现野生型hHBRK1蛋白在胞浆中弥散分布,在细胞运动前沿富集,与细胞片状伪足的微丝共定位.hHBRK1- R54和hHBRK1-S56 G57蛋白在胞浆弥散分布,但失去了在细胞运动前沿富集的特征.hHBRK1ΔN(1-45)在细胞内弥散分布,而hHBRK1ΔC(46-75)选择性地在高尔基体富集.研究提示,hHBRK1蛋白为微丝相关蛋白,结构的完整性是其发挥功能的前提.hHBRK1蛋白可能通过HR结构域调控微丝聚合,从而参与微丝依赖性的细胞运动或物质运输.     

C-MET在大鼠胰腺发育过程中的表达

目的:研究CMET在大鼠胰腺发育阶段的表达和细胞定位.方法:运用RT-PCR和WesternBlot技术分别检测C-MET在大鼠胰腺发育阶段的mRNA和蛋白表达水平;运用免疫组化和免疫荧光技术检测不同时期C-MET在胰腺的组织细胞学定位.结果:RT-PCR结果显示E18.5 C-METmRNA高表达.Western Blot结果显示其蛋白在P14,P21高表达,并存在两种亚型,分子量分别为190KD和170KD.免疫组化和免疫荧光结果显示在不同发育时期C-MET在胰岛B细胞和间充质细胞都有表达.结论:C-MET在大鼠胚胎发育后期及生后出现高表达,并表达于胰岛B细胞和间充质细胞,可能参与了胰岛形成、结构重塑和功能维持.     

Protein Subcellular Location: The Gap Between Prediction and Experimentation

Newly synthesized proteins in eukaryotic cells can only function well after they are accurately transported to specific organelles. The establishment of protein databases and the development of programs have accelerated the study of protein subcellular locations, but their comparisons and evaluations of the prediction accuracy of subcellular location programs in plants are lacking. In this study, we built a random test set of maize proteins to evaluate the accuracy of six commonly used programs of subcellular locations: iLoc-Plant, Plant-mPLoc, CELLO, WoLF PSORT, SherLoc2, and Predotar. Our results showed that the accuracy of prediction varied greatly depending on the programs and subcellular locations involved. The programs using homology search methods (iLoc-Plant and Plant-mPLoc) performed better than those using feature search methods (CELLO, WoLF PSORT, SherLoc2, and Predotar). In particular, iLoc-Plant achieved an 84.9 % accuracy for proteins whose subcellular locations have been experimentally determined and a 74.3 % accuracy for all of the proteins in the test set. Regarding locations, the highest prediction accuracies for subcellular locations were obtained for the nucleus, followed by the cytoplasm, mitochondria, plastids, endoplasmic reticulum, and vacuoles, while the lowest were obtained for cell membrane, secreted, and multiple-location proteins. We discussed the accuracy of the six programs in this article. This study will assist plant biologists in choosing appropriate programs to predict the location of proteins and provide clues regarding their function, especially for hypothetical or novel proteins.     

Subcellular Location of Phosphoglucomutase and UDP Glucose Pyrophosphorylase in Avena Coleoptiles

Extraction of phosphoglucomutuse and UDP glucose pyrophosphorylase from oat coleoptiles using both aqueous and non-aqueous systems showed that both enzymes are largely soluble. Phosphoglucomutase was completely absent from the cell wall fraction. The inhibition of phosphoglucomutase by peroxyacetyl nitrate was confirmed and the enzyme in the subcellular participate fractions was shown to be more inhibited than that in the soluble fraction. UDP glucose pyrophosphorylase was not inhibited by peroxyacetyl nitrate or ozone in vivo but was inhibited by peroxyacetyl nitrate in vitro. The SH reagents, iodoacetamide and p-chloromercuribenzoate, inhibited phosphoglucomutase severely but UDP glucose pyrophosphorylase moderately or not at all.     

The Temporal Expression Pattern of Alpha-Synuclein Modulates Olfactory Neurogenesis in Transgenic Mice

Background

Adult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS) under the murine Thy1 (mThy1) promoter, a model known to have a particularly high tg expression associated with impaired olfaction.

Results

Survival of newly generated neurons (NeuN-positive) in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF) promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.

Conclusions

The dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.     

水稻叶绿体蛋白质在生长发育过程中的表达研究

在植物中,叶绿体是负责光合作用的细胞器,对叶绿体内的各种生物过程人们已经积累了很多知识,但对叶绿体蛋白质的表达还所知甚少．为了解水稻叶绿体蛋白质在正常生长发育过程中的表达情况,尝试基于抗体的水稻蛋白质组学策略．选取了10个水稻叶绿体基因,利用表达的蛋白质或合成的抗原决定簇片段制备了抗体,用Western blotting检测了相应蛋白质在5个发育时期的根、茎、叶及穗组织中的表达．发现10个蛋白质均在叶片中表达,在根中不表达．与原初反应相关的叶绿素A/B结合蛋白1和2(CAB1和CAB2)、与电子传递相关的放氧增强蛋白1(OEE1)及与活性氧清除相关的过氧还蛋白过氧化物酶(2-CysP)和硫氧还蛋白(Trx)在茎中表达．而在卡尔文循环中发挥作用的Rubisco活化酶(RCA)、甘油醛-3-磷酸脱氢酶(GAPDH)、果糖二磷酸醛缩酶(FBPA)和景天庚酮糖-1, 7-二磷酸酶(SBPase)蛋白质在茎中不表达．在穗中,这些蛋白质的表达时序不同,CAB2和2-CysP在穗发育的全程表达,CAB1和OEE1在中后期表达,而卡尔文循环中的蛋白质只在中期表达．有意思的是,卡尔文循环中的蛋白质表达模式相似,这一结果从蛋白质表达水平支持它们之间的相互衔接关系．此外,实验还揭示了可能的蛋白质修饰、二聚体及不同的转录本现象．将目标基因的表达谱与转录谱进行比较,发现二者间有一定的平行性,但也有明显的区别．以水稻叶绿体蛋白质为对象,直观并相对定量地揭示了它们的表达模式,为阐释其功能提供了信息,也为基于抗体的水稻蛋白质组学策略提供了一个初步数据．     

Effects of Alpha-Synuclein on Primary Spinal Cord Neurons Associated with Apoptosis and CNTF Expression

Spinal cord injury (SCI) often causes neurological deficits with poor recovery; the treatment, however, is far from satisfaction, and the mechanisms remain unclear. Using immunohistochemistry and western blotting analysis, we found α-synuclein (SNCA) was significantly up-regulated in the spinal caudal segment of rats subjected to spinal cord transection at 3 days post-operation. Moreover, the role of SNCA on neuronal growth and apoptosis in vitro was determined by using overexpressing and interfering SNCA recombined plasmid vectors, and the underlying mechanism was detected by QRT-PCR and western blotting. Spinal neurons transfected with SNCA-shRNA lentivirus gave rise to an optimal neuronal survival, while it results in cell apoptosis in SNCA-ORF group. In molecular level, SNCA silence induced the up-regulation of CNTF and down-regulation of Caspase7/9. Together, endogenous SNCA plays a crucial role in spinal neuronal survival, in which the underlying mechanism may be linked to the regulation both apoptotic genes (Caspase7/9) and CNTF. The present findings therefore provide novel insights into the role of SNCA in spinal cord and associated mechanism, which may provide novel cue for the treatment of SCI in future clinic trials.     

Prion Properties of Alpha-Synuclein

Molecular Biology - The prion properties of alpha-synuclein, a key aggregating protein involved in the pathogenesis of so-called synucleinopathies, including Parkinson’s disease (PD),...