Pannexin 2 Is Expressed by Postnatal Hippocampal Neural Progenitors and Modulates Neuronal Commitment

The pannexins (Panx1, -2, and -3) are a mammalian family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins, the innexins. Because connexin gap junction proteins are known regulators of neural stem and progenitor cell proliferation, migration, and specification, we asked whether pannexins, specifically Panx2, play a similar role in the postnatal hippocampus. We show that Panx2 protein is differentially expressed by multipotential progenitor cells and mature neurons. Both in vivo and in vitro, Type I and IIa stem-like neural progenitor cells express an S-palmitoylated Panx2 species localizing to Golgi and endoplasmic reticulum membranes. Protein expression is down-regulated during neurogenesis in neuronally committed Type IIb and III progenitor cells and immature neurons. Panx2 is re-expressed by neurons following maturation. Protein expressed by mature neurons is not palmitoylated and localizes to the plasma membrane. To assess the impact of Panx2 on neuronal differentiation, we used short hairpin RNA to suppress Panx2 expression in Neuro2a cells. Knockdown significantly accelerated the rate of neuronal differentiation. Neuritic extension and the expression of antigenic markers of mature neurons occurred earlier in stable lines expressing Panx2 short hairpin RNA than in controls. Together, these findings describe an endogenous post-translational regulation of Panx2, specific to early neural progenitor cells, and demonstrate that this expression plays a role in modulating the timing of their commitment to a neuronal lineage.     

Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development.     

Identification of Differentially Expressed Genes in Breast Muscle and Skin Fat of Postnatal Pekin Duck

Lean-type Pekin duck is a commercial breed that has been obtained through long-term selection. Investigation of the differentially expressed genes in breast muscle and skin fat at different developmental stages will contribute to a comprehensive understanding of the potential mechanisms underlying the lean-type Pekin duck phenotype. In the present study, RNA-seq was performed on breast muscle and skin fat at 2-, 4- and 6-weeks of age. More than 89% of the annotated duck genes were covered by our RNA-seq dataset. Thousands of differentially expressed genes, including many important genes involved in the regulation of muscle development and fat deposition, were detected through comparison of the expression levels in the muscle and skin fat of the same time point, or the same tissue at different time points. KEGG pathway analysis showed that the differentially expressed genes clustered significantly in many muscle development and fat deposition related pathways such as MAPK signaling pathway, PPAR signaling pathway, Calcium signaling pathway, Fat digestion and absorption, and TGF-beta signaling pathway. The results presented here could provide a basis for further investigation of the mechanisms involved in muscle development and fat deposition in Pekin duck.     

microRNA在小鼠乳腺不同发育时期差异表达谱及作用

microRNA是一类大小约22个核苷酸的非编码RNA分子,是一种广泛存在的对基因表达进行微调的分子。microRNA可以通过与靶基因mRNA的特定位点结合,抑制该蛋白的合成或诱导该mRNA的降解,从而参与基因的表达调控。一般来源于染色体的非编码区域,由大约70个核苷酸大小的可形成发夹结构的前体经Dicer酶加工而来。这类小RNA在表达上具有组织和时间的特异性,是调节其他功能基因表达的重要调控分子,在生物的生长发育过程中发挥着重要作用。因此,虽然microRNA的研究仅有很短的历史,但已成为基因表达调控研究的热点领域。以中国昆明小鼠不同发育时期的乳腺组织为实验材料,应用芯片技术及荧光定量PCR技术,分析发育不同时期的乳腺组织microRNA差异表达图谱。本文研究发现microRNA在乳腺不同的发育时期表达图谱不同;与青春期、退化期比较,妊娠期、哺乳期有十余种microRNAs表达上调,20余种microRNAs表达下调;microRNAs在乳腺发育和泌乳周期中发挥重要的作用。     

应用LCM和蛋白质组学技术筛选肺腺癌的差异表达蛋白质

为筛选肺腺癌(lung adenocarcinoma,AdC)发病相关蛋白质,首先采用激光捕获显微切割技术(laser capture microdissection,LCM)分别从AdC组织和正常支气管上皮(normal bronchial epithelium,NBE)组织中切割并收集AdC细胞和NBE细胞,再应用双向凝胶电泳技术(two-dimensional gel electrophoresis,2-DE)分离经LCM收集的细胞蛋白质,通过PDQuest软件分析差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定差异表达蛋白质,组织芯片免疫组化方法检测差异蛋白质膜联蛋白A4(annexin A4)在30例AdC组织、配对的癌旁组织和淋巴结转移癌组织中的表达水平。研究结果显示,通过蛋白质组学方法建立了LCM收集的AdC和NBE细胞的2-DE图谱,质谱鉴定得到了33个差异表达蛋白质,其中21个蛋白质在AdC细胞中表达上调,12个蛋白质在AdC细胞中表达下调。组织芯片免疫组化结果显示,与癌旁肺组织相比,annexin A4在AdC组织中的表达水平显著上调,且在AdC淋巴结转移癌组织中的表达明显高于其原发癌组织。上述结果提示annexin A4与AdC的发病及淋巴结转移相关,有望成为诊断AdC及预测AdC转移的分子标志物。     

Identification of Streptomyces coelicolor Proteins That Are Differentially Expressed in the Presence of Plant Material

Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions. S. coelicolor was grown in minimal medium with and without L. minor fronds. Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material. One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions. The repressed proteins were proteins synthesized under starvation stress conditions. These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants.     

Bayesian Analysis of iTRAQ Data with Nonrandom Missingness: Identification of Differentially Expressed Proteins

iTRAQ (isobaric Tags for Relative and Absolute Quantitation) is a technique that allows simultaneous quantitation of proteins in multiple samples. In this paper, we describe a Bayesian hierarchical model-based method to infer the relative protein expression levels and hence to identify differentially expressed proteins from iTRAQ data. Our model assumes that the measured peptide intensities are affected by both protein expression levels and peptide specific effects. The values of these two effects across experiments are modeled as random effects. The nonrandom missingness of peptide data is modeled with a logistic regression which relates the missingness probability for a peptide with the expression level of the protein that produces this peptide. We propose a Markov chain Monte Carlo method for the inference of model parameters, including the relative expression levels across samples. Our simulation results suggest that the estimates of relative protein expression levels based on the MCMC samples have smaller bias than those estimated from ANOVA models or fold changes. We apply our method to an iTRAQ dataset studying the roles of Caveolae for postnatal cardiovascular function.     

人胚胎干细胞定向分化为神经前体细胞

采用单层贴壁分化的方法在无血清条件下诱导同源饲养层培养的人胚胎干细胞定向分化,得到了高比例的神经前体细胞(97.5±0.83)%(P<0.05)。这些神经前体细胞具有分化为神经元、星形胶质细胞和少突胶质细胞的能力。在长期的传代培养中发现,随着培养时间的延长,nestin阳性的神经前体细胞比例下降,同时发育能力也发生了变化。在传代培养的早期,神经前体细胞发育为神经元的比例很高,几乎没有胶质细胞分化出来。随着培养时间的延长,胶质细胞的比例逐渐上升。这与体内神经系统的发育过程非常相似。进一步研究发现具有bHLH(basic helix-loop-helix)结构域的转录因子neurogenein2(Ngn2)和Olig2可能在这一变化中起重要作用。因此,人胚胎干细胞来源的神经前体细胞能够模拟体内神经发育的模式,为在体外研究人的神经发育和再生医学奠定了基础。     

Identification of Differentially Expressed Proteins in Direct Expressed Prostatic Secretions of Men with Organ-confined Versus Extracapsular Prostate Cancer

Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n = 8) or organ-confined (n = 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n = 5) versus patients with no evidence of recurrence upon follow-up (n = 10). Lastly, we performed proof-of-concept SRM-MS-based relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS.Prostate cancer is the most common malignancy to affect men in the Western world, but only 15–20% of these men will present with aggressive, lethal disease (1, 2) whereas the majority of patients will die of other causes. Although the implementation of large-scale screening for prostate cancer using serum prostate-specific antigen (PSA) has dramatically improved early detection of disease, unnecessary biopsies and patient overtreatment are becoming increasingly evident (2, 3). Consequently, there has been a shift in emphasis away from detection of prostate cancer and toward identification of lethal disease. Currently, Gleason grading is considered to be one of the best outcome predictors; however, patients with Gleason 7 tumors are in the clinical “gray zone,” whereby the predictive ability of Gleason grading is mixed (4, 5). A recent study constructed a 157-gene signature based on the comparison of Gleason score ≤6 and ≥8 patients, and could show that their panel could predict lethality in the cohort of Gleason 7 patients (5). Nonetheless, the development and large-scale implementation of prognostic markers of prostate cancer has been hampered by numerous factors owing, in part, to the heterogeneous and multifocal nature of the disease (6). Although the widely used Gleason grading system attempts to control for heterogeneity of the glands and multifocality of cancerous lesions by summing the 2–3 most commonly observed histological patterns via inspection of multiple (typically 8–12) core biopsies, cancerous foci are still often missed (2, 6) providing only partial information that can lead to imprecise diagnoses and prognoses. Pathologic staging remains the gold standard for disease staging and risk assessment (7, 8); however, this process lacks timeliness in discriminating organ-confined from extracapsular disease. Indeed, one-third of individuals with nonorgan-confined disease are identified only after surgery (9). Furthermore, ∼35% of men treated with radical prostatectomy with curative intent subsequently develop biochemical recurrence (10–13) and the mean time from surgery to recurrence is 3.5 years (4). Significant risk factors for time to prostate-specific mortality following biochemical recurrence after radical prostatectomy are PSA doubling time, pathological Gleason score, and time from surgery to biochemical recurrence (4). Estimates place the percent of lethal cases at 20–25% of all patients that show biochemical recurrence, suggesting that nearly 75–80% of patients in this group may be overtreated (14).There is an emerging trend toward recruitment of men with perceived low-risk disease to an “active surveillance” monitoring approach. This is based on the supposition that most prostate cancers are slow growing, and that the more aggressive forms can be identified during a period of observation with little increased risk of death. Although a consensus may not exist for defining the disease stage where active surveillance is warranted, there is considerable agreement that men who have a PSA level less than 10 ng/ml, impalpable disease (clinical stage T1c) and only 1 biopsy core out of 12 or more that show Gleason 6 cancer are most likely to harbor indolent disease (15). Even so, these candidates for active surveillance will still contain individuals who will have disease progression and die from their cancer. Thus, despite efforts to recruit individuals to active surveillance protocols, overtreatment of prostate cancer is fueled by the lack of reliable means to accurately discriminate between men with clinically indolent prostate cancer from those with more aggressive disease (16, 17). This inability to accurately predict prostate cancer aggressiveness based solely on standard clinicopathologic features clearly underscores the need to explore the ability of additional biomarkers to enhance outcome prediction for men with prostate cancer. Furthermore, it is important to acknowledge that a single biomarker alone is unlikely to have sufficient prognostic power; rather, the integration of a panel of biomarkers hold the promise for improved prostate cancer detection and prognosis (2).Fluids that are proximal to the prostate are attractive sources of potential prostate cancer biomarkers (2, 18), as they house secreted proteins and sloughed cells that provide a presumably more comprehensive assessment of the organ and extent of disease. Further, fluids such as urine are clinically favorable for their ease of collection, the volume and frequency at which they can be obtained, and their adaptability to routine clinical assays. Prostate-proximal fluids include seminal fluid, semen, and expressed prostatic secretions (EPS)¹. Here, we focus on the analysis of EPS as our biological specimen, using direct-EPS samples for the discovery of candidate prognostic biomarkers and both direct-EPS and pooled EPS-urines derived from independent sets of patients for candidate biomarker verification. Direct-EPS is a prostatic fluid that is collected from patients undergoing prostatectomy by massaging the organ and expelling 0.5–1 ml of the fluid just prior to surgical removal. It was chosen as our discovery fluid as it is expected to house prostate-secreted proteins at a higher concentration and purity, and we have developed a workflow for the in-depth proteomic analysis of this fluid (19). Following discovery proteomics in 16 clinically stratified direct-EPS samples, verification studies were performed using independent sample sets of direct-EPS. Next, we focused our attention on the verification and quantitative analysis of candidate proteins in pooled EPS-urines. Before EPS-urine collection, men undergo digital rectal examination (DRE), often as part of a routine procedure, which causes direct-EPS to be expelled from the prostate and subsequently voided in urine. Because EPS-urine can be collected with substantial ease and in greater volumes and frequencies than direct-EPS, much attention has been paid to this fluid as a valuable resource of prostate cancer biomarkers amenable to routine clinical analysis. Following the recent FDA approval of the EPS-urine assay for prostate cancer gene 3 (PCA3), standardized clinical collection protocols will be widely implemented and easier access to this fluid is expected. Moreover, we have recently identified a number of prostate-enriched proteins in EPS-urine by comparing its proteome to a urine background (20).The present study used multidimensional protein identification technology (MudPIT) coupled with bioinformatics to first catalog and comparatively analyze the direct-EPS proteomes from a small cohort of patients with extracapsular versus organ-confined prostate cancers. A semiquantitative algorithm based on spectral counts (QSpec) (21) and an integrative data mining strategy led to the selection of a number of putative biomarkers that were verified by Western blotting in direct-EPS. Lastly, to demonstrate accurate quantitative measurements of verified candidates in EPS-urine, a pilot study utilizing SRM-MS was undertaken as a proof-of-concept.     

Transcriptional Dysregulation in Postnatal Glutamatergic Progenitors Contributes to Closure of the Cortical Neurogenic Period

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Mass Spectrometric Identification of In Vivo Phosphorylation Sites of Differentially Expressed Proteins in Elongating Cotton Fiber Cells

Two-dimensional gel electrophoresis (2-DE)-based proteomics approach was applied to extensively explore the molecular basis of plant development and environmental adaptation. These proteomics analyses revealed thousands of differentially expressed proteins (DEPs) closely related to different biological processes. However, little attention has been paid to how peptide mass fingerprinting (PMF) data generated by the approach can be directly utilized for the determination of protein phosphorylation. Here, we used the software tool FindMod to predict the peptides that might carry the phosphorylation modification by examining their PMF data for mass differences between the empirical and theoretical peptides and then identified phosphorylation sites using MALDI TOF/TOF according to predicted peptide data from these DEP spots in the 2-D gels. As a result, a total of 48 phosphorylation sites of 40 DEPs were successfully identified among 235 known DEPs previously revealed in the 2-D gels of elongating cotton fiber cells. The 40 phosphorylated DEPs, including important enzymes such as enolase, transketolase and UDP-L-rhamnose synthase, are presumed to participate in the functional regulation of numerous metabolic pathways, suggesting the reverse phosphorylation of these proteins might play important roles in elongating cotton fibers. The results also indicated that some different isoforms of the identical DEP revealed in our 2-DE-based proteomics analysis could be annotated by phosphorylation events. Taken together, as the first report of large-scale identification of phosphorylation sites in elongating cotton fiber cells, our study provides not only an excellent example of directly identifying phosphorylation sites from known DEPs on 2-D gels but also provides a valuable resource for future functional studies of phosphorylated proteins in this field.     

Detection of Differentially Expressed Basal Cell Proteins by Mass Spectrometry

The ability of cells to modulate interactions with each other and the substrate is essential for epithelial tissue remodeling during processes such as wound healing and tumor progression. However, despite strides made in the field of proteomics, proteins involved in adhesion have been difficult to study. Here, we report a method for the enrichment and analysis of proteins associated with the basal surface of the cell and its underlying matrix. The enrichment involves deroofing the cells with 20 mm ammonium hydroxide and the removal of cytosolic and organellar proteins by stringent water wash. Proteomic profiling was achieved by LC-FTMS, which allowed comparison of differentially expressed or shared proteins under different cell states. First, we analyzed and compared the basal cell components of mouse keratinocytes lacking the cell-cell junction molecule plakoglobin with their control counterparts. Changes in the molecules involved in motility and invasion were detected in plakoglobin-deficient cells, including decreased detection of fibronectin, integrin β₄, and FAT tumor suppressor. Second, we assessed the differences in basal cell components between two human oral squamous cell carcinoma lines originating from different sites in the oral cavity (CAL33 and UM-SCC-1). The data show differences between the two lines in the type and abundance of proteins specific to cell adhesion, migration, and angiogenesis. Therefore, the method described here has the potential to serve as a platform to assess proteomic changes in basal cell components including extracellular and adhesion-specific proteins involved in wound healing, cancer, and chronic and acquired adhesion-related disorders.There is an urgent need for tools to comprehensively identify markers of normal and pathological processes at the molecular level. DNA microarrays have enabled researchers to follow gene expression changes with respect to many of these processes, including individual tumors in the case of cancer (1). Direct detection of proteins is typically required to validate changes at the gene product level; however, the changes in protein levels do not always reflect changes in gene expression because of post-translational modifications, differential compartmentalization, recycling, and degradation. Because it is ultimately the proteins that convey cellular phenotypes, it is necessary to develop methods for direct screening of proteins, and mass spectrometry shows promise for this purpose. However, the usefulness of mass spectrometry as an analytical tool to detect proteins in cells or tissue is limited to the extent to which the sample is sufficiently enriched for the specific fraction of interest. It is still challenging to identify molecules involved in specific normal or pathological processes because the relevant proteins are often difficult to isolate from the majority of cellular proteins that are not correlated to the process of interest. In this context, an ideal proteomics approach would require a minimal amount of starting material, be amenable to an efficient enrichment strategy, and would provide results quickly.It has been well established that molecules directly involved in cell-cell and cell-substrate adhesions are critical for processes such as epithelial to mesenchymal transition and wound healing. Their further role in regulation of tissue integrity, cell polarity, motility, and invasion is emphasized by a variety of disorders stemming from their inappropriate expression and mutations (2, 3). Selectins, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 have been established both as biomarkers (4) and predictive factors (5, 6) for the development of accelerated atherosclerosis and heart disease. In epithelial tissues, reduced expression of the cell-cell adhesion molecule E-cadherin correlates with epithelial to mesenchymal transition, tissue invasion, and metastasis and is a prognostic biomarker of poor clinical outcome in many cell types (7–9). Furthermore, up-regulating E-cadherin is considered as a treatment option in several types of cancer (10). Therefore, methods are also needed to not only identify adhesion molecules as disease markers but to also understand the pathology of underlying medical problems caused by impairment in adhesion molecule function (e.g. inability to heal chronic wounds (11)). However, the lack of knowledge about regulation and functional interactions of the specific adhesion-related proteins has so far thwarted the attempts at direct targeting of these molecules in basic and clinical research (12, 13). Therefore, a comprehensive understanding of how proteins that function in adhesive processes work together to maintain proper tissue form and function is critical.Some of the same barriers to effective application of mass spectrometry as an analytical tool (as discussed above) have impeded analysis of cell-cell and cell-matrix adhesion-dependent processes such as wound healing and cancer (14). The study of extracellular matrix (ECM)1 and adhesion-related proteins is further complicated by the difficulty in sample preparation because compared with cytosolic proteins basal cell proteins are often highly insoluble (e.g. transmembrane and plaque components) and difficult to isolate from intracellular proteins. One general strategy involves using ECM-specific enzymes to dislodge the cells at their points of attachment (15). The supernatant from the partial digest is collected for further proteomics analysis. However, most mass spectrometric analyses depend on detection of peptides with specific ionization and fragmentation properties that are most readily achieved using trypsin as the sole enzyme. The use of ECM-specific enzymes may result in a distribution of peptides that are not optimal for detection (i.e. the generation of non-tryptic termini). The other general approach to isolate components of the ECM involves using detergents to lyse cells on the surfaces to which they are attached and collect the remaining cell debris for analysis (15). Although progress has been made with respect to the creation of “mass spectrometry-friendly” detergents (16), the use of chemicals for the purpose of protein solubilization is generally not ideal. To overcome these problems, we adapted a fast, simple method of isolating extracellular, transmembrane, and associated proteins (from here on collectively referred to as “basal cell proteins”) from cells attached to a solid substrate. The method consists of “deroofing” the cells attached to glass coverslips by 20 mm NH₄OH solution followed by rapid water rinses to remove the bulk of the cell and its remaining debris (17). Our results show efficient removal of cytoplasm and organelles and detection of basal cell proteins by mass spectrometry, including those involved in cell-cell and cell-extracellular matrix interactions. These proteins were liberated from the surface with trypsin, and the subsequently generated peptides were detected and profiled for differences using LC-FTMS.The approach was first validated by comparing basal cell protein composition in mouse keratinocytes with or without a critical cell-cell junction protein called plakoglobin (PG). This desmosomal protein is required for cell-cell adhesion and maintenance of tissue integrity (18). Plakoglobin inhibits keratinocyte motility (19) and is down-regulated in several distinct tumor types, including bladder, breast, and cervical cancers (20–22). Moreover, we were able to dissect the molecular differences between an independent isolate of PG^−/− keratinocytes that behaved differently in motility assays from the rest of the PG-null cells, further emphasizing the potential for using the method to differentiate between cells with distinct adhesive and motile behaviors. The method was then evaluated in clinically relevant human tumor cell lines by extending the analysis to include two human oral squamous cancers of different origin. Because they lack precisely defined changes in cell adhesion molecules and phenotype, we compared the basal cell protein expression of UM-SCC-1 (23) and CAL33 (24) cell lines isolated from the roof of the mouth and tongue, respectively. These experiments revealed 40 proteins differentially expressed between the cell lines among over 100 detected. Moreover, the proteomic profile reveals a set of motility- and invasion-related genes unique to tongue-derived CAL33 cells. This could indicate the difference between oral cancers derived from different parts of the mouth, or it may indicate a potential difference in aggressiveness between these cell lines. These results show that our detection method is applicable for both detection and comparative studies in human cancer model systems.     

五倍体草莓及其十倍体的叶片差异表达蛋白分析

以二倍体绿色草莓(Fragaria viridis Duch.)为父本、八倍体栽培草莓‘房香’(F.×ananassa‘Fusanoka’)为母本杂交所得的五倍体草莓(株系代号:FxLs-11-37,2n=5x=35)及其染色体加倍而成的十倍体(株系代号:A3,2n=10x=70)草莓为试材,观察记载其部分农艺性状、SPAD值以及净光合速率,利用双向凝胶电泳技术对草莓染色体加倍后叶片蛋白质进行了分析,获得了分辨率高、重复性好的电泳图谱。结果表明:(1)与五倍体FxLs-11-37相比,十倍体A3的株型明显矮化、植株冠径变大、叶长叶宽增大、叶片增厚以及叶色浓绿,其叶片的SPAD值与净光合速率显著高于FxLs-11-37。(2)通过PDQuest软件对图谱分析表明,两者在等电点4~7、分子量14.4~66.2kD范围内蛋白质斑点分布最多,可识别的总蛋白质斑点数超过700个,其中蛋白质表达差异水平在1.5倍以上的有18个,2.0倍以上的有4个。利用MALDI-TOF-MS/MS质谱技术鉴定了这4个差异蛋白质,分别是草莓主要过敏原a 1-E、核内不均一核糖核蛋白1、叶绿体硫辛酰基合酶1、NAD(P)H脱氢酶(醌)FQR1类似蛋白质。这些差异蛋白质主要与抗逆、mRNA转运、物质与能量代谢相关。荧光定量PCR对上述4个蛋白编码基因的转录表达水平检测表明,与蛋白质表达差异的趋势一致。该研究获得了草莓染色体加倍后差异表达的蛋白质,为深入研究提供了线索。     

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.     

Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

Background

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1.

Methods

The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1⁺NIL were also compared to identify pathogen-responsive proteins.

Results

Eight proteins were either induced or upregulated in inoculated Fhb1⁺NIL when compared with mock-inoculated Fhb1⁺NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1⁺NIL when compared with Fusarium-inoculated Fhb1⁻NIL. Proteins that were differentially expressed in the Fhb1⁺NIL, not in the Fhb1⁻NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification.

Conclusions

Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1⁺NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance.     

Identification of Differentially Expressed Proteins from Leishmania amazonensis Associated with the Loss of Virulence of the Parasites

Background

The present study analyzed whether or not the in vitro cultivation for long periods of time of pre-isolated Leishmania amazonensis from lesions of chronically infected BALB/c mice was able to interfere in the parasites'' infectivity using in vivo and in vitro experiments. In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach.

Methodology/Principal Findings

Parasites were cultured in vitro for 150 days. Aliquots were collected on the day 0 of culture (R0), as well as after ten (R10; 50 days of culture), twenty (R20; 100 days of culture), and thirty (R30; 150 days of culture) passages, and were used to analyze the parasites'' in vitro and in vivo infectivity, as well as to perform the proteomic approach. Approximately 837, 967, 935, and 872 spots were found in 2-DE gels prepared from R0, R10, R20, and R30 samples, respectively. A total of 37 spots presented a significant decrease in their intensity of expression, whereas a significant increase in protein content during cultivation could be observed for 19 proteins (both cases >2.0 folds). Some of these identified proteins can be described, such as diagnosis and/or vaccine candidates, while others are involved in the infectivity of Leishmania. It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified.

Conclusions/Significance

The present study contributes to the understanding that the cultivation of parasites over long periods of time may well be related to the possible loss of infectivity of L. amazonensis. The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites'' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis.     

筛选差异表达基因和蛋白质的方法进展

分离和鉴定差异表达基因和蛋白质不仅有助于发现基因和蛋白质的功能,更有助于揭示某些疾病的发生机理.目前筛选差异表达基因的方法主要有差异显示PCR方法(differential display RT-PCR,DDRT-PCR)、消减杂交法(subtractive hybridization,SH)、基因芯片技术(DNA chip technique)和基因表达的系统分析(serial analysis of gene expression,SAGE)等,其中消减杂交法中又先后建立了代表性差异分析技术(representational difference analysis,RDA)、抑制消减杂交法(suppression subtractive hybridization,SSH)和获得全长基因的消减杂交法(full-length-gene-obtainable subtractive hybridization).筛选差异表达蛋白质的方法主要有双向电泳技术(two-dimentional gel electrophoresis)和噬菌体全套抗体库技术(phage display antibody repertoire library technique).这些方法各有特点,各有利弊,研究者可根据自己的需要选择适合于自己的方法.     

喉癌差异表达cDNA序列的分离与初步鉴定

分离和克隆人喉癌中新的相关基因将有助于提示喉癌的易感性与癌变机制。运用ｍＲＮＡ差异显示法对２例成人喉癌组织及配对癌旁正常组织的基因表达进行研究,分离到３５个差异显示片段;用反向Ｎｏｒｔｈｅｒｎ点杂交筛选到６个差异片段,经克隆、测序和匹配分析,得１２条不同ｃＤＮＡ序列,其中４条为新基因序列,另外８条与已知基因高度同源。将１２条ｃＤＮＡ序列固定在膜上,用来自喉癌和配对正常组织的总ｃＤＮＡ探针与其杂交和