Sample records for aberrant protein expression

  1. Aberrant ATRX protein expression is associated with poor overall survival in NF1-MPNST

    PubMed Central

    Lu, Hsiang-Chih; Eulo, Vanessa; Apicelli, Anthony J.; Pekmezci, Melike; Tao, Yu; Luo, Jingqin; Hirbe, Angela C.; Dahiya, Sonika

    2018-01-01

    Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive soft tissue sarcomas that can occur sporadically or in the setting of the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome. These tumors carry a dismal overall survival. Previous work in our lab had identified ATRX chromatin remodeler (ATRX), previously termed, Alpha Thalassemia/Mental Retardation Syndrome X Linked as a gene mutated in a subset of MPNSTs. Given the great need for novel biomarkers and therapeutic targets for MPNSTs, we sought to determine the expression of ATRX in a larger subset of sporadic and NF1 associated MPNSTs (NF1-MPNSTs). We performed immunohistochemistry (IHC) on 74 MPNSTs (43 NF1-associated and 31 sporadic), 21 plexiform neurofibromas, and 9 atypical neurofibromas. Using this approach, we have demonstrated that 58% (43/74) of MPNSTs have aberrant ATRX expression (<80% nuclear expression) compared to only 7% (2/30) of benign (plexiform and atypical) neurofibromas. Second, we demonstrated that 65% (28/43) of NF1-MPNSTs displayed aberrant ATRX expression as did 48% (15/31) of sporadic MPNSTs. Finally, we show that aberrant ATRX expression was associated with a significantly decreased overall survival for patients with NF1-MPNST (median OS of 17.9 months for aberrant expression and median OS not met (>120 months) for intact expression, p = 0.0276). In summary, we demonstrate that ATRX is aberrantly expressed in the majority of NF1-MPNSTs, but not plexiform or atypical neurofibromas. Additionally, aberrant ATRX expression is associated with decreased overall survival in NF1-MPNST, but not sporadic MPNST and may serve as a prognostic marker for patients with NF1-MPNST. PMID:29796169

  2. Aberrant expression of genes and proteins in pterygium and their implications in the pathogenesis

    PubMed Central

    Feng, Qing-Yang; Hu, Zi-Xuan; Song, Xi-Ling; Pan, Hong-Wei

    2017-01-01

    Pterygium is a common ocular surface disease induced by a variety of factors. The exact pathogenesis of pterygium remains unclear. Numbers of genes and proteins are discovered in pterygium and they function differently in the occurrence and development of this disease. We searched the Web of Science and PubMed throughout history for literatures about the subject. The keywords we used contain pterygium, gene, protein, angiogenesis, fibrosis, proliferation, inflammation, pathogenesis and therapy. In this review, we summarize the aberrant expression of a range of genes and proteins in pterygium compared with normal conjunctiva or cornea, including growth factors, matrix metalloproteinases and tissue inhibitors of metalloproteinases, interleukins, tumor suppressor genes, proliferation related proteins, apoptosis related proteins, cell adhesion molecules, extracellular matrix proteins, heat shock proteins and tight junction proteins. We illustrate their possible mechanisms in the pathogenesis of pterygium as well as the related intervention based on them for pterygium therapy. PMID:28730091

  3. Aberration hubs in protein interaction networks highlight actionable targets in cancer.

    PubMed

    Karimzadeh, Mehran; Jandaghi, Pouria; Papadakis, Andreas I; Trainor, Sebastian; Rung, Johan; Gonzàlez-Porta, Mar; Scelo, Ghislaine; Vasudev, Naveen S; Brazma, Alvis; Huang, Sidong; Banks, Rosamonde E; Lathrop, Mark; Najafabadi, Hamed S; Riazalhosseini, Yasser

    2018-05-18

    Despite efforts for extensive molecular characterization of cancer patients, such as the international cancer genome consortium (ICGC) and the cancer genome atlas (TCGA), the heterogeneous nature of cancer and our limited knowledge of the contextual function of proteins have complicated the identification of targetable genes. Here, we present Aberration Hub Analysis for Cancer (AbHAC) as a novel integrative approach to pinpoint aberration hubs, i.e. individual proteins that interact extensively with genes that show aberrant mutation or expression. Our analysis of the breast cancer data of the TCGA and the renal cancer data from the ICGC shows that aberration hubs are involved in relevant cancer pathways, including factors promoting cell cycle and DNA replication in basal-like breast tumors, and Src kinase and VEGF signaling in renal carcinoma. Moreover, our analysis uncovers novel functionally relevant and actionable targets, among which we have experimentally validated abnormal splicing of spleen tyrosine kinase as a key factor for cell proliferation in renal cancer. Thus, AbHAC provides an effective strategy to uncover novel disease factors that are only identifiable by examining mutational and expression data in the context of biological networks.

  4. Aberrant expression of the PHF14 gene in biliary tract cancer cells

    PubMed Central

    AKAZAWA, TAKAKO; YASUI, KOHICHIROH; GEN, YASUYUKI; YAMADA, NOBUHISA; TOMIE, AKIRA; DOHI, OSAMU; MITSUYOSHI, HIRONORI; YAGI, NOBUAKI; ITOH, YOSHITO; NAITO, YUJI; YOSHIKAWA, TOSHIKAZU

    2013-01-01

    DNA copy number aberrations in human biliary tract cancer (BTC) cell lines were investigated using a high-density oligonucleotide microarray. A novel homozygous deletion was detected at chromosomal region 7p21.3 in the OZ cell line. Further validation experiments using genomic PCR revealed a homozygous deletion of a single gene, plant homeodomain (PHD) finger protein 14 (PHF14). No PHF14 mRNA or protein expression was detected, thus demonstrating the absence of PHF14 expression in the OZ cell line. Although the PHD finger protein is considered to be involved in chromatin-mediated transcriptional regulation, little is known about the function of PHF14 in cancer. The present study observed that the knock down of PHF14 using small interfering RNA (siRNA) enhanced the growth of the BTC cells. These observations suggest that aberrant PHF14 expression may have a role in the tumorigenesis of BTC. PMID:23833654

  5. Aberrant expression of long noncoding RNAs in autistic brain.

    PubMed

    Ziats, Mark N; Rennert, Owen M

    2013-03-01

    The autism spectrum disorders (ASD) have a significant hereditary component, but the implicated genetic loci are heterogeneous and complex. Consequently, there is a gap in understanding how diverse genomic aberrations all result in one clinical ASD phenotype. Gene expression studies from autism brain tissue have demonstrated that aberrantly expressed protein-coding genes may converge onto common molecular pathways, potentially reconciling the strong heritability and shared clinical phenotypes with the genomic heterogeneity of the disorder. However, the regulation of gene expression is extremely complex and governed by many mechanisms, including noncoding RNAs. Yet no study in ASD brain tissue has assessed for changes in regulatory long noncoding RNAs (lncRNAs), which represent a large proportion of the human transcriptome, and actively modulate mRNA expression. To assess if aberrant expression of lncRNAs may play a role in the molecular pathogenesis of ASD, we profiled over 33,000 annotated lncRNAs and 30,000 mRNA transcripts from postmortem brain tissue of autistic and control prefrontal cortex and cerebellum by microarray. We detected over 200 differentially expressed lncRNAs in ASD, which were enriched for genomic regions containing genes related to neurodevelopment and psychiatric disease. Additionally, comparison of differences in expression of mRNAs between prefrontal cortex and cerebellum within individual donors showed ASD brains had more transcriptional homogeneity. Moreover, this was also true of the lncRNA transcriptome. Our results suggest that further investigation of lncRNA expression in autistic brain may further elucidate the molecular pathogenesis of this disorder.

  6. The study of the relationship between aberrant expression of hot shock protein 70 (HSP70) and spontaneous abortion.

    PubMed

    Peng, Y-B; Liu, H; Huang, S-H; Lai, H; Zhou, Q; Luo, Y; Zhang, Z-Y; Xi, B-R; Ouyang, X

    2017-02-01

    The present study is aimed to explore the relationship between aberrant expression of heat shock protein 70 (HSP) and spontaneous abortion. 50 patients with spontaneous abortion and 50 patients with induced abortion were continuously selected based on the nearest matching principle, and the proportion of age and gestational age was 1:1. The decidual tissues were obtained, and the cell apoptosis was determined by TUNEL assay. Further, the expression of HSP70 was assayed by immune-histochemical staining, and the expression of HSP70 mRNA was detected by the RT-PCR approach. Apoptosis rate, HSP70 expression and HSP70 mRNA expression in the observation group were significantly higher than the control group. HSP70 might induce apoptosis so as to cause spontaneous abortion.

  7. Aberrant rhythmic expression of cryptochrome2 regulates the radiosensitivity of rat gliomas.

    PubMed

    Fan, Wang; Caiyan, Li; Ling, Zhu; Jiayun, Zhao

    2017-09-29

    In this study, we investigated the role of the clock regulatory protein cryptochrome 2 (Cry2) in determining the radiosensitivity of C6 glioma cells in a rat model. We observed that Cry2 mRNA and protein levels showed aberrant rhythmic periodicity of 8 h in glioma tissues, compared to 24 h in normal brain tissue. Cry2 mRNA and protein levels did not respond to irradiation in normal tissues, but both were increased at the ZT4 (low Cry2) and ZT8 (high Cry2) time points in gliomas. Immunohistochemical staining of PCNA and TUNEL assays demonstrated that high Cry2 expression in glioma tissues was associated with increased cell proliferation and decreased apoptosis. Western blot analysis showed that glioma cell fate was independent of p53, but was probably dependent on p73, which was more highly expressed at ZT4 (low Cry2) than at ZT8 (high Cry2). Levels of both p53 and p73 were unaffected by irradiation in normal brain tissues. These findings suggest aberrant rhythmic expression of Cry2 influence on radiosensitivity in rat gliomas.

  8. Aberrant Gene Expression in Humans

    PubMed Central

    Yang, Ence; Ji, Guoli; Brinkmeyer-Langford, Candice L.; Cai, James J.

    2015-01-01

    Gene expression as an intermediate molecular phenotype has been a focus of research interest. In particular, studies of expression quantitative trait loci (eQTL) have offered promise for understanding gene regulation through the discovery of genetic variants that explain variation in gene expression levels. Existing eQTL methods are designed for assessing the effects of common variants, but not rare variants. Here, we address the problem by establishing a novel analytical framework for evaluating the effects of rare or private variants on gene expression. Our method starts from the identification of outlier individuals that show markedly different gene expression from the majority of a population, and then reveals the contributions of private SNPs to the aberrant gene expression in these outliers. Using population-scale mRNA sequencing data, we identify outlier individuals using a multivariate approach. We find that outlier individuals are more readily detected with respect to gene sets that include genes involved in cellular regulation and signal transduction, and less likely to be detected with respect to the gene sets with genes involved in metabolic pathways and other fundamental molecular functions. Analysis of polymorphic data suggests that private SNPs of outlier individuals are enriched in the enhancer and promoter regions of corresponding aberrantly-expressed genes, suggesting a specific regulatory role of private SNPs, while the commonly-occurring regulatory genetic variants (i.e., eQTL SNPs) show little evidence of involvement. Additional data suggest that non-genetic factors may also underlie aberrant gene expression. Taken together, our findings advance a novel viewpoint relevant to situations wherein common eQTLs fail to predict gene expression when heritable, rare inter-individual variation exists. The analytical framework we describe, taking into consideration the reality of differential phenotypic robustness, may be valuable for investigating

  9. Aberrant p53 protein expression is associated with an increased risk of neoplastic progression in patients with Barrett's oesophagus.

    PubMed

    Kastelein, Florine; Biermann, Katharina; Steyerberg, Ewout W; Verheij, Joanne; Kalisvaart, Marit; Looijenga, Leendert H J; Stoop, Hans A; Walter, Laurens; Kuipers, Ernst J; Spaander, Manon C W; Bruno, Marco J

    2013-12-01

    The value of surveillance for patients with Barrett's oesophagus (BO) is under discussion given the overall low incidence of neoplastic progression and lack of discriminative tests for risk stratification. Histological diagnosis of low-grade dysplasia (LGD) is the only accepted predictor for progression to date, but has a low predictive value. The aim of this study was therefore to evaluate the value of p53 immunohistochemistry for predicting neoplastic progression in patients with BO. We conducted a case-control study within a prospective cohort of 720 patients with BO. Patients who developed high-grade dysplasia (HGD) or oesophageal adenocarcinoma (OAC) were classified as cases and patients without neoplastic progression were classified as controls. P53 protein expression was determined by immunohistochemistry in more than 12 000 biopsies from 635 patients and was scored independently by two expert pathologists who were blinded to long-term outcome. During follow-up, 49 (8%) patients developed HGD or OAC. P53 overexpression was associated with an increased risk of neoplastic progression in patients with BO after adjusting for age, gender, Barrett length and oesophagitis (adjusted relative risks (RR(a)) 5.6; 95% CI 3.1 to 10.3), but the risk was even higher with loss of p53 expression (RR(a) 14.0; 95% CI 5.3 to 37.2). The positive predictive value for neoplastic progression increased from 15% with histological diagnosis of LGD to 33% with LGD and concurrent aberrant p53 expression. Aberrant p53 protein expression is associated with an increased risk of neoplastic progression in patients with BO and appears to be a more powerful predictor of neoplastic progression than histological diagnosis of LGD.

  10. Aberrant protein expression and frequent allelic loss of MSH3 in colorectal cancer with low-level microsatellite instability.

    PubMed

    Plaschke, Jens; Preußler, Mark; Ziegler, Andreas; Schackert, Hans K

    2012-07-01

    High level of microsatellite instability (MSI-H) in colorectal cancer (CRC) is caused by the inactivation of mismatch repair (MMR) genes; however, it is unknown for tumors with low level MSI (MSI-L). The protein complex involving MSH3 preferentially recognizes insertion/deletion loops (IDLs) of two to eight bases and di- and tetranucleotide repeats are affected in the majority of MSI-L CRC. We selected 10 and eight MSI-L CRCs from 228 and 204 patients with sporadic and hereditary disease, respectively. The tumors were analyzed for protein expression of MSH3, MSH2, MSH6, MLH1, and PMS2, and for mutations and loss of heterozygosity (LOH) in MSH3. Four tumors showed a markedly reduced MSH3 expression, whereas all 18 tumors had normal expression of the remaining MMR proteins. Twenty-five different sequence variants were identified. None of these results in a truncated protein, though L902W represents the first constitutional missense mutation in MSH3 predicted to be functional based on conservation among mutS homologues. All variants have also been found in normal DNA of the patients and in controls. LOH intragenic to MSH3 was evident for 12 of 16 (75%) informative tumors. Occurrence of sequence variants in normal DNA of the patients and in controls excludes somatic mutations and mutations specific to the CRC patient population, respectively. In contrast, the high frequency of LOH as well as the aberrant protein expression in some tumors indicates an involvement of MSH3 impairment in MSI-L CRC.

  11. Aberrant expression of NKL homeobox gene HLX in Hodgkin lymphoma.

    PubMed

    Nagel, Stefan; Pommerenke, Claudia; Meyer, Corinna; Kaufmann, Maren; MacLeod, Roderick A F; Drexler, Hans G

    2018-03-06

    NKL homeobox genes are basic regulators of cell and tissue differentiation, many acting as oncogenes in T-cell leukemia. Recently, we described an hematopoietic NKL-code comprising six particular NKL homeobox genes expressed in hematopoietic stem cells and lymphoid progenitors, unmasking their physiological roles in the development of these cell types. Hodgkin lymphoma (HL) is a B-cell malignancy showing aberrant activity of several developmental genes resulting in disturbed B-cell differentiation. To examine potential concordances in abnormal lymphoid differentiation of T- and B-cell malignancies we analyzed the expression of the hematopoietic NKL-code associated genes in HL, comprising HHEX, HLX, MSX1, NKX2-3, NKX3-1 and NKX6-3. Our approach revealed aberrant HLX activity in 8 % of classical HL patients and additionally in HL cell line L-540. Accordingly, to identify upstream regulators and downstream target genes of HLX we used L-540 cells as a model and performed chromosome and genome analyses, comparative expression profiling and functional assays via knockdown and overexpression experiments therein. These investigations excluded chromosomal rearrangements of the HLX locus at 1q41 and demonstrated that STAT3 operated directly as transcriptional activator of the HLX gene. Moreover, subcellular analyses showed highly enriched STAT3 protein in the nucleus of L-540 cells which underwent cytoplasmic translocation by repressing deacetylation. Finally, HLX inhibited transcription of B-cell differentiation factors MSX1, BCL11A and SPIB and of pro-apoptotic factor BCL2L11/BIM, thereby suppressing Etoposide-induced cell death. Collectively, we propose that aberrantly expressed NKL homeobox gene HLX is part of a pathological gene network in HL, driving deregulated B-cell differentiation and survival.

  12. KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast cancer.

    PubMed

    Alshareeda, A T; Negm, O H; Green, A R; Nolan, C C; Tighe, P; Albarakati, N; Sultana, R; Madhusudan, S; Ellis, I O; Rakha, E A

    2015-06-09

    It is recognised that modulations of the nuclear import of macromolecules have a role in changing cellular phenotypes and carcinogenesis. We and others have noticed that aberrant subcellular localisation of DNA damage response (DDR) proteins in breast cancer (BC) is associated with loss-of-function phenotype. This study aims to investigate the biological and clinical significance of the nucleocytoplasmic transport protein karyopherin α-2 (KPNA2), and its role in controlling DDR proteins subcellular localisation in BC. A large (n=1494) and well-characterised series of early-stage invasive BC with a long-term follow-up was assessed for KPNA2 protein by using immunohistochemistry. KPNA2 expression was associated with the subcellular localisation of key DDR proteins that showed cytoplasmic expression including BRCA1, RAD51, SMC6L1, γH2AX, BARD1, UBC9, PIAS1 and CHK1. High level of KPNA2 was associated not only with cytoplasmic localisation of these proteins but also with their low/negative nuclear expression. Positive KPNA2 expression was associated with negative oestrogen receptor and triple-negative phenotype. Survival analysis showed that KPNA2 was associated with poor outcome (P<0.0001), but this effect was not independent of other prognostic variables. This study provides further evidence for the complexity of DDR mechanism in BC, and that KNPA2 has a role in the aberrant subcellular localisation of DDR proteins with subsequent impaired function.

  13. Human cytomegalovirus UL76 induces chromosome aberrations

    PubMed Central

    2009-01-01

    Background Human cytomegalovirus (HCMV) is known to induce chromosome aberrations in infected cells, which can lead to congenital abnormalities in infected fetuses. HCMV UL76 belongs to a conserved protein family from herpesviruses. Some reported roles among UL76 family members include involvement in virulence determination, lytic replication, reactivation of latent virus, modulation of gene expression, induction of apoptosis, and perturbation of cell cycle progression, as well as potential nuclease activity. Previously, we have shown that stable expression of UL76 inhibits HCMV replication in glioblastoma cells. Methods To examine chromosomal integrity and the DNA damage signal γ-H2AX in cells constitutively expressing UL76, immunofluorescent cell staining and Western blotting were performed. The comet assay was employed to assess DNA breaks in cells transiently expressing UL76. Results We report that stably transfected cells expressing UL76 developed chromosome aberrations including micronuclei and misaligned chromosomes, lagging and bridging. In mitotic cells expressing UL76, aberrant spindles were increased compared to control cells. However, cells with supernumerary centrosomes were marginally increased in UL76-expressing cells relative to control cells. We further demonstrated that UL76-expressing cells activated the DNA damage signal γ-H2AX and caused foci formation in nuclei. In addition, the number of cells with DNA breaks increased in proportion to UL76 protein levels. Conclusion Our findings suggest that the virus-associated protein UL76 induces DNA damage and the accumulation of chromosome aberrations. PMID:19930723

  14. Identification of aberrant gene expression associated with aberrant promoter methylation in primordial germ cells between E13 and E16 rat F3 generation vinclozolin lineage.

    PubMed

    Taguchi, Y-h

    2015-01-01

    Transgenerational epigenetics (TGE) are currently considered important in disease, but the mechanisms involved are not yet fully understood. TGE abnormalities expected to cause disease are likely to be initiated during development and to be mediated by aberrant gene expression associated with aberrant promoter methylation that is heritable between generations. However, because methylation is removed and then re-established during development, it is not easy to identify promoter methylation abnormalities by comparing normal lineages with those expected to exhibit TGE abnormalities. This study applied the recently proposed principal component analysis (PCA)-based unsupervised feature extraction to previously reported and publically available gene expression/promoter methylation profiles of rat primordial germ cells, between E13 and E16 of the F3 generation vinclozolin lineage that are expected to exhibit TGE abnormalities, to identify multiple genes that exhibited aberrant gene expression/promoter methylation during development. The biological feasibility of the identified genes were tested via enrichment analyses of various biological concepts including pathway analysis, gene ontology terms and protein-protein interactions. All validations suggested superiority of the proposed method over three conventional and popular supervised methods that employed t test, limma and significance analysis of microarrays, respectively. The identified genes were globally related to tumors, the prostate, kidney, testis and the immune system and were previously reported to be related to various diseases caused by TGE. Among the genes reported by PCA-based unsupervised feature extraction, we propose that chemokine signaling pathways and leucine rich repeat proteins are key factors that initiate transgenerational epigenetic-mediated diseases, because multiple genes included in these two categories were identified in this study.

  15. Identification of aberrant gene expression associated with aberrant promoter methylation in primordial germ cells between E13 and E16 rat F3 generation vinclozolin lineage

    PubMed Central

    2015-01-01

    Background Transgenerational epigenetics (TGE) are currently considered important in disease, but the mechanisms involved are not yet fully understood. TGE abnormalities expected to cause disease are likely to be initiated during development and to be mediated by aberrant gene expression associated with aberrant promoter methylation that is heritable between generations. However, because methylation is removed and then re-established during development, it is not easy to identify promoter methylation abnormalities by comparing normal lineages with those expected to exhibit TGE abnormalities. Methods This study applied the recently proposed principal component analysis (PCA)-based unsupervised feature extraction to previously reported and publically available gene expression/promoter methylation profiles of rat primordial germ cells, between E13 and E16 of the F3 generation vinclozolin lineage that are expected to exhibit TGE abnormalities, to identify multiple genes that exhibited aberrant gene expression/promoter methylation during development. Results The biological feasibility of the identified genes were tested via enrichment analyses of various biological concepts including pathway analysis, gene ontology terms and protein-protein interactions. All validations suggested superiority of the proposed method over three conventional and popular supervised methods that employed t test, limma and significance analysis of microarrays, respectively. The identified genes were globally related to tumors, the prostate, kidney, testis and the immune system and were previously reported to be related to various diseases caused by TGE. Conclusions Among the genes reported by PCA-based unsupervised feature extraction, we propose that chemokine signaling pathways and leucine rich repeat proteins are key factors that initiate transgenerational epigenetic-mediated diseases, because multiple genes included in these two categories were identified in this study. PMID:26677731

  16. Aberrant membranous expression of β-catenin predicts poor prognosis in patients with craniopharyngioma.

    PubMed

    Li, Zongping; Xu, Jianguo; Huang, Siqing; You, Chao

    2015-12-01

    The objective of this study is to investigate β-catenin expression in craniopharyngioma patients and determine its significance in predicting the prognosis of this disease. Fifty craniopharyngioma patients were enrolled in this study. Expression of β-catenin in tumor specimens collected from these patients was examined through immunostaining. In addition, mutation of exon 3 in the β-catenin gene, CTNNB1, was analyzed using polymerase chain reaction, denaturing high-pressure liquid chromatography, and DNA sequencing. Based on these results, we explored the association between membranous β-catenin expression, clinical and pathologic characteristics, and prognoses in these patients. Of all craniopharyngioma specimens, 31 (62.0%) had preserved membranous β-catenin expression, whereas the remaining 19 specimens (38.0%) displayed aberrant expression. Statistical analysis showed a significant correlation between aberrant membranous β-catenin expression and CTNNB1 exon 3 mutation, as well as between aberrant membranous β-catenin expression and the histopathologic type of craniopharyngioma and type of resection in our patient population. Furthermore, aberrant membranous β-catenin expression was found to be associated with poor patient survival. Results of Kaplan-Meier survival analysis and Cox regression analysis further confirmed this finding. In conclusion, our study demonstrated that aberrant membranous β-catenin expression was significantly correlated with poor survival in patients with craniopharyngioma. This raises the possibility for use of aberrant membranous β-catenin expression as an independent risk factor in predicting the prognosis of this disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis.

    PubMed

    Liu, Na; Enkemann, Steven A; Liang, Ping; Hersmus, Remko; Zanazzi, Claudia; Huang, Junjiu; Wu, Chao; Chen, Zhisheng; Looijenga, Leendert H J; Keefe, David L; Liu, Lin

    2010-12-01

    Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos.

  18. From DNA Copy Number to Gene Expression: Local aberrations, Trisomies and Monosomies

    NASA Astrophysics Data System (ADS)

    Shay, Tal

    The goal of my PhD research was to study the effect of DNA copy number changes on gene expression. DNA copy number aberrations may be local, encompassing several genes, or on the level of an entire chromosome, such as trisomy and monosomy. The main dataset I studied was of Glioblastoma, obtained in the framework of a collaboration, but I worked also with public datasets of cancer and Down's Syndrome. The molecular basis of expression changes in Glioblastoma. Glioblastoma is the most common and aggressive type of primary brain tumors in adults. In collaboration with Prof. Hegi (CHUV, Switzerland), we analyzed a rich Glioblastoma dataset including clinical information, DNA copy number (array CGH) and expression profiles. We explored the correlation between DNA copy number and gene expression at the level of chromosomal arms and local genomic aberrations. We detected known amplification and over expression of oncogenes, as well as deletion and down-regulation of tumor suppressor genes. We exploited that information to map alterations of pathways that are known to be disrupted in Glioblastoma, and tried to characterize samples that have no known alteration in any of the studied pathways. Identifying local DNA aberrations of biological significance. Many types of tumors exhibit chromosomal losses or gains and local amplifications and deletions. A region that is aberrant in many tumors, or whose copy number change is stronger, is more likely to be clinically relevant, and not just a by-product of genetic instability. We developed a novel method that defines and prioritizes aberrations by formalizing these intuitions. The method scores each aberration by the fraction of patients harboring it, its length and its amplitude, and assesses the significance of the score by comparing it to a null distribution obtained by permutations. This approach detects genetic locations that are significantly aberrant, generating a 'genomic aberration profile' for each sample. The 'genomic

  19. Aberrant Expression of COT Is Related to Recurrence of Papillary Thyroid Cancer

    PubMed Central

    Lee, Jandee; Jeong, Seonhyang; Park, Jae Hyun; Lee, Cho Rok; Ku, Cheol Ryong; Kang, Sang-Wook; Jeong, Jong Ju; Nam, Kee-Hyun; Shin, Dong Yeob; Lee, Eun Jig; Chung, Woong Youn; Jo, Young Suk

    2015-01-01

    Abstract Aberrant expression of Cancer Osaka Thyroid Oncogene mitogen-activated protein kinase kinase kinase 8 (COT) (MAP3K8) is a driver of resistance to B-RAF inhibition. However, the de novo expression and clinical implications of COT in papillary thyroid cancer (PTC) have not been investigated. The aim of this study is to investigate the expression of A-, B-, C-RAF, and COT in PTC (n = 167) and analyze the clinical implications of aberrant expression of these genes. Quantitative polymerase chain reaction (qPCR) and immunohistochemical staining (IHC) were performed on primary thyroid cancers. Expression of COT was compared with clinicopathological characteristics including recurrence-free survival. Datasets from public repository (NCBI) were subjected to Gene Set Enrichment Analysis (GSEA). qPCR data showed that the relative mRNA expression of A-, B-, C-RAF and COT of PTC were higher than normal tissues (all P < 0.01). In addition, the expression of COT mRNA in PTC showed positive correlation with A- (r = 0.4083, P < 0.001), B- (r = 0.2773, P = 0.0003), and C-RAF (r = 0.5954, P < 0.001). The mRNA expressions of A-, B,- and C-RAF were also correlated with each other (all P < 0.001). In IHC, the staining intensities of B-RAF and COT were higher in PTC than in normal tissue (P < 0.001). Interestingly, moderate-to-strong staining intensities of B-RAF and COT were more frequent in B-RAFV600E-positive PTC (P < 0.001, P = 0.013, respectively). In addition, aberrant expression of COT was related to old age at initial diagnosis (P = 0.045) and higher recurrence rate (P = 0.025). In multivariate analysis, tumor recurrence was persistently associated with moderate-to-strong staining of COT after adjusting for age, sex, extrathyroidal extension, multifocality, T-stage, N-stage, TNM stage, and B-RAFV600E mutation (odds ratio, 4.662; 95% confidence interval 1.066 − 21.609; P = 0.045). Moreover, moderate

  20. Aberrant expression of COT is related to recurrence of papillary thyroid cancer.

    PubMed

    Lee, Jandee; Jeong, Seonhyang; Park, Jae Hyun; Lee, Cho Rok; Ku, Cheol Ryong; Kang, Sang-Wook; Jeong, Jong Ju; Nam, Kee-Hyun; Shin, Dong Yeob; Lee, Eun Jig; Chung, Woong Youn; Jo, Young Suk

    2015-02-01

    Aberrant expression of Cancer Osaka Thyroid Oncogene mitogen-activated protein kinase kinase kinase 8 (COT) (MAP3K8) is a driver of resistance to B-RAF inhibition. However, the de novo expression and clinical implications of COT in papillary thyroid cancer (PTC) have not been investigated.The aim of this study is to investigate the expression of A-, B-, C-RAF, and COT in PTC (n = 167) and analyze the clinical implications of aberrant expression of these genes.Quantitative polymerase chain reaction (qPCR) and immunohistochemical staining (IHC) were performed on primary thyroid cancers. Expression of COT was compared with clinicopathological characteristics including recurrence-free survival. Datasets from public repository (NCBI) were subjected to Gene Set Enrichment Analysis (GSEA).qPCR data showed that the relative mRNA expression of A-, B-, C-RAF and COT of PTC were higher than normal tissues (all P < 0.01). In addition, the expression of COT mRNA in PTC showed positive correlation with A- (r = 0.4083, P < 0.001), B- (r = 0.2773, P = 0.0003), and C-RAF (r = 0.5954, P < 0.001). The mRNA expressions of A-, B,- and C-RAF were also correlated with each other (all P < 0.001). In IHC, the staining intensities of B-RAF and COT were higher in PTC than in normal tissue (P < 0.001). Interestingly, moderate-to-strong staining intensities of B-RAF and COT were more frequent in B-RAF-positive PTC (P < 0.001, P = 0.013, respectively). In addition, aberrant expression of COT was related to old age at initial diagnosis (P = 0.045) and higher recurrence rate (P = 0.025). In multivariate analysis, tumor recurrence was persistently associated with moderate-to-strong staining of COT after adjusting for age, sex, extrathyroidal extension, multifocality, T-stage, N-stage, TNM stage, and B-RAF mutation (odds ratio, 4.662; 95% confidence interval 1.066 - 21.609; P = 0.045). Moreover, moderate-to-strong COT expression in PTC

  1. Aberrant lymphoid antigen expression in acute myeloid leukemia in Saudi Arabia.

    PubMed

    El-Sissy, Azza H; El-Mashari, May A; Bassuni, Wafaa Y; El-Swaayed, Aziza F

    2006-09-01

    Immunophenotyping improves both accuracy and reproducibility of acute leukemia classification and is considered particularly useful for identifying aberrant lineage association of acute leukemia, biphenotypic and bilineal acute leukemia, as well as monitoring minimal residual disease. Some immunophenotypes correlate with cytogenetic abnormalities and prognosis. Is to determine aberrant lymphoid antigen expression in Saudi acute myeloid leukemia (AML), correlate them with FAB subtypes, evaluate early surface markers CD7 and CD56, and to investigate the role of cytoplasmic CD79a (a B cell marker that is assigned a high score of 2.0 in the WHO classification). Thirty four newly diagnosed AML cases were included in this study, 47% showed aberrant lymphoid antigen expression. CD9 was the most frequently expressed lymphoid antigen (29.4%) followed by CD7 & CD19 (11.8%), CD4 (8.8%) and CD22 (2.9%). CD9 was expressed in 3/6 (50%) of M3 cases, CD7 was expressed in 11.8% and was mostly confined to FAB M1 and M2 and associated with immature antigens CD34, HLA-DR and TdT. CD56 was expressed in 7/34 (20.6%) cases, three of these cases (42.9%) belonged to the monocytic group. CD56 was also detected in 2 cases with 11q23 rearrangement. CD56 was expressed in 2/7 (28.6%) M2 cases, and was associated with t (8;21) (q22;q22) together with CD19. Co-expression of CD56 and CD7 was detected in 2.9% of the cases. CD79a was expressed in one case together with CD19, diagnosed as acute biphenotypic leukemia, and was associated with t(8;21) (q22;q22). Minimal residual disease in AML is very difficult to trace, detection of aberrant expression of lymphoid antigens will make it easier. The high score given to CD79a by EGIL is questionable based on cytogenetic classification.

  2. Aberrant Expression of Retinoic Acid Signaling Molecules Influences Patient Survival in Astrocytic Gliomas

    PubMed Central

    Campos, Benito; Centner, Franz-Simon; Bermejo, Justo Lorenzo; Ali, Ramadan; Dorsch, Katharina; Wan, Feng; Felsberg, Jörg; Ahmadi, Rezvan; Grabe, Niels; Reifenberger, Guido; Unterberg, Andreas; Burhenne, Jürgen; Herold-Mende, Christel

    2011-01-01

    Undifferentiated cell populations may influence tumor growth in malignant glioma. We investigated potential disruptions in the retinoic acid (RA) differentiation pathway that could lead to a loss of differentiation capacity, influencing patient prognosis. Expression of key molecules belonging to the RA differentiation pathway was analyzed in 283 astrocytic gliomas and was correlated with tumor proliferation, tumor differentiation, and patient survival. In addition, in situ concentrations of retinoids were measured in tumors, and RA signaling events were studied in vitro. Unlike other tumors, in gliomas expression of most RA signaling molecules increased with malignancy and was associated with augmented intratumoral retinoid levels in high-grade gliomas. Aberrantly expressed RA signaling molecules included i) the retinol-binding protein CRBP1, which facilitates cellular retinoid uptake; ii) ALDH1A1, capable of activating RA precursors; iii) the RA-degrading enzyme CYP26B1; and iv) the RA-binding protein FABP5, which can inhibit RA-induced differentiation. In contrast, expression of the RA-binding protein CRABP2, which fosters differentiation, was decreased in high-grade tumors. Moreover, expression of CRBP1 correlated with tumor proliferation, and FABP5 expression correlated with an undifferentiated tumor phenotype. CRBP1 and ALDH1A1 were independent prognostic markers for adverse patient survival. Our data indicate a complex and clinically relevant deregulation of RA signaling, which seems to be a central event in glioma pathogenesis. PMID:21514413

  3. Aberrant calreticulin expression is involved in the dedifferentiation of dedifferentiated liposarcoma.

    PubMed

    Hisaoka, Masanori; Matsuyama, Atsuji; Nakamoto, Mitsuhiro

    2012-05-01

    Liposarcomas are a representative group of soft tissue sarcomas with variably hampered adipogenesis, which is most exemplified by its dedifferentiated subtype. However, the factor(s) responsible for inhibiting adipocyte differentiation remains unknown. A recent gene expression profiling study identified several unique genes that were highly expressed in dedifferentiated liposarcoma, and the gene encoding calreticulin (CALR), a major Ca(2+)-buffering protein that can inhibit adipocyte differentiation, was found to be overexpressed. Thus, we investigated the expression of calreticulin in 45 cases of liposarcomas, including 15 dedifferentiated tumors, at both the protein and mRNA levels. Immunohistochemically, calreticulin was consistently expressed in the dedifferentiated areas of dedifferentiated liposarcomas and commonly observed in atypical stromal cells and/or lipoblasts in the well-differentiated areas (87%), whereas large vacuolated adipocytic cells in either the tumors or normal fat were essentially negative. These results were further supported by the findings of Western blot and quantitative RT-PCR analyses. Although abnormalities in 19p13.1-13.2 where CALR is localized were uncommon in the dedifferentiated liposarcomas examined by fluorescence in situ hybridization, expression of miR-1257, a putative microRNA that targets calreticulin, was suppressed in the dedifferentiated subtype. The down-regulation of calreticulin by small-interfering RNA could induce adipogenesis in dedifferentiated liposarcoma cells and reduce cell proliferation. Our results therefore suggest that aberrantly expressed calreticulin in dedifferentiated liposarcoma is involved in its dedifferenitation and/or tumor progression. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  4. Aberrant expressions of c-KIT and DOG-1 in mucinous and nonmucinous colorectal carcinomas and relation to clinicopathologic features and prognosis.

    PubMed

    Foda, Abd Al-Rahman Mohammad; Mohamed, Mie Ali

    2015-10-01

    c-KIT and DOG-1 are 2 highly expressed proteins in gastrointestinal stromal tumors. Few studies had investigated c-KIT, but not DOG-1, expression in colorectal carcinoma (CRC). This study aims to investigate expressions of c-KIT and DOG-1 in colorectal mucinous carcinoma and nonmucinous carcinoma using manual tissue microarray technique. In this work, we studied tumor tissue specimens from 150 patients with colorectal mucinous (MA) and nonmucinous adenocarcinoma (NMA). High-density manual tissue microarrays were constructed using modified mechanical pencil tip technique, and immunohistochemistry for c-KIT and DOG-1 was done. We found that aberrant c-KIT expression was detected in 12 cases (8%); 6 cases (4%) showed strong expression. Aberrant DOG-1 expression was detected in 15 cases (10%); among them, only 4 cases (2.7%) showed strong expression. Nonmucinous adenocarcinoma showed a significantly high expression of c-KIT, but not DOG-1, than MA. Aberrant c-KIT and DOG-1 expressions were significantly unrelated but were associated with excessive microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. In conclusion, aberrant c-KIT and DOG-1 expressions in CRC are rare events, either in NMA or MA. Nonmucinous adenocarcinoma showed a significantly higher expression of c-KIT, but not DOG-1, than MA. The expressions of both in CRC are significantly unrelated but are associated with microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. So, c-KIT and DOG-1 immunostaining is not a cost-effective method of identifying patients with CRC who may benefit from treatment with tyrosine kinase inhibitors. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Identification of Proteins Related to Epigenetic Regulation in the Malignant Transformation of Aberrant Karyotypic Human Embryonic Stem Cells by Quantitative Proteomics

    PubMed Central

    Sun, Yi; Yang, Yixuan; Zeng, Sicong; Tan, Yueqiu; Lu, Guangxiu; Lin, Ge

    2014-01-01

    Previous reports have demonstrated that human embryonic stem cells (hESCs) tend to develop genomic alterations and progress to a malignant state during long-term in vitro culture. This raises concerns of the clinical safety in using cultured hESCs. However, transformed hESCs might serve as an excellent model to determine the process of embryonic stem cell transition. In this study, ITRAQ-based tandem mass spectrometry was used to quantify normal and aberrant karyotypic hESCs proteins from simple to more complex karyotypic abnormalities. We identified and quantified 2583 proteins, and found that the expression levels of 316 proteins that represented at least 23 functional molecular groups were significantly different in both normal and abnormal hESCs. Dysregulated protein expression in epigenetic regulation was further verified in six pairs of hESC lines in early and late passage. In summary, this study is the first large-scale quantitative proteomic analysis of the malignant transformation of aberrant karyotypic hESCs. The data generated should serve as a useful reference of stem cell-derived tumor progression. Increased expression of both HDAC2 and CTNNB1 are detected as early as the pre-neoplastic stage, and might serve as prognostic markers in the malignant transformation of hESCs. PMID:24465727

  6. Expression of AID, P53, and Mlh1 proteins in endoscopically resected differentiated-type early gastric cancer

    PubMed Central

    Takeda, Yohei; Yashima, Kazuo; Hayashi, Akihiro; Sasaki, Shuji; Kawaguchi, Koichiro; Harada, Kenichi; Murawaki, Yoshikazu; Ito, Hisao

    2012-01-01

    AIM: To analyze the expression of the tumor-related proteins in differentiated-type early gastric carcinoma (DEGC) samples. METHODS: Tumor specimens were obtained from 102 patients (75 males and 27 females) who had received an endoscopic tumor resection at Tottori University Hospital between 2007 and 2009. Ninety-one cancer samples corresponded to noninvasive or intramucosal carcinoma according to the Vienna classification system, and 11 samples were submucosal invasive carcinomas. All of the EGCs were histologically differentiated carcinomas. All patients were classified as having Helicobacter pylori (H. pylori) infections by endoscopic atrophic changes or by testing seropositive for H. pylori IgG. All of the samples were histopathologically classified as either tubular or papillary adenocarcinoma according to their structure. The immunohistochemical staining was performed in a blinded manner with respect to the clinical information. Two independent observers evaluated protein expression. All data were statistically analyzed then. RESULTS: The rates of aberrant activation-induced cytidine deaminase (AID) expression and P53 overexpression were both 34.3% in DEGCs. The expression of Mlh1 was lost in 18.6% of DEGCs. Aberrant AID expression was not significantly associated with P53 overexpression in DEGCs. However, AID expression was associated with the severity of mononuclear cell activity in the non-cancerous mucosa adjacent to the tumor (P = 0.064). The rate of P53 expression was significantly greater in flat or depressed tumors than in elevated tumors. The frequency of Mlh1 loss was significantly increased in distal tumors, elevated gross-type tumors, papillary histological-type tumors, and tumors with a severe degree of endoscopic atrophic gastritis (P < 0.05). CONCLUSION: Aberrant AID expression, P53 overexpression, and the loss of Mlh1 were all associated with clinicopathological features and gastric mucosal alterations in DEGCs. The aberrant expression of AID

  7. Mice with Inactivation of Aryl Hydrocarbon Receptor-Interacting Protein (Aip) Display Complete Penetrance of Pituitary Adenomas with Aberrant ARNT Expression

    PubMed Central

    Raitila, Anniina; Lehtonen, Heli J.; Arola, Johanna; Heliövaara, Elina; Ahlsten, Manuel; Georgitsi, Marianthi; Jalanko, Anu; Paetau, Anders; Aaltonen, Lauri A.; Karhu, Auli

    2010-01-01

    Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been shown to predispose to pituitary adenoma predisposition, a condition characterized by growth hormone (GH)-secreting pituitary tumors. To study AIP-mediated tumorigenesis, we generated an Aip mouse model. Heterozygous mice developed normally but were prone to pituitary adenomas, in particular to those secreting GH. A complete loss of AIP was detected in these lesions, and full penetrance was reached at the age of 15 months. No excess of any other tumor type was found. Ki-67 analysis indicated that Aip-deficient tumors have higher proliferation rates compared with Aip-proficient tumors, suggesting a more aggressive disease. Similar to human AIP-deficient pituitary adenomas, immunohistochemical studies showed that expression of aryl hydrocarbon receptor nuclear translocator 1 or 2 (ARNT or ARNT2) protein was lost in the mouse tumors, suggesting that mechanisms of AIP-related tumorigenesis involve aberrant ARNT function. The Aip+/− mouse appears to be an excellent model for the respective human disease phenotype. This model constitutes a tool to further study AIP-associated pituitary tumorigenesis and may be potentially valuable in efforts to develop therapeutic strategies to treat pituitary adenomas. PMID:20709796

  8. Aberrant alternative splicing is another hallmark of cancer.

    PubMed

    Ladomery, Michael

    2013-01-01

    The vast majority of human genes are alternatively spliced. Not surprisingly, aberrant alternative splicing is increasingly linked to cancer. Splice isoforms often encode proteins that have distinct and even antagonistic properties. The abnormal expression of splice factors and splice factor kinases in cancer changes the alternative splicing of critically important pre-mRNAs. Aberrant alternative splicing should be added to the growing list of cancer hallmarks.

  9. How and why do toxic conformers of aberrant proteins accumulate during ageing?

    PubMed

    Josefson, Rebecca; Andersson, Rebecca; Nyström, Thomas

    2017-07-15

    Ageing can be defined as a gradual decline in cellular and physical functions accompanied by an increased sensitivity to the environment and risk of death. The increased risk of mortality is causally connected to a gradual, intracellular accumulation of so-called ageing factors, of which damaged and aggregated proteins are believed to be one. Such aggregated proteins also contribute to several age-related neurodegenerative disorders e.g. Alzheimer's, Parkinson's, and Huntington's diseases, highlighting the importance of protein quality control (PQC) in ageing and its associated diseases. PQC consists of two interrelated systems: the temporal control system aimed at refolding, repairing, and/or removing aberrant proteins and their aggregates and the spatial control system aimed at harnessing the potential toxicity of aberrant proteins by sequestering them at specific cellular locations. The accumulation of toxic conformers of aberrant proteins during ageing is often declared to be a consequence of an incapacitated temporal PQC system-i.e. a gradual decline in the activity of chaperones and proteases. Here, we review the current knowledge on PQC in relation to ageing and highlight that the breakdown of both temporal and spatial PQC may contribute to ageing and thus comprise potential targets for therapeutic interventions of the ageing process. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  10. Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

    PubMed

    Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

  11. Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype

    PubMed Central

    Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis. PMID:27031510

  12. Dysregulation of miRNAs in bladder cancer: altered expression with aberrant biogenesis procedure

    PubMed Central

    Dong, Fan; Xu, Tianyuan; Shen, Yifan; Zhong, Shan; Chen, Shanwen; Ding, Qiang; Shen, Zhoujun

    2017-01-01

    Aberrant expression profiles of miRNAs are widely observed in the clinical tissue specimens and urine samples as well as the blood samples of bladder cancer patients. These profiles are closely related to the pathological features of bladder cancer, such as the tumour stage/grade, metastasis, recurrence and chemo-sensitivity. MiRNA biogenesis forms the basis of miRNA expression and function, and its dysregulation has been shown to be essential for variations in miRNA expression profiles as well as tumourigenesis and cancer progression. In this review, we summarize the up-to-date and widely reported miRNAs in bladder cancer that display significantly altered expression. We then compare the miRNA expression profiles among three different sample types (tissue, urine and blood) from patients with bladder cancer. Moreover, for the first time, we outline the dysregulated miRNA biogenesis network in bladder cancer from different levels and analyse its possible relationship with aberrant miRNA expression and the pathological characteristics of the disease. PMID:28187437

  13. Lack of Correlation between Aberrant p16, RAR-β2, TIMP3, ERCC1, and BRCA1 Protein Expression and Promoter Methylation in Squamous Cell Carcinoma Accompanying Candida albicans-Induced Inflammation

    PubMed Central

    Terayama, Yui; Matsuura, Tetsuro; Ozaki, Kiyokazu

    2016-01-01

    Hyperplastic candidiasis is characterized by thickening of the mucosal epithelia with Candida albicans infection with occasional progression to squamous cell carcinoma (SCC). C. albicans is a critical factor in tumor development; however, the oncogenic mechanism is unclear. We have previously produced an animal model for hyperplastic candidiasis in the rat forestomach. In the present study, we investigate whether impaired DNA methylation and associated protein expression of tumor suppressor and DNA repair genes are involved in the SCC carcinogenesis process using this hyperplastic candidiasis model. Promoter methylation and protein expression were analyzed by methylation specific PCR and immunohistochemical staining, respectively, of 5 areas in the forestomachs of alloxan-induced diabetic rats with hyperplastic candidiasis: normal squamous epithelia, squamous hyperplasia, squamous hyperplasia adjacent to SCC, squamous hyperplasia transitioning to SCC, and SCC. We observed nuclear p16 overexpression despite increases in p16 gene promoter methylation during the carcinogenic process. TIMP3 and RAR-β2 promoter methylation progressed until the precancerous stage but disappeared upon malignant transformation. In comparison, TIMP3 protein expression was suppressed during carcinogenesis and RAR-β2 expression was attenuated in the cytoplasm but enhanced in nuclei. ERCC1 and BRCA1 promoters were not methylated at any stage; however, their protein expression disappeared beginning at hyperplasia and nuclear protein re-expression in SCC was observed only for ERCC1. These results suggest that aberrant p16, RAR-β2, TIMP3, ERCC1, and BRCA1 expression might occur that is inconsistent with the respective gene promoter methylation status, and that this overexpression might serve to promote the inflammatory carcinogenesis caused by C. albicans infection. PMID:27410681

  14. Enhanced Skeletal Muscle Expression of EcSOD Mitigates Streptozotocin-Induced Diabetic Cardiomyopathy by Reducing Oxidative Stress and Aberrant Cell Signaling

    PubMed Central

    Call, Jarrod A.; Chain, Kristopher H.; Martin, Kyle S.; Lira, Vitor A.; Okutsu, Mitsuharu; Zhang, Mei; Yan, Zhen

    2015-01-01

    Background Exercise training enhances extracellular superoxide dismutase (EcSOD) expression in skeletal muscle and elicits positive health outcomes in individuals with diabetes. The goal of this study was to determine if enhanced skeletal muscle expression of EcSOD is sufficient to mitigate streptozotocin (STZ)-induced diabetic cardiomyopathy (DCM). Methods and Results Exercise training promotes EcSOD expression in skeletal muscle and provides protection against DCM; however, it is not known if enhanced EcSOD expression in skeletal muscle plays a functional role in this protection. Here, we show that skeletal muscle-specific EcSOD transgenic mice (TG) are protected from cardiac hypertrophy, fibrosis and dysfunction under the condition of type-1 diabetes induced by STZ injection. We also show that both exercise training and muscle-specific transgenic expression of EcSOD result in elevated EcSOD protein in the blood and heart without increased transcription in the heart, suggesting enhanced expression of EcSOD from skeletal muscle redistributes to the heart. Importantly, cardiac tissue in TG mice displayed significantly reduced oxidative stress, aberrant cell signaling and inflammatory cytokine expression compared with wild type mice under the same diabetic condition. Conclusions Enhanced expression of EcSOD in skeletal muscle is sufficient to mitigate STZ-induced DCM through attenuation of oxidative stress, aberrant cell signaling and inflammation, suggesting a cross-organ mechanism by which exercise training improves cardiac function in diabetes. PMID:25504759

  15. Multiparameter flow cytometry reveals myelodysplasia-related aberrant antigen expression in myelodysplastic/myeloproliferative neoplasms.

    PubMed

    Kern, Wolfgang; Bacher, Ulrike; Schnittger, Susanne; Alpermann, Tamara; Haferlach, Claudia; Haferlach, Torsten

    2013-05-01

    Within the myelodysplastic/myeloproliferative neoplasm (MDS/MPN) category of the WHO (2008), only chronic myelomonocytic leukemia was so far evaluated by multiparameter flow cytometry (MFC). To investigate the potential of MFC for MDS/MPNs, unclassifiable (MDS/MPNu), and refractory anemia associated with ring sideroblasts and marked thrombocytosis (RARS-T), we studied 91 patients with these entities (60 males/31 females; 35.3-87.4 years) for MDS-related aberrant immunophenotypes (≥ 2 different cell lineages with ≥ 3 aberrantly expressed antigens). Data were correlated with cytomorphology and cytogenetics. MFC identified MDS-related immunophenotypes in 54/91 (59.3%) of patients. Patients with or without MDS-related immunophenotype did not differ significantly by demographic characteristics, blood values, or median overall survival. MDS-related immunophenotype cases showed a higher number of aberrantly expressed antigens (mean ± SD, 4.9 ± 2.4 vs. 2.0 ± 1.4; P < 0.001). Aberrant karyotypes showed a similar frequency in patients with and without MDS-related immunophenotype (11/54; 20.4% vs. 7/37; 18.9%; P = n.s.). MDS-related immunophenotype are present in more than half of patients with MDS/MPNu and RARS-T. MFC therefore may be helpful to separate cases into more "MDS-like" or "MPN-like" subgroups. Copyright © 2012 International Clinical Cytometry Society.

  16. The Mechanisms of Aberrant Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Cohen, Samuel; Vendruscolo, Michele; Dobson, Chris; Knowles, Tuomas

    2012-02-01

    We discuss the development of a kinetic theory for understanding the aberrant loss of solubility of proteins. The failure to maintain protein solubility results often in the assembly of organized linear structures, commonly known as amyloid fibrils, the formation of which is associated with over 50 clinical disorders including Alzheimer's and Parkinson's diseases. A true microscopic understanding of the mechanisms that drive these aggregation processes has proved difficult to achieve. To address this challenge, we apply the methodologies of chemical kinetics to the biomolecular self-assembly pathways related to protein aggregation. We discuss the relevant master equation and analytical approaches to studying it. In particular, we derive the underlying rate laws in closed-form using a self-consistent solution scheme; the solutions that we obtain reveal scaling behaviors that are very generally present in systems of growing linear aggregates, and, moreover, provide a general route through which to relate experimental measurements to mechanistic information. We conclude by outlining a study of the aggregation of the Alzheimer's amyloid-beta peptide. The study identifies the dominant microscopic mechanism of aggregation and reveals previously unidentified therapeutic strategies.

  17. Aberrant Expression of PIWIL1 and PIWIL2 and Their Clinical Significance in Ductal Breast Carcinoma.

    PubMed

    Litwin, Monika; Szczepańska-Buda, Anna; Michałowska, Dagmara; Grzegrzółka, Jędrzej; Piotrowska, Aleksandra; Gomułkiewicz, Agnieszka; Wojnar, Andrzej; Dzięgiel, Piotr; Witkiewicz, Wojciech

    2018-04-01

    P-Element-induced wimpy testis (PIWI) proteins in complex with PIWI-interacting RNA (piRNA) are involved in epigenetic regulation of gene expression in germline cells. Aberrant expression of piRNA and PIWI proteins have been identified in various types of tumour cells. The aim of this study was to evaluate the expression profiles of PIWI-like protein-1, -2 (PIWIL1 and PIWIL2), their immunohistochemical (IHC) characteristics in ductal breast cancer, and determine their correlation with clinicopathological parameters of this type of cancer. Material for IHC studies comprised of 101 invasive ductal carcinoma (IDC) cases and 31 mastopathy tissues. Frozen fragments of paired tissue specimens (tumour and adjacent non-malignant breast tissue) taken from 55 patients with IDC and 18 samples of mastopathy were used for molecular studies using real-time polymerase chain reaction (RT-PCR). A statistically significantly higher level of PIWIL1 and PIWIL2 was found in IDC compared to mastopathy samples (p≤0.0001). Increased expression of PIWIL1 was correlated with increased PIWIL2 expression in breast cancer tissue. Surprisingly, PIWIL1 mRNA was detected only in cancer and mastopathy, but was not found in most normal breast tissues, although it is noteworthy that the PIWIL2 mRNA level was statistically significantly lower in mastopathy and IDC samples compared to normal breast tissues. Our results affirm the hypothesis that reactivation of PIWI expression in various caner types is crucial for cancer development. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  18. Structural centrosome aberrations promote non-cell-autonomous invasiveness.

    PubMed

    Ganier, Olivier; Schnerch, Dominik; Oertle, Philipp; Lim, Roderick Yh; Plodinec, Marija; Nigg, Erich A

    2018-05-02

    Centrosomes are the main microtubule-organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein-like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP-induced structural centrosome aberrations trigger the escape ("budding") of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP-induced centrosome aberrations trigger a re-organization of the cytoskeleton, which stabilizes microtubules and weakens E-cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non-cell-autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  19. Plum polyphenols inhibit colorectal aberrant crypt foci formation in rats: potential role of the miR-143/protein kinase B/mammalian target of rapamycin axis.

    PubMed

    Banerjee, Nivedita; Kim, Hyemee; Talcott, Stephen T; Turner, Nancy D; Byrne, David H; Mertens-Talcott, Susanne U

    2016-10-01

    The nutritional prevention of aberrant crypt foci by polyphenols may be a crucial step to dietary cancer prevention. The objective of this study was to determine the underlying mechanisms that contribute to the anti-inflammatory and antitumorigenic properties of plum (Prunus salicina L.) polyphenols, including chlorogenic acid and neochlorogenic acid, in azoxymethane (AOM)-treated rats. The hypothesis was that plum polyphenolics suppress AOM-induced aberrant crypt foci formation through alterations in the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway and relative micro-RNA expressions. Sprague-Dawley rats (n=10/group) received plum beverage (1346mg gallic acid equivalents/L) or a control beverage ad libitum for 10 weeks with subcutaneous injections of AOM (15mg/kg) at weeks 2 and 3. Results show that the consumption of the plum beverage decreased the number of dysplastic aberrant crypt foci by 48% (P<.05) and lowered proliferation of mucosal cells by 24% (P<.05). The plum beverage decreased the activity of glutathione peroxidase, superoxide dismutase, and catalase in mucosal scrapings, as well as the superoxide dismutase activity in serum. The results were accompanied by a down-regulation of proinflammatory enzymes nuclear factor κB, nitric oxide synthase, cyclooxygenase-2, and vascular cell adhesion molecule 1 messenger RNA. Plum inhibited the expression of AKT and mTOR messenger RNA, phosphorylated AKT, mTOR, and hypoxia-inducible factor-1α protein levels, and the ratio of the phosphorylated/total protein expression of mTOR. Also, the plum beverage increased the expression of miR-143, which is involved in the regulation of AKT. These results suggest that plum polyphenols may exhibit a chemopreventive potential against colon carcinogenesis by impacting the AKT/mTOR pathway and miR-143. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Enhanced neuronal expression of major histocompatibility complex class I leads to aberrations in neurodevelopment and neurorepair

    PubMed Central

    Wu, Zhongqi-Phyllis; Washburn, Lorraine; Bilousova, Tina V.; Boudzinskaia, Maia; Escande-Beillard, Nathalie; Querubin, Jyes; Dang, Hoa; Xie, Cui-Wei; Tian, Jide; Kaufman, Daniel L.

    2012-01-01

    Mice deficient in classical major histocompatibility complex class I (MHCI) have aberrations in neurodevelopment. The consequences of up-regulated neuronal MHCI expression have not been examined. We found that transgenic C57Bl/6 mice that are engineered to express higher levels of self-Db on their CNS neurons have alterations in their hippocampal morphology and retinogeniculate projections, as well as impaired neurorepair responses. Thus, enhanced neuronal classical MHCI expression can lead to aberrations in neural circuitry and neurorepair. These findings complement a growing body of knowledge concerning the neurobiological activities of MHCI and may have potential clinical relevance. PMID:20950866

  1. Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

    2016-01-01

    Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

  2. Aberrant DNA methylation associated with silencing BNIP3 gene expression in haematopoietic tumours

    PubMed Central

    Murai, M; Toyota, M; Satoh, A; Suzuki, H; Akino, K; Mita, H; Sasaki, Y; Ishida, T; Shen, L; Garcia-Manero, G; Issa, J-P J; Hinoda, Y; Tokino, T; Imai, K

    2005-01-01

    Hypoxia is a key factor contributing to the progression of human neoplasias and to the development of resistance to chemotherapy. BNIP3 is a proapoptotic member of the Bcl-2 protein family involved in hypoxia-induced cell death. We evaluated the expression and methylation status of BNIP3 gene to better understand the role of epigenetic alteration of its expression in haematopoietic tumours. Methylation of the region around the BNIP3 transcription start site was detected in four acute lymphocytic leukaemia, one multiple myeloma and one Burkitt lymphoma cell lines, and was closely associated with silencing the gene. That expression of BNIP3 was restored by treatment with 5-aza2′-deoxycytidine (5-aza-dC), a methyltransferase inhibitor, which confirmed the gene to be epigenetically inactivated by methylation. Notably, re-expression of BNIP3 using 5-aza2-dC also restored hypoxia-mediated cell death in methylated cell lines. Acetylation of histone H3 in the 5′ region of the gene, which was assessed using chromatin immunoprecipitation assays, correlated directly with gene expression and inversely with DNA methylation. Among primary tumours, methylation of BNIP3 was detected in five of 34 (15%) acute lymphocytic leukaemias, six of 35 (17%) acute myelogenous leukaemias and three of 14 (21%) multiple myelomas. These results suggest that aberrant DNA methylation of the 5′ CpG island and histone deacetylation play key roles in silencing BNIP3 expression in haematopoietic tumours. PMID:15756280

  3. NCAM (CD56) expression in keratin-producing odontogenic cysts: aberrant expression in KCOT.

    PubMed

    Vera-Sirera, Beatriz; Forner-Navarro, Leopoldo; Vera-Sempere, Francisco

    2015-02-12

    To investigate immunohistochemically the expression of neural cell adhesion molecule (NCAM), which has been identified as a signaling receptor with frequent reactivity in ameloblastomas (AB), in a series of keratin-producing odontogenic cysts (KPOCs). Immunohistochemical expression of NCAM, using a monoclonal antibody, was determined in a series of 58 KPOCs comprising 12 orthokeratinized odontogenic cysts (OOCs) and 46 keratocystic odontogenic tumors (KCOTs), corresponding to 40 non-syndromic KCOT (NS-KCOTs) and 6 syndromic KCOT (S-KCOTs), associated with nevic basocellular syndrome (NBCS). NCAM expression was negative in all OOCs, but 36.45% of KCOTs exhibited focal and heterogeneous expression at the basal cell level, as well as in basal budding areas and the basal cells of daughter cysts. The latter two locations were especially applicable to S-KCOTs, with focal NCAM reactivity occurring in 66.66% of cases. Aberrant NCAM expression, in KCOTs but especially in S-KCOTs, together with its immunomorphological location, suggests that this adhesion molecule and signaling receptor plays a role in the pathogenesis of KCOTs, with a probable impact on lesional recurrence.

  4. TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology inmore » pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful

  5. Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs

    PubMed Central

    Soheilypour, M.; Mofrad, M. R. K.

    2016-01-01

    Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus. PMID:27805000

  6. Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs.

    PubMed

    Soheilypour, M; Mofrad, M R K

    2016-11-02

    Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus.

  7. Aberrantly Expressed OTX Homeobox Genes Deregulate B-Cell Differentiation in Hodgkin Lymphoma.

    PubMed

    Nagel, Stefan; Ehrentraut, Stefan; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

    2015-01-01

    In Hodgkin lymphoma (HL) we recently reported that deregulated homeobox gene MSX1 mediates repression of the B-cell specific transcription factor ZHX2. In this study we investigated regulation of MSX1 in this B-cell malignancy. Accordingly, we analyzed expression and function of OTX homeobox genes which activate MSX1 transcription during embryonal development in the neural plate border region. Our data demonstrate that OTX1 and OTX2 are aberrantly expressed in both HL patients and cell lines. Moreover, both OTX loci are targeted by genomic gains in overexpressing cell lines. Comparative expression profiling and subsequent pathway modulations in HL cell lines indicated that aberrantly enhanced FGF2-signalling activates the expression of OTX2. Downstream analyses of OTX2 demonstrated transcriptional activation of genes encoding transcription factors MSX1, FOXC1 and ZHX1. Interestingly, examination of the physiological expression profile of ZHX1 in normal hematopoietic cells revealed elevated levels in T-cells and reduced expression in B-cells, indicating a discriminatory role in lymphopoiesis. Furthermore, two OTX-negative HL cell lines overexpressed ZHX1 in correlation with genomic amplification of its locus at chromosomal band 8q24, supporting the oncogenic potential of this gene in HL. Taken together, our data demonstrate that deregulated homeobox genes MSX1 and OTX2 respectively impact transcriptional inhibition of (B-cell specific) ZHX2 and activation of (T-cell specific) ZHX1. Thus, we show how reactivation of a specific embryonal gene regulatory network promotes disturbed B-cell differentiation in HL.

  8. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche.

    PubMed

    Davis, Hayley; Irshad, Shazia; Bansal, Mukesh; Rafferty, Hannah; Boitsova, Tatjana; Bardella, Chiara; Jaeger, Emma; Lewis, Annabelle; Freeman-Mills, Luke; Giner, Francesc Castro; Rodenas-Cuadrado, Pedro; Mallappa, Sreelakshmi; Clark, Susan; Thomas, Huw; Jeffery, Rosemary; Poulsom, Richard; Rodriguez-Justo, Manuel; Novelli, Marco; Chetty, Runjan; Silver, Andrew; Sansom, Owen James; Greten, Florian R; Wang, Lai Mun; East, James Edward; Tomlinson, Ian; Leedham, Simon John

    2015-01-01

    Hereditary mixed polyposis syndrome (HMPS) is characterized by the development of mixed-morphology colorectal tumors and is caused by a 40-kb genetic duplication that results in aberrant epithelial expression of the gene encoding mesenchymal bone morphogenetic protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell fate that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem cell properties in Lgr5-negative progenitor cells that have exited the stem cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem cell is not the sole cell of origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic premalignant lesions with a hitherto unknown pathogenesis, and these lesions can be considered the sporadic equivalents of HMPS polyps.

  9. Oligoamine analogues in combination with 2-difluoromethylornithine synergistically induce re-expression of aberrantly silenced tumour-suppressor genes

    PubMed Central

    Wu, Yu; Steinbergs, Nora; Murray-Stewart, Tracy; Marton, Laurence J.; Casero, Robert A.

    2011-01-01

    Epigenetic gene silencing is an important mechanism in the initiation and progression of cancer. Abnormal DNA CpG island hypermethylation and histone modifications are involved in aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) was the first enzyme identified to specifically demethylate H3K4 (Lys4 of histone H3). Methylated H3K4 is an important mark associated with transcriptional activation. The flavin adenine dinucleotide-binding amine oxidase domain of LSD1 is homologous with two polyamine oxidases, SMO (spermine oxidase) and APAO (N1-acetylpolyamine oxidase). We have demonstrated previously that long-chain polyamine analogues, the oligoamines, are inhibitors of LSD1. In the present paper we report the synergistic effects of specific oligoamines in combination with DFMO (2-difluoromethylornithine), an inhibitor of ornithine decarboxylase, in human colorectal cancer cells. DFMO treatment depletes natural polyamines and increases the uptake of exogenous polyamines. The combination of oligoamines and DFMO results in a synergistic re-expression of aberrantly silenced tumour-suppressor genes, including SFRP2 (secreted frizzled-related protein 2), which encodes a Wnt signalling pathway antagonist and plays an anti-tumorigenic role in colorectal cancer. The treatment-induced re-expression of SFRP2 is associated with increased H3K4me2 (di-methyl H3K4) in the gene promoter. The combination of LSD1-inhibiting oligoamines and DFMO represents a novel approach to epigenetic therapy of cancer. PMID:22132744

  10. Drosophila Suppressor of Sable Protein [Su(s)] Promotes Degradation of Aberrant and Transposon-Derived RNAs▿

    PubMed Central

    Kuan, Yung-Shu; Brewer-Jensen, Paul; Bai, Wen-Li; Hunter, Cedric; Wilson, Carrie B.; Bass, Sarah; Abernethy, John; Wing, James S.; Searles, Lillie L.

    2009-01-01

    RNA-binding proteins act at various stages of gene expression to regulate and fine-tune patterns of mRNA accumulation. One protein in this class is Drosophila Su(s), a nuclear protein that has been previously shown to inhibit the accumulation of mutant transcripts by an unknown mechanism. Here, we have identified several additional RNAs that are downregulated by Su(s). These Su(s) targets include cryptic wild-type transcripts from the developmentally regulated Sgs4 and ng1 genes, noncoding RNAs derived from tandemly repeated αβ/αγ elements within an Hsp70 locus, and aberrant transcripts induced by Hsp70 promoter transgenes inserted at ectopic sites. We used the αβ RNAs to investigate the mechanism of Su(s) function and obtained evidence that these transcripts are degraded by the nuclear exosome and that Su(s) promotes this process. Furthermore, we showed that the RNA binding domains of Su(s) are important for this effect and mapped the sequences involved to a 267-nucleotide region of an αβ element. Taken together, these results suggest that Su(s) binds to certain nascent transcripts and stimulates their degradation by the nuclear exosome. PMID:19687295

  11. A Evaluation of Optical Aberrations in Underwater Hologrammetry

    NASA Astrophysics Data System (ADS)

    Kilpatrick, J. M.

    Available from UMI in association with The British Library. An iterative ray-trace procedure is developed in conjunction with semi-analytic expressions for spherical aberration, coma, and astigmatism in the reconstructed holographic images of underwater objects. An exact expression for the astigmatic difference is obtained, based on the geometry of the caustic for refraction. The geometrical characteristics of the aberrated images associated with axial and non-axial field positions are represented by ray intersection diagrams. A third order expression for the wavefront aberration introduced at a planar air/water boundary is given. The associated third order aberration coefficients are used to obtain analytic expressions for the aberrations observed in underwater hologrammetry. The results of the third order treatment are shown to give good agreement with the results obtained by geometrical ray tracing and by direct measurement on the reconstructed real image. The third order aberration coefficients are employed to estimate the limit of resolution in the presence of the aberrations associated with reconstruction in air. In concurrence with practical observations it is found that the estimated resolution is primarily limited by astigmatism. The limitations of the planar window in underwater imaging applications are outlined and various schemes are considered to effect a reduction in the extent of aberration. The analogous problems encountered in underwater photography are examined in order to establish the grounds for a common solution based on a conventional optical corrector. The performance of one such system, the Ivanoff Corrector, is investigated. The spherical aberration associated with axial image formation is evaluated. The equivalence of the third order wavefront aberration introduced at a planar air/water boundary to that introduced upon reconstruction by an appropriate wavelength change is shown to provide a basis for the compensation of aberrations in

  12. Protein regulator of cytokinesis-1 expression: prognostic value in lung squamous cell carcinoma patients

    PubMed Central

    Zhan, Ping; Xi, Guang-Min; Liu, Hong-Bing; Liu, Ya-Fang; Xu, Wu-Jian; Zhu, Qingqing; Zhou, Ze-Jun; Miao, Ying-Ying; Wang, Xiao-Xia; Jin, Jia-Jia

    2017-01-01

    Background Protein regulator of cytokinesis-1 (PRC1) has been shown to participate in the completion of cytokinesis, and it is dysregulated in cancer processes. However, its relevance in lung squamous cell carcinoma (SCC) remained largely unknown. We aimed to study the expression pattern of PRC1 and assess its clinical significance in lung SCC. Methods PRC1 protein expression in human lung SCC and adjacent normal lung tissues was detected by immunohistochemistry. PRC1 expression was assessed in association with clinicopathological features and clinical outcomes of lung SCC patients. Results In lung SCC tissues, PRC1 protein expression was significantly higher than those in paired normal lung tissues. The lung SCC patients with PRC1 overexpression had an advanced pathological stage (TNM stage), positive lymph node metastasis, and a shorter overall survival (OS) time more frequently than patients with low PRC1 expression. Additional, PRC1 expression was also shown to be poor as a prognostic factor for OS in patients with lung SCC. Conclusions Our study indicated that aberrant expression of PRC1 may point to biochemical recurrence in lung SCC. This highlights its potential as a valuable prognostic marker for lung SCC. PMID:28840006

  13. Quantitative analysis of aberrant protein glycosylation in liver cancer plasma by AAL-enrichment and MRM mass spectrometry.

    PubMed

    Ahn, Yeong Hee; Shin, Park Min; Kim, Yong-Sam; Oh, Na Ree; Ji, Eun Sun; Kim, Kwang Hoe; Lee, Yeon Jung; Kim, Sung Ho; Yoo, Jong Shin

    2013-11-07

    A lectin-coupled mass spectrometry (MS) approach was employed to quantitatively monitor aberrant protein glycosylation in liver cancer plasma. To do this, we compared the difference in the total protein abundance of a target glycoprotein between hepatocellular carcinoma (HCC) plasmas and hepatitis B virus (HBV) plasmas, as well as the difference in lectin-specific protein glycoform abundance of the target glycoprotein. Capturing the lectin-specific protein glycoforms from a plasma sample was accomplished by using a fucose-specific aleuria aurantia lectin (AAL) immobilized onto magnetic beads via a biotin-streptavidin conjugate. Following tryptic digestion of both the total plasma and its AAL-captured fraction of each HCC and HBV sample, targeted proteomic mass spectrometry was conducted quantitatively by a multiple reaction monitoring (MRM) technique. From the MRM-based analysis of the total plasmas and AAL-captured fractions, differences between HCC and HBV plasma groups in fucosylated glycoform levels of target glycoproteins were confirmed to arise from both the change in the total protein abundance of the target proteins and the change incurred by aberrant fucosylation on target glycoproteins in HCC plasma, even when no significant change occurs in the total protein abundance level. Combining the MRM-based analysis method with the lectin-capturing technique proved to be a successful means of quantitatively investigating aberrant protein glycosylation in cancer plasma samples. Additionally, it was elucidated that the differences between HCC and control groups in fucosylated biomarker candidates A1AT and FETUA mainly originated from an increase in fucosylation levels on these target glycoproteins, rather than an increase in the total protein abundance of the target glycoproteins.

  14. Aberrant expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice.

    PubMed

    Hwang, Soo Seok; Kim, Kiwan; Lee, Wonyong; Lee, Gap Ryol

    2012-08-03

    The Th2 locus control region (LCR) has been shown to be a crucial cis-acting element for Th2 cytokine expression and Th2 cell differentiation. To study the role of Th2 LCR in ifng locus regulation, we examined the expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice. We found IFN-γ to be aberrantly up-regulated. In addition, histone 3(H3)-acetylation and histone 3 lysine 4 (H3-K4)-methylation greatly increased at the ifng locus of the Th2 cells. GATA-3 and STAT6 bound to the ifng promoter in Th2 cells from the wild type but not from the Th2 LCR-deficient mice, and they directly repressed ifng expression in transient reporter assay. Moreover, ectopic expression of GATA-3 and STAT6-VT repressed the aberrant expression of the ifng gene and restored repressive chromatin state at the ifng locus in Th2 cells from Th2 LCR-deficient mice. These results suggest that expression of the ifng gene and chromatin remodeling of the ifng locus are under the control of a Th2 LCR-mediated Th2 differentiation program. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Aberrant expression of decoy receptor 3 in human breast cancer: relevance to lymphangiogenesis.

    PubMed

    Wu, Qiuwan; Zheng, Yahong; Chen, Donghan; Li, Xiaohong; Lu, Chuanhui; Zhang, Zhiming

    2014-05-15

    Decoy receptor 3 (DcR3), a decoy receptor against Fas ligand belonging to the tumor necrosis factor receptor superfamily, is overexpressed in some forms of cancer. It was recently reported that DcR3 could protect endothelial cells from apoptosis, implying a potential role in the development of vessels, whereas its role in the lymphangiogenesis remains unclear. In the present study, we studied the DcR3 expression and its relationship with the lymphatic microvessel density (LMVD) to investigate if it played a role in the lymph metastasis of human breast cancer. Real-time polymerase chain reaction and immunohistochemistry were performed to measure the messenger RNA and protein expression of DcR3 in the breast cancer tissues, noncancerous counterparts, and axillary lymph node from 63 patients. LMVD in these specimens was assessed by counting the D2-40 labeled-microvessels. Furthermore, the correlations between DcR3 expression and LMVD and other clinicopathologic parameters were analyzed. DcR3 was overexpressed in the breast cancer tissue of 58 patients (92.1%) and was also expressed in vascular endothelial cells and tumor cells in the lymph nodes. LMVD in cancer tissue and lymph nodes were both positively correlated to the aberrant expression of DcR3. The relevance between DcR3 overexpression and LMVD revealed the existence of possible links between DcR3 and lymphangiogenesis. Based on these findings, it is important to further explore the regulation of lymphangiogenesis operated by the reverse tumor necrosis factor signaling of DcR3. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Aberrant gonadotropin-releasing hormone receptor (GnRHR) expression and its regulation of CYP11B2 expression and aldosterone production in adrenal aldosterone-producing adenoma (APA).

    PubMed

    Nakamura, Yasuhiro; Hattangady, Namita G; Ye, Ping; Satoh, Fumitoshi; Morimoto, Ryo; Ito-Saito, Takako; Sugawara, Akira; Ohba, Koji; Takahashi, Kazuhiro; Rainey, William E; Sasano, Hironobu

    2014-03-25

    Aberrant expression of gonadotropin-releasing hormone receptor (GnRHR) has been reported in human adrenal tissues including aldosterone-producing adenoma (APA). However, the details of its expression and functional role in adrenals are still not clear. In this study, quantitative RT-PCR analysis revealed the mean level of GnRHR mRNA was significantly higher in APAs than in human normal adrenal (NA) (P=0.004). GnRHR protein expression was detected in human NA and neoplastic adrenal tissues. In H295R cells transfected with GnRHR, treatment with GnRH resulted in a concentration-dependent increase in CYP11B2 reporter activity. Chronic activation of GnRHR with GnRH (100nM), in a cell line with doxycycline-inducible GnRHR (H295R-TR/GnRHR), increased CYP11B2 expression and aldosterone production. These agonistic effects were inhibited by blockers for the calcium signaling pathway, KN93 and calmidazolium. These results suggest GnRH, through heterotopic expression of its receptor, may be a potential regulator of CYP11B2 expression levels in some cases of APA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. Aberrant expression of miR-141 and nuclear receptor small heterodimer partner in clinical samples of prostate cancer.

    PubMed

    Khorasani, Maryam; Teimoori-Toolabi, Ladan; Farivar, Taghi Naserpour; Asgari, Mojgan; Abolhasani, Maryam; Shahrokh, Hossein; Afgar, Ali; Kalantari, Elham; Peymani, Amir; Mahdian, Reza

    2018-01-01

    Prostate cancer (PCa) is the second most common cancer in men worldwide. Currently, prostate-specific antigen (PSA) test and digital rectal exam are the main screening tests used for PCa diagnosis. However, due to the low specificity of these tests, new alternative biomarkers such as deregulated RNAs and microRNAs have been implemented. Aberrant expressions of small heterodimer partner gene (SHP, NR0B2) and mir-141 are reported in various cancers. The aim of this study was to investigate the SHP and miR-141 expression level in tissue samples of prostate cancer. The expression level of SHP gene and miR-141 was assessed by real time PCR and their relative amounts were calculated by the Δ⁢ΔCT method. Also, IHC technique was used to determine the expression level of SHP protein. The miR-141 was significantly up-regulated in the samples of metastatic tumors compared to localized tumor samples (P< 0.001, 31.17-fold change). Tumor samples showed lower SHP mRNA expression levels than BPH samples (p= 0.014, 4.7-fold change). The results of paired t-test analysis showed there was no significant difference between the SHP gene expression in PCa samples and their matched tumor-adjacent normal tissue (p= 0.5). The data obtained in our study confirm the involvement of miR-141 in PCa progression and metastasis. These effects could be mediated by AR via down-regulation of its co-repressor protein, i.e., SHP.

  18. Microarray-based genomic profiling reveals novel genomic aberrations in follicular lymphoma which associate with patient survival and gene expression status.

    PubMed

    Schwaenen, Carsten; Viardot, Andreas; Berger, Hilmar; Barth, Thomas F E; Bentink, Stefan; Döhner, Hartmut; Enz, Martina; Feller, Alfred C; Hansmann, Martin-Leo; Hummel, Michael; Kestler, Hans A; Klapper, Wolfram; Kreuz, Markus; Lenze, Dido; Loeffler, Markus; Möller, Peter; Müller-Hermelink, Hans-Konrad; Ott, German; Rosolowski, Maciej; Rosenwald, Andreas; Ruf, Sandra; Siebert, Reiner; Spang, Rainer; Stein, Harald; Truemper, Lorenz; Lichter, Peter; Bentz, Martin; Wessendorf, Swen

    2009-01-01

    Follicular lymphoma (FL) is characterized by a large number of chromosomal aberrations. However, their exact genomic extension and involved target genes remain to be determined. For this purpose, we used array-based intermediate-high resolution genomic profiling in combination with Affymetrix gene expression analysis. Tumor specimens from 128 FL patients were analyzed for the presence of genomic aberrations and the results were correlated to clinical data sets and mRNA expression levels. In 114 (89%) of the 128 analyzed cases, a total of 688 genomic aberrations (384 gains/amplifications and 304 losses) were detected. Frequent genomic aberrations were: -1p36 (18%), +2p15 (24%), -3q (14%), -6q (25%), +7p (19%), +7q (23%), +8q (14%), -9p (16%), -11q (15%), +12q (20%), -13q (11%), -17p (16%), +18p (18%), and +18q (28%). Critical segments of these imbalances were delineated to genomic fragments with a minimum size down to 0.2 Mb. By comparison of these with mRNA gene expression data, putative candidate genes were identified. Moreover, we found that deletions affecting the tumor suppressor gene CDKN2A/B on 9p21 were detected in nontransformed FL grade I-II. For this aberration as well as for -6q25 and -6q26, an association with inferior survival was observed.

  19. Immunohistochemical panel to characterize canine prostate carcinomas according to aberrant p63 expression.

    PubMed

    Fonseca-Alves, Carlos Eduardo; Kobayashi, Priscila Emiko; Rivera Calderón, Luis Gabriel; Felisbino, Sérgio Luis; Rinaldi, Jaqueline de Carvalho; Drigo, Sandra Aparecida; Rogatto, Silvia Regina; Laufer-Amorim, Renée

    2018-01-01

    An unusual variant of prostate adenocarcinoma (PC) expressing nuclear p63 in secretory cells instead of the typical basal expression has been reported in men. Nevertheless, the biological behavior and clinical significance of this phenomenon is unknown. In dogs, this unusual PC subtype has not been described. In this study, p63 immunoexpression was investigated in 90 canine PCs and 20 normal prostate tissues (NT). The p63 expression pattern in luminal or basal cells was confirmed in a selected group of 26 PCs and 20 NT by immunohistochemistry and/or Western blotting assays. Eleven canine PC samples aberrantly expressing p63 (p63+) in secretory cells were compared with 15 p63 negative (p63-) cases in the context of several molecular markers (high molecular weight cytokeratin-HMWC, CK8/18, CK5, AR, PSA, chromogranin, NKX3.1, PTEN, AKT and C-MYC). P63+ samples were positive for CK5, HMWC and CK8/18 and negative for PSA, NKX3.1, PTEN and chromogranin. Five p63+ PCs were negative for AR, and the remaining six samples had low AR expression. In contrast, p63- PC showed AR and PSA positive expression in all 15 samples. Only five p63- PCs were positive for CK5. Both p63+ and p63- PC samples showed higher cytoplasmic AKT expression and nuclear C-MYC staining in comparison with normal tissues. Metastatic (N = 12) and non-metastatic (N = 14) PCs showed similar immunoexpression for all markers tested. In contrast to human PC, canine PC aberrantly expressing p63 showed higher expression levels of HMWC and CK5 and lower levels of NKX3.1. Canine p63+ PC is a very rare PC group showing a distinct phenotype compared to typical canine PC, including AR and PSA negative expression. Although in a limited number of cases, p63 expression was not associated with metastasis in canine PC, and cytoplasmic p63 expression was observed in animals with shorter survival time, similar to human PC cases.

  20. Aberrant glycosylation of plasma proteins in severe preeclampsia promotes monocyte adhesion.

    PubMed

    Flood-Nichols, Shannon K; Kazanjian, Avedis A; Tinnemore, Deborah; Gafken, Philip R; Ogata, Yuko; Napolitano, Peter G; Stallings, Jonathan D; Ippolito, Danielle L

    2014-02-01

    Glycosylation of plasma proteins increases during pregnancy. Our objectives were to investigate an anti-inflammatory role of these proteins in normal pregnancies and determine whether aberrant protein glycosylation promotes monocyte adhesion in preeclampsia. Plasma was prospectively collected from nonpregnant controls and nulliparous patients in all 3 trimesters. Patients were divided into cohorts based on the applicable postpartum diagnosis. U937 monocytes were preconditioned with enzymatically deglycosylated plasma, and monocyte adhesion to endothelial cell monolayers was quantified by spectrophotometry. Plasma from nonpregnant controls, first trimester normotensives, and first trimester patients with mild preeclampsia inhibited monocyte-endothelial cell adhesion (P < .05), but plasma from first trimester patients with severe preeclampsia and second and third trimester normotensives did not. Deglycosylating plasma proteins significantly increased adhesion in all the cohorts. These results support a role of plasma glycoprotein interaction in monocyte-endothelial cell adhesion and could suggest a novel therapeutic target for severe preeclampsia.

  1. Elucidation of the Molecular Mechanisms for Aberrant Expression of Breast Cancer Specific Gene 1 in Invasive and Metastatic Breast Carcinomas

    DTIC Science & Technology

    2004-06-01

    cells in mitosis. Mutations in any of these genes result in failure to arrest Keywords: BCSG I: BubRl; mitotic checkpoint; yeast the cell cycle at G2...AD Award Number: DAMD17-02-1-0534 TITLE: Elucidation of the Molecular Mechanisms for Aberrant Expression of Breast Cancer Specific Gene 1 in Invasive...SUBTITLE 5. FUNDING NUMBERS Elucidation of the Molecular Mechanisms for Aberrant DAMD17-02-1-0534 Expression of Breast Cancer Specific Gene 1 in Invasive

  2. Pervasive transcription read-through promotes aberrant expression of oncogenes and RNA chimeras in renal carcinoma

    PubMed Central

    Grosso, Ana R; Leite, Ana P; Carvalho, Sílvia; Matos, Mafalda R; Martins, Filipa B; Vítor, Alexandra C; Desterro, Joana MP; Carmo-Fonseca, Maria; de Almeida, Sérgio F

    2015-01-01

    Aberrant expression of cancer genes and non-canonical RNA species is a hallmark of cancer. However, the mechanisms driving such atypical gene expression programs are incompletely understood. Here, our transcriptional profiling of a cohort of 50 primary clear cell renal cell carcinoma (ccRCC) samples from The Cancer Genome Atlas (TCGA) reveals that transcription read-through beyond the termination site is a source of transcriptome diversity in cancer cells. Amongst the genes most frequently mutated in ccRCC, we identified SETD2 inactivation as a potent enhancer of transcription read-through. We further show that invasion of neighbouring genes and generation of RNA chimeras are functional outcomes of transcription read-through. We identified the BCL2 oncogene as one of such invaded genes and detected a novel chimera, the CTSC-RAB38, in 20% of ccRCC samples. Collectively, our data highlight a novel link between transcription read-through and aberrant expression of oncogenes and chimeric transcripts that is prevalent in cancer. DOI: http://dx.doi.org/10.7554/eLife.09214.001 PMID:26575290

  3. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside of the crypt base stem cell niche

    PubMed Central

    Bansal, Mukesh; Rafferty, Hannah; Boitsova, Tatjana; Bardella, Chiara; Jaeger, Emma; Lewis, Annabelle; Freeman-Mills, Luke; Giner, Francesc Castro; Rodenas-Cuadrado, Pedro; Mallappa, Sreelakshmi; Clark, Susan; Thomas, Huw; Jeffery, Rosemary; Poulsom, Richard; Rodriguez-Justo, Manuel; Novelli, Marco; Chetty, Runjan; Silver, Andrew; Sansom, Owen James; Greten, Florian R; Wang, Lai Mun; East, James Edward; Tomlinson, Ian; Leedham, Simon John

    2015-01-01

    Hereditary mixed polyposis syndrome (HMPS) is characterised by the development of mixed morphology colorectal tumours and is caused by a 40 kb duplication that results in aberrant epithelial expression of the mesenchymal Bone Morphogenetic Protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell-fate, that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem-cell properties in Lgr5 negative (non-expressing) progenitor cells that have exited the stem-cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem-cell is not the sole cell-of-origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic pre-malignant lesions with a hitherto unknown pathogenesis and these lesions can be considered the sporadic equivalents of HMPS polyps. PMID:25419707

  4. RSRC1 mutation affects intellect and behaviour through aberrant splicing and transcription, downregulating IGFBP3.

    PubMed

    Perez, Yonatan; Menascu, Shay; Cohen, Idan; Kadir, Rotem; Basha, Omer; Shorer, Zamir; Romi, Hila; Meiri, Gal; Rabinski, Tatiana; Ofir, Rivka; Yeger-Lotem, Esti; Birk, Ohad S

    2018-04-01

    RSRC1, whose polymorphism is associated with altered brain function in schizophrenia, is a member of the serine and arginine rich-related protein family. Through homozygosity mapping and whole exome sequencing we show that RSRC1 mutation causes an autosomal recessive syndrome of intellectual disability, aberrant behaviour, hypotonia and mild facial dysmorphism with normal brain MRI. Further, we show that RSRC1 is ubiquitously expressed, and that the RSRC1 mutation triggers nonsense-mediated mRNA decay of the RSRC1 transcript in patients' fibroblasts. Short hairpin RNA (shRNA)-mediated lentiviral silencing and overexpression of RSRC1 in SH-SY5Y cells demonstrated that RSRC1 has a role in alternative splicing and transcription regulation. Transcriptome profiling of RSRC1-silenced cells unravelled specific differentially expressed genes previously associated with intellectual disability, hypotonia and schizophrenia, relevant to the disease phenotype. Protein-protein interaction network modelling suggested possible intermediate interactions by which RSRC1 affects gene-specific differential expression. Patient-derived induced pluripotent stem cells, differentiated into neural progenitor cells, showed expression dynamics similar to the RSRC1-silenced SH-SY5Y model. Notably, patient neural progenitor cells had 9.6-fold downregulated expression of IGFBP3, whose brain expression is affected by MECP2, aberrant in Rett syndrome. Interestingly, Igfbp3-null mice have behavioural impairment, abnormal synaptic function and monoaminergic neurotransmission, likely correlating with the disease phenotype.

  5. Fetal Onset of Aberrant Gene Expression Relevant to Pulmonary Carcinogenesis in Lung Adenocarcinoma Development Induced by In Utero Arsenic Exposure

    PubMed Central

    Shen, Jun; Liu, Jie; Xie, Yaxiong; Diwan, Bhalchandra A.; Waalkes, Michael P.

    2009-01-01

    Arsenic is a human pulmonary carcinogen. Our work indicates that in utero arsenic exposure in mice can induce or initiate lung cancer in female offspring. To define early molecular changes, pregnant C3H mice were given 85 ppm arsenic in drinking water from days 8 to 18 of gestation and expression of selected genes in the fetal lung or in lung tumors developing in adults was examined. Transplacental arsenic exposure increased estrogen receptor-α (ER-α) transcript and protein levels in the female fetal lung. An overexpression of various estrogen-regulated genes also occurred, including trefoil factor-3, anterior gradient-2, and the steroid metabolism genes 17-β-hydroxysteroid dehydrogenase type 5 and aromatase. The insulin growth factor system, which can be influenced by ER and has been implicated in the pulmonary oncogenic process, was activated in fetal lung after gestational arsenic exposure. in utero arsenic exposure also induced overexpression of α-fetoprotein, epidermal growth factor receptor, L-myc, and metallothionein-1 in fetal lung, all of which are associated with lung cancer. Lung adenoma and adenocarcinoma from adult female mice exposed to arsenic in utero showed widespread, intense nuclear ER-α expression. In contrast, normal adult lung and diethylnitrosamine-induced lung adenocarcinoma showed little evidence of ER-α expression. Thus, transplacental arsenic exposure at a carcinogenic dose produced aberrant estrogen-linked pulmonary gene expression. ER-α activation was specifically associated with arsenic-induced lung adenocarcinoma and adenoma but not with nitrosamine-induced lung tumors. These data provide evidence that arsenic-induced aberrant ER signaling could disrupt early life stage genetic programing in the lung leading eventually to lung tumor formation much later in adulthood. PMID:17077188

  6. Fetal onset of aberrant gene expression relevant to pulmonary carcinogenesis in lung adenocarcinoma development induced by in utero arsenic exposure.

    PubMed

    Shen, Jun; Liu, Jie; Xie, Yaxiong; Diwan, Bhalchandra A; Waalkes, Michael P

    2007-02-01

    Arsenic is a human pulmonary carcinogen. Our work indicates that in utero arsenic exposure in mice can induce or initiate lung cancer in female offspring. To define early molecular changes, pregnant C3H mice were given 85 ppm arsenic in drinking water from days 8 to 18 of gestation and expression of selected genes in the fetal lung or in lung tumors developing in adults was examined. Transplacental arsenic exposure increased estrogen receptor-alpha (ER-alpha) transcript and protein levels in the female fetal lung. An overexpression of various estrogen-regulated genes also occurred, including trefoil factor-3, anterior gradient-2, and the steroid metabolism genes 17-beta-hydroxysteroid dehydrogenase type 5 and aromatase. The insulin growth factor system, which can be influenced by ER and has been implicated in the pulmonary oncogenic process, was activated in fetal lung after gestational arsenic exposure. In utero arsenic exposure also induced overexpression of alpha-fetoprotein, epidermal growth factor receptor, L-myc, and metallothionein-1 in fetal lung, all of which are associated with lung cancer. Lung adenoma and adenocarcinoma from adult female mice exposed to arsenic in utero showed widespread, intense nuclear ER-alpha expression. In contrast, normal adult lung and diethylnitrosamine-induced lung adenocarcinoma showed little evidence of ER-alpha expression. Thus, transplacental arsenic exposure at a carcinogenic dose produced aberrant estrogen-linked pulmonary gene expression. ER-alpha activation was specifically associated with arsenic-induced lung adenocarcinoma and adenoma but not with nitrosamine-induced lung tumors. These data provide evidence that arsenic-induced aberrant ER signaling could disrupt early life stage genetic programing in the lung leading eventually to lung tumor formation much later in adulthood.

  7. Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

    PubMed

    Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

    2015-02-23

    A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

  8. Proteins interacting with cloning scars: a source of false positive protein-protein interactions

    PubMed Central

    Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

    2015-01-01

    A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine “cloning scar” present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected. PMID:25704442

  9. Sixth-order wave aberration theory of ultrawide-angle optical systems.

    PubMed

    Lu, Lijun; Cao, Yiqing

    2017-10-20

    In this paper, we develop sixth-order wave aberration theory of ultrawide-angle optical systems like fisheye lenses. Based on the concept and approach to develop wave aberration theory of plane-symmetric optical systems, we first derive the sixth-order intrinsic wave aberrations and the fifth-order ray aberrations; second, we present a method to calculate the pupil aberration of such kind of optical systems to develop the extrinsic aberrations; third, the relation of aperture-ray coordinates between adjacent optical surfaces is fitted with the second-order polynomial to improve the calculation accuracy of the wave aberrations of a fisheye lens with a large acceptance aperture. Finally, the resultant aberration expressions are applied to calculate the aberrations of two design examples of fisheye lenses; the calculation results are compared with the ray-tracing ones with Zemax software to validate the aberration expressions.

  10. Proteomic Analysis Reveals Aberrant O-GlcNAcylation of Extracellular Proteins from Breast Cancer Cell Secretion.

    PubMed

    Netsirisawan, Pukkavadee; Chokchaichamnankit, Daranee; Srisomsap, Chantragan; Svasti, Jisnuson; Champattanachai, Voraratt

    2015-01-01

    O-GlcNAcylation is a unique intracellular protein modification; however, few extracellular O-GlcNAc-modified proteins have been discovered. We have previously demonstrated that many cellular proteins were aberrant in O-GlcNAcylation in breast cancer tissues. In the present study, therefore, we investigated whether O-GlcNAc-modified proteins were abnormally secreted from breast cancer cells. Intracellular and extracellular proteins were prepared from cell lysates of breast cancer cells (MCF-7 and MDA-MB-231) and normal breast cells (HMEC) and from their serum-free media (SFM), respectively. O-GlcNAcylation level was examined by immunoblotting. O-GlcNAc-Modified proteins were identified using two-dimensional gel electrophoresis and Liquid Chromatography-tandem Mass Spectrometry. O-GlcNAcylation level was significantly increased in the extracellular compartment of both types of cancer cells compared to normal cells. Interestingly, O-GlcNAc patterns differed between intracellular and extracellular proteins. Proteomic analysis revealed that many O-GlcNAc spots in MCF-7 secretions were abnormally increased in comparison to those in HMEC secretions. Among these, transitional endoplasmic reticulum ATPase (TER ATPase) and heat-shock 70 kDa (HSP70) were confirmed to be O-GlcNAc-modified. The levels of O-GlcNAc-HSP70 and O-GlcNAc-TER ATPase were higher in SFM from MCF-7 cells than in that from HMEC. O-GlcNAcomic study of the extracellular compartments reveals aberrant O-GlcNAc-secreted proteins, which may be of interest as potential biomarkers in breast cancer. Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  11. Aberrant expression of long noncoding RNAs in cumulus cells isolated from PCOS patients.

    PubMed

    Huang, Xin; Hao, Cuifang; Bao, Hongchu; Wang, Meimei; Dai, Huangguan

    2016-01-01

    To describe the long noncoding RNA (lncRNA) profiles in cumulus cells isolated from polycystic ovary syndrome (PCOS) patients by employing a microarray and in-depth bioinformatics analysis. This information will help us understand the occurrence and development of PCOS. In this study, we used a microarray to describe lncRNA profiles in cumulus cells isolated from ten patients (five PCOS and five normal women). Several differentially expressed lncRNAs were chosen to validate the microarray results by quantitative RT-PCR (qRT-PCR). Then, the differentially expressed lncRNAs were classified into three subgroups (HOX loci lncRNA, enhancer-like lncRNA, and lincRNA) to deduce their potential features. Furthermore, a lncRNA/mRNA co-expression network was constructed by using the Cytoscape software (V2.8.3, http://www.cytoscape.org/ ). We observed that 623 lncRNAs and 260 messenger RNAs (mRNAs) were significantly up- or down-regulated (≥2-fold change), and these differences could be used to discriminate cumulus cells of PCOS from those of normal patients. Five differentially expressed lncRNAs (XLOC_011402, ENST00000454271, ENST00000433673, ENST00000450294, and ENST00000432431) were selected to validate the microarray results using quantitative RT-PCR (qRT-PCR). The qRT-PCR results were consistent with the microarray data. Further analysis indicated that many differentially expressed lncRNAs were transcribed from chromosome 2 and may act as enhancers to regulate their neighboring protein-coding genes. Forty-three lncRNAs and 29 mRNAs were used to construct the coding-non-coding gene co-expression network. Most pairs positively correlated, and one mRNA correlated with one or more lncRNAs. Our study is the first to determine genome-wide lncRNA expression patterns in cumulus cells isolated from PCOS patients by microarray. The results show that clusters of lncRNAs were aberrantly expressed in cumulus cells of PCOS patients compared with those of normal women, which revealed

  12. Differentially expressed microRNAs in lung adenocarcinoma invert effects of copy number aberrations of prognostic genes

    PubMed Central

    Tokar, Tomas; Pastrello, Chiara; Ramnarine, Varune R.; Zhu, Chang-Qi; Craddock, Kenneth J.; Pikor, Larrisa A.; Vucic, Emily A.; Vary, Simon; Shepherd, Frances A.; Tsao, Ming-Sound; Lam, Wan L.; Jurisica, Igor

    2018-01-01

    In many cancers, significantly down- or upregulated genes are found within chromosomal regions with DNA copy number alteration opposite to the expression changes. Generally, this paradox has been overlooked as noise, but can potentially be a consequence of interference of epigenetic regulatory mechanisms, including microRNA-mediated control of mRNA levels. To explore potential associations between microRNAs and paradoxes in non-small-cell lung cancer (NSCLC) we curated and analyzed lung adenocarcinoma (LUAD) data, comprising gene expressions, copy number aberrations (CNAs) and microRNA expressions. We integrated data from 1,062 tumor samples and 241 normal lung samples, including newly-generated array comparative genomic hybridization (aCGH) data from 63 LUAD samples. We identified 85 “paradoxical” genes whose differential expression consistently contrasted with aberrations of their copy numbers. Paradoxical status of 70 out of 85 genes was validated on sample-wise basis using The Cancer Genome Atlas (TCGA) LUAD data. Of these, 41 genes are prognostic and form a clinically relevant signature, which we validated on three independent datasets. By meta-analysis of results from 9 LUAD microRNA expression studies we identified 24 consistently-deregulated microRNAs. Using TCGA-LUAD data we showed that deregulation of 19 of these microRNAs explains differential expression of the paradoxical genes. Our results show that deregulation of paradoxical genes is crucial in LUAD and their expression pattern is maintained epigenetically, defying gene copy number status. PMID:29507679

  13. Increased 5-hydroxymethylcytosine and Ten-eleven Translocation Protein Expression in Ultraviolet B-irradiated HaCaT Cells

    PubMed Central

    Wang, Dan; Huang, Jin-Hua; Zeng, Qing-Hai; Gu, Can; Ding, Shu; Lu, Jian-Yun; Chen, Jing; Yang, Sheng-Bo

    2017-01-01

    Background: DNA hydroxymethylation refers to a chemical modification process in which 5-methylcytosine (5mC) is catalyzed to 5- hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) family proteins. Recent studies have revealed that aberrant TETs expression or 5hmC level may play important roles in the occurrence and development of various pathological and physiological processes including cancer and aging. This study aimed to explore the relation between aberrant DNA hydroxymethylation with skin photoaging and to investigate the levels of TETs, 5mC, and 5hmC expression 24 h after 40 mJ/cm2 and 80 mJ/cm2 doses of ultraviolet B (UVB) irradiation to HaCaT cells. Methods: To explore whether aberrant DNA hydroxymethylation is also related to skin photoaging, 40 mJ/cm2 and 80 mJ/cm2 doses of UVB were chosen to treat keratinocytes (HaCaT cells). After 24 h of UVB irradiation, 5mC and 5hmC levels were determined by immunohistochemistry (IHC) and immunofluorescence (IF), and at the same time, the expression levels of matrix metalloproteinase 1 (MMP-1) and TETs were assessed by reverse transcription-polymerase chain reaction or Western blot analysis. Results: After 40 mJ/cm2 and 80 mJ/cm2 doses of UVB exposure, both IHC and IF results showed that 5hmC levels increased significantly, while the 5mC levels did not exhibit significant changes in HaCaT cells, compared with HaCat cells without UVB exposure. Moreover, compared with HaCat cells without UVB exposure, the levels of TET1, TET2, and TET3 mRNA and protein expression were significantly upregulated (mRNA: P = 0.0022 and 0.0043 for TET1; all P < 0.0001 for TET2; all P = 0.0006 for TET3; protein: P = 0.0012 and 0.0006 for TET1; all P = 0.0022 for TET2; and all P = 0.0002 for TET3), and the levels of MMP-1 mRNA expression increased dose dependently in 40 mJ/cm2 and 80 mJ/cm2 UVB-irradiated groups. Conclusion: UVB radiation could cause increased 5hmC and TET expression, which might become a novel biomarker in UVB

  14. Altered Expression of Polycomb Group Genes in Glioblastoma Multiforme

    PubMed Central

    Li, Gang; Warden, Charles; Zou, Zhaoxia; Neman, Josh; Krueger, Joseph S.; Jain, Alisha; Jandial, Rahul; Chen, Mike

    2013-01-01

    The Polycomb group (PcG) proteins play a critical role in histone mediated epigenetics which has been implicated in the malignant evolution of glioblastoma multiforme (GBM). By systematically interrogating The Cancer Genome Atlas (TCGA), we discovered widespread aberrant expression of the PcG members in GBM samples compared to normal brain. The most striking differences were upregulation of EZH2, PHF19, CBX8 and PHC2 and downregulation of CBX7, CBX6, EZH1 and RYBP. Interestingly, changes in EZH2, PHF19, CBX7, CBX6 and EZH1 occurred progressively as astrocytoma grade increased. We validated the aberrant expression of CBX6, CBX7, CBX8 and EZH2 in GBM cell lines by Western blotting and qRT-PCR, and further the aberrant expression of CBX6 in GBM tissue samples by immunohistochemical staining. To determine if there was functional significance to the diminished CBX6 levels in GBM, CBX6 was overexpressed in GBM cells resulting in decreased proliferative capacity. In conclusion, aberrant expression of PcG proteins in GBMs may play a role in the development or maintenance of the malignancy. PMID:24260522

  15. Altered expression of polycomb group genes in glioblastoma multiforme.

    PubMed

    Li, Gang; Warden, Charles; Zou, Zhaoxia; Neman, Josh; Krueger, Joseph S; Jain, Alisha; Jandial, Rahul; Chen, Mike

    2013-01-01

    The Polycomb group (PcG) proteins play a critical role in histone mediated epigenetics which has been implicated in the malignant evolution of glioblastoma multiforme (GBM). By systematically interrogating The Cancer Genome Atlas (TCGA), we discovered widespread aberrant expression of the PcG members in GBM samples compared to normal brain. The most striking differences were upregulation of EZH2, PHF19, CBX8 and PHC2 and downregulation of CBX7, CBX6, EZH1 and RYBP. Interestingly, changes in EZH2, PHF19, CBX7, CBX6 and EZH1 occurred progressively as astrocytoma grade increased. We validated the aberrant expression of CBX6, CBX7, CBX8 and EZH2 in GBM cell lines by Western blotting and qRT-PCR, and further the aberrant expression of CBX6 in GBM tissue samples by immunohistochemical staining. To determine if there was functional significance to the diminished CBX6 levels in GBM, CBX6 was overexpressed in GBM cells resulting in decreased proliferative capacity. In conclusion, aberrant expression of PcG proteins in GBMs may play a role in the development or maintenance of the malignancy.

  16. Increased expression of HOXB2 and HOXB13 proteins is associated with HPV infection and cervical cancer progression.

    PubMed

    Gonzalez-Herrera, Al; Salgado-Bernabe, M; Velazquez-Velazquez, Ck; Salcedo-Vargas, M; Andrade-Manzano, A; Avila-Moreno, F; Pina-Sanchez, P

    2015-01-01

    Cervical cancer (CeCa) is the second most common cancer in women in developing countries, and human papilloma virus (HPV) is the primary etiological factor. Aberrant expression of HOX transcription factors has been observed in several types of cancer. To date, however, no reports exist on the expression of HOXB2 and HOXB13 proteins during neoplastic progression in CeCa and its correlation with HPV infection. Expression of HOXB2 and HOXB13 proteins was assessed in tissue microarrays from normal cervical epithelium, cervical intraepithelial neoplasias grade 1-3, and CeCa. HPV was detected by PCR and sequencing. Expression of HOX-positive cells was determined in each diagnostic group. Percentage of HOXB2- and HOXB13-positive cells gradually increased from means of 10.9% and 16.7%, respectively, in samples from healthy women, to 75.2% and 88.6% in those from CeCa patients. Frequency of HPV infection also increased from 13% in healthy tissue samples to 92.3% in CeCa. Both HOXB2 and HOXB13 proteins were preferentially expressed in HPV+ samples. The present study represents the first report on the expression of both HOXB2 and HOXB13 proteins through cervix tumorigenesis, providing evidence that increased expression of such proteins is a common event during progression to CeCa.

  17. Aberrant proteins featured in the saliva of habitual betel quid chewers: an indication of early oral premalignancy?

    PubMed

    Jessie, Kala; Jayapalan, Jaime Jacqueline; Rahim, Zubaidah Haji Abdul; Hashim, Onn Haji

    2014-12-01

    Prolonged chewing of betel quid is known to cause oral diseases, including cancer. The present study was performed to screen for aberrant proteins in the saliva of habitual betel quid chewers compared to nonchewers. Saliva of female subjects (n = 10) who had been chewing betel quid for more than 20 years and nonbetel quid chewers (n = 10) of the same gender and range of age was analyzed by gel-based proteomics. Increased structural microheterogeneity of saliva haptoglobin beta chains indicated by shifts of focused spots similar to that earlier reported in patients with oral squamous cell carcinoma, and their relatively higher abundance compared to nonbetel quid chewers, were detected in saliva protein profiles of all chewers. In addition, the majority of the betel quid chewers also showed significant higher abundance of hemopexin, alpha-1B glycoprotein, alpha1-antitrypsin, complement C3, and transthyretin. These proteins had previously been associated with several different cancers. Our data demonstrated different forms of protein aberration in the saliva of betel quid chewers, which may be indicative of early oral precancerous conditions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2) is a novel biomarker of myxofibrosarcoma invasion identified by global protein expression profiling.

    PubMed

    Kikuta, Kazutaka; Kubota, Daisuke; Yoshida, Akihiko; Qiao, Zhiwei; Morioka, Hideo; Nakamura, Masaya; Matsumoto, Morio; Chuman, Hirokazu; Kawai, Akira; Kondo, Tadashi

    2017-09-01

    Myxofibrosarcoma (MFS) is a mesenchymal malignancy characterized by frequent recurrence even after radical wide resection. To optimize therapy for MFS patients, we aimed to identify candidate tissue biomarkers of MFS invasion potential. Invasion characteristics of MFS were evaluated by magnetic resonance imaging and protein expression profiling of primary tumor tissues performed using two-dimensional difference gel electrophoresis (2D-DIGE). Protein expression profiles were compared between invasive and non-invasive tumors surgically resected from 11 patients. Among the 3453 protein spots observed, 59 demonstrated statistically significant difference in intensity (≥2-fold) between invasive and non-invasive tumors (p<0.01 by Wilkoxon test), and were identified by mass spectrometry as 47 individual proteins. Among them, we further focused on discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2), a receptor tyrosine kinase with aberrant expression in malignant tumors. Immunohistochemistry analysis of 21 additional MFS cases revealed that higher DCBLD2 expression was significantly associated with invasive properties of tumor cells. DCBLD2 sensitivity and specificity, and positive and negative predictive values for MFS invasion were 69.2%, 87.5%, 90%, and 63.6%, respectively. The expression level of DCBLD2 was consistent in different portions of tumor tissues. Thus, DCBLD2 expression can be a useful biomarker to evaluate invasive properties of MFS. Further validation studies based on multi-institutional collaboration and comprehensive analysis of DCBLD2 biological functions in MFS are required to confirm its prognostic utility for clinical application. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Aberrant expression of epithelial leucine-rich repeat containing G protein-coupled receptor 5-positive cells in the eutopic endometrium in endometriosis and implications in deep-infiltrating endometriosis.

    PubMed

    Vallvé-Juanico, Júlia; Suárez-Salvador, Elena; Castellví, Josep; Ballesteros, Agustín; Taylor, Hugh S; Gil-Moreno, Antonio; Santamaria, Xavier

    2017-11-01

    To characterize leucine-rich repeat containing G protein-coupled receptor 5-positive (LGR5 + ) cells from the endometrium of women with endometriosis. Prospective experimental study. University hospital/fertility clinic. Twenty-seven women with endometriosis who underwent surgery and 12 healthy egg donors, together comprising 39 endometrial samples. Obtaining of uterine aspirates by using a Cornier Pipelle. Immunofluorescence in formalin-fixed paraffin-embedded tissue from mice and healthy and pathologic human endometrium using antibodies against LGR5, E-cadherin, and cytokeratin, and epithelial and stromal LGR5 + cells isolated from healthy and pathologic human eutopic endometrium by fluorescence-activated cell sorting and transcriptomic characterization by RNA high sequencing. Immunofluorescence showed that LGR5 + cells colocalized with epithelial markers in the stroma of the endometrium only in endometriotic patients. The results from RNA high sequencing of LGR5 + cells from epithelium and stroma did not show any statistically significant differences between them. The LGR5 + versus LGR5 - cells in pathologic endometrium showed 394 differentially expressed genes. The LGR5 + cells in deep-infiltrating endometriosis expressed inflammatory markers not present in the other types of the disease. Our results revealed the presence of aberrantly located LGR5 + cells coexpressing epithelial markers in the stromal compartment of women with endometriosis. These cells have a statistically significantly different expression profile in deep-infiltrating endometriosis in comparison with other types of endometriosis, independent of the menstrual cycle phase. Further studies are needed to elucidate their role and influence in reproductive outcomes. Copyright © 2017. Published by Elsevier Inc.

  20. Placental expression of myostatin and follistatin-like-3 protein in a model of developmental programming.

    PubMed

    Peiris, Hassendrini N; Ponnampalam, Anna P; Osepchook, Claire C; Mitchell, Murray D; Green, Mark P

    2010-04-01

    Maternal undernutrition during gestation is known to be detrimental to fetal development, leading to a propensity for metabolic disorders later in the adult lives of the offspring. Identifying possible mediators and physiological processes involved in modulating nutrient transport within the placenta is essential to prevent and/or develop treatments for the effects of aberrant nutrition, nutrient transfer, and detrimental changes to fetal development. A potential role for myostatin as a mediator of nutrient uptake and transport from the mother to the fetus was shown through the recent finding that myostatin acts within the human placenta to modulate glucose uptake and therefore homeostasis. The mRNA and protein expression of myostatin and its inhibitor, follistatin-like-3 (FSTL3), was studied in the placenta and skeletal muscle of a transgenerational Wistar rat model of gestational maternal undernutrition in which the F2 offspring postweaning consumed a high-fat (HF) diet. Alterations in placental characteristics and offspring phenotype, specifically glucose homeostasis, were evident in the transgenerationally undernourished (UNAD) group. Myostatin and FSTL3 protein expression were also higher (P < 0.05) in the placentae of the UNAD compared with the control group. At maturity, UNAD HF-fed animals had higher (P < 0.05) skeletal muscle expression of FSTL3 than control animals. In summary, maternal undernutrition during gestation results in the aberrant regulation of myostatin and FSTL3 in the placenta and skeletal muscle of subsequent generations. Myostatin, through the disruption of maternal nutrient supply to the fetus, may thus be a potential mediator of offspring phenotype.

  1. G Protein-Coupled Estrogen Receptor (GPER) Expression in Normal and Abnormal Endometrium

    PubMed Central

    Lessey, Bruce A.; Taylor, Robert N.; Wang, Wei; Bagchi, Milan K.; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A.; Young, Steven L.

    2012-01-01

    Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen’s importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis. PMID:22378861

  2. G protein-coupled estrogen receptor (GPER) expression in normal and abnormal endometrium.

    PubMed

    Plante, Beth J; Lessey, Bruce A; Taylor, Robert N; Wang, Wei; Bagchi, Milan K; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A; Young, Steven L

    2012-07-01

    Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen's importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis.

  3. ALK amplification and protein expression predict inferior prognosis in neuroblastomas.

    PubMed

    Wang, Miao; Zhou, Chunju; Sun, Qinnuan; Cai, Rongqin; Li, Yong; Wang, Daye; Gong, Liping

    2013-10-01

    ALK gene has been identified as a major neuroblastoma (NBL) predisposition gene. But ALK gene copy number and protein expression in ganglioneuroblastoma (GNBL) and ganglioneuroma (GN) are poorly described in the literature. Furthermore, there are controversies on the correlation between ALK protein expression and clinical outcome in NBL. We evaluated MYCN/ALK gene copy number by fluorescence in situ hybridization (FISH) and detected ALK protein expression by immunohistochemistry (IHC) in 188 NBL, 52 GNBL and 6 GN samples and analyzed their association with clinical outcome of the patients. Although ALK gene copy number increase is a recurrent genetic aberration of neuroblastic tumors (NTs) (39.1%, 96/246), ALK amplification was only present in three NBLs (1.2%, 3/246). The frequency of ALK positivity in NBL (50.5%, 51/101) was significantly higher than in GNBL (22.6%, 7/31) and in GN (0.0%, 0/4) (P<0.05). In addition, ALK positivity also significantly correlates with MYCN/ALK gene copy number increases (P<0.05). Kaplan-Meier survival analysis indicated that MYCN/ALK amplification is correlated with decreased overall survival in NBL. A better prognosis trend was observed in patients with MYCN/ALK gain tumors compared with those with MYCN/ALK normal tumors. Furthermore, ALK positivity significantly correlated with inferior survival in NBL (P=0.044). ALK positivity in NTs correlated with advanced tumor types and MYCN/ALK gene copy number increases. ALK positivity predicts inferior prognosis in NBL and IHC is a simplified strategy to screen ALK positivity in clinical practice. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Connexin 26 correlates with Bcl-xL and Bax proteins expression in colorectal cancer

    PubMed Central

    Kanczuga-Koda, Luiza; Sulkowski, Stanislaw; Koda, Mariusz; Skrzydlewska, Elzbieta; Sulkowska, Mariola

    2005-01-01

    AIM: To evaluate of Cx26 in correlation with Bcl-xL and Bax proteins in colorectal cancer. METHODS: Immunohistochemical staining using specific antibodies was performed to evaluate the protein expression of Cx26, Bax and Bcl-xL in 152 colorectal cancer samples and the correlations among studied proteins as well as the relationships between the expression of Cx26, Bax, Bcl-xL and clinicopathological features were analyzed. RESULTS: Both normal epithelial cells and carcinoma cells expressed Cx26, Bax and Bcl-xL, but Cx26 in cancer cells showed aberrant, mainly cytoplasmic staining. Expression of Cx26, Bax and Bcl-xL was observed in 55.9%, 55.5% and 72.4% of evaluated colorectal cancers respectively. We found the positive correlation between Cx26 and Bax expression (r = 0.561, P<0.0001), Cx26 and Bcl-xL (r = 0.409, P<0.0001) as well as between Bax and Bcl-xL (r = 0.486, P<0.0001). Association of Cx26, Bax and Bcl-xL expression with histological G2 grade of tumors was noted (P<0.005, P<0.001 and P<0.002 respectively). CONCLUSION: Cytoplasmic presence of Cx26 and its association with apoptotic markers could indicate a distinct role from physiological functions of Cx26 in cancer cells and it could suggest that connexins might be a target point for modulations of apoptosis with therapeutic implications. PMID:15770735

  5. Expression of aberrant CD markers in acute leukemia: a study of 100 cases with immunophenotyping by multiparameter flowcytometry.

    PubMed

    Sarma, Anupam; Hazarika, Munlima; Das, Debabrata; Kumar Rai, Avdhesh; Sharma, Jagannath Dev; Bhuyan, Chidananda; Kataki, Amal Chandra

    2015-01-01

    Acute leukemia is a heterogenous disease having diverse phenotypes. Immunophenotyping by flowcytometry is essential for diagnosis of myeloid and lymphoid subtypes. Aberrant phenotype incidence is controversial and dissimilar results have been reported by different groups. Purpose of the study was to determine the incidence of aberrant phenotypes in North East Indian patients with acute leukemia. We analysed a total of 100 cases (AML = 36, ALL = 61, MPAL = 3) by multiparametric flow cytometry using an acute panel of monoclonal antibodies (MoAbs). The MoAbs were selected to identify differentiation-associated antigens of both myeloid and lymphoid lineages. Aberrant phenotypes were found in 21 (58.3%) cases of AML, 36 (59.2%) cases of B-ALL and 6 (66.7%) cases of T-ALL. CD7 was the most frequent lymphoid associated antigen found in 33% of AML cases while CD117 was the myeloid antigen most frequently detected in ALL (54%) cases. Aberrant expression of CD 117 is highly significant by Fischer's exact test (P< 0.0001). We conclude that aberrant phenotypes are present in a great majority of acute leukemia patients of North East India. Future studies will be directed to correlate of these markers with prognosis and therapeutic response.

  6. Aberrant expression and hormonal regulation of Galectin-3 in endometriosis women with infertility.

    PubMed

    Yang, H; Yin, J; Ficarrotta, K; Hsu, S H; Zhang, W; Cheng, C

    2016-07-01

    To investigate the role and potential molecular mechanism of Galectin-3 (Gal-3) in the etiology of endometriosis-associated infertility. We detected Gal-3 expression in eutopic endometrium from women with endometriosis-associated infertility and healthy women without endometriosis or infertility. We then evaluated Gal-3 expression in endometrial glandular epithelial cells (EECs) and endometrial stromal cells (ESCs) and investigated its response to hormone stimulation in EECs and ESCs from both groups of women. Results of real-time PCR and western blot analysis showed Gal-3 expression in both proliferative and secretory stages of the menstrual cycle decreased significantly in women with endometriosis-associated infertility compared to healthy women. The changes in expression of Gal-3 were more dramatic in EECs than ESCs. Moreover, estrogen (E2) and progesterone (P4) induced Gal-3 expression in EECs of healthy groups, and P4 was more significant than E2 and combined E2 and P4 (E2P4). However, in the endometriosis group, P4 failed to induce a similar increase in Gal-3 expression. Our results suggest that aberrant expression of Gal-3 might contribute to infertility in patients with endometriosis due to progesterone resistance.

  7. From General Aberrant Alternative Splicing in Cancers and Its Therapeutic Application to the Discovery of an Oncogenic DMTF1 Isoform

    PubMed Central

    Tian, Na; Li, Jialiang; Shi, Jinming; Sui, Guangchao

    2017-01-01

    Alternative pre-mRNA splicing is a crucial process that allows the generation of diversified RNA and protein products from a multi-exon gene. In tumor cells, this mechanism can facilitate cancer development and progression through both creating oncogenic isoforms and reducing the expression of normal or controllable protein species. We recently demonstrated that an alternative cyclin D-binding myb-like transcription factor 1 (DMTF1) pre-mRNA splicing isoform, DMTF1β, is increasingly expressed in breast cancer and promotes mammary tumorigenesis in a transgenic mouse model. Aberrant pre-mRNA splicing is a typical event occurring for many cancer-related functional proteins. In this review, we introduce general aberrant pre-mRNA splicing in cancers and discuss its therapeutic application using our recent discovery of the oncogenic DMTF1 isoform as an example. We also summarize new insights in designing novel targeting strategies of cancer therapies based on the understanding of deregulated pre-mRNA splicing mechanisms. PMID:28257090

  8. Heritable Transmission of Diabetic Metabolic Memory in Zebrafish Correlates With DNA Hypomethylation and Aberrant Gene Expression

    PubMed Central

    Olsen, Ansgar S.; Sarras, Michael P.; Leontovich, Alexey; Intine, Robert V.

    2012-01-01

    Metabolic memory (MM) is the phenomenon whereby diabetes complications persist and progress after glycemic recovery is achieved. Here, we present data showing that MM is heritable and that the transmission correlates with hyperglycemia-induced DNA hypomethylation and aberrant gene expression. Streptozocin was used to induce hyperglycemia in adult zebrafish, and then, following streptozocin withdrawal, a recovery phase was allowed to reestablish a euglycemic state. Blood glucose and serum insulin returned to physiological levels during the first 2 weeks of the recovery phase as a result of pancreatic β-cell regeneration. In contrast, caudal fin regeneration and skin wound healing remained impaired to the same extent as in diabetic fish, and this impairment was transmissible to daughter cell tissue. Daughter tissue that was never exposed to hyperglycemia, but was derived from tissue that was, did not accumulate AGEs or exhibit increased levels of oxidative stress. However, CpG island methylation and genome-wide microarray expression analyses revealed the persistence of hyperglycemia-induced global DNA hypomethylation that correlated with aberrant gene expression for a subset of loci in this daughter tissue. Collectively, the data presented here implicate the epigenetic mechanism of DNA methylation as a potential contributor to the MM phenomenon. PMID:22228713

  9. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales

    PubMed Central

    Margres, Mark J.; Wray, Kenneth P.; Seavy, Margaret; McGivern, James J.; Herrera, Nathanael D.; Rokyta, Darin R.

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our

  10. Increased expression of argininosuccinate synthetase protein predicts poor prognosis in human gastric cancer

    PubMed Central

    SHAN, YAN-SHEN; HSU, HUI-PING; LAI, MING-DERG; YEN, MENG-CHI; LUO, YI-PEY; CHEN, YI-LING

    2015-01-01

    Aberrant expression of argininosuccinate synthetase (ASS1, also known as ASS) has been found in cancer cells and is involved in the carcinogenesis of gastric cancer. The aim of the present study was to investigate the level of ASS expression in human gastric cancer and to determine the possible correlations between ASS expression and clinicopathological findings. Immunohistochemistry was performed on paraffin-embedded tissues to determine whether ASS was expressed in 11 of 11 specimens from patients with gastric cancer. The protein was localized primarily to the cytoplasm of cancer cells and normal epithelium. In the Oncomine cancer microarray database, expression of the ASS gene was significantly increased in gastric cancer tissues. To investigate the clinicopathological and prognostic roles of ASS expression, we performed western blot analysis of 35 matched specimens of gastric adenocarcinomas and normal tissue obtained from patients treated at the National Cheng Kung University Hospital. The ratio of relative ASS expression (expressed as the ASS/β-actin ratio) in tumor tissues to that in normal tissues was correlated with large tumor size (P=0.007) and with the tumor, node, metastasis (TNM) stage of the American Joint Committee on Cancer staging system (P=0.031). Patients whose cancer had increased the relative expression of ASS were positive for perineural invasion and had poor recurrence-free survival. In summary, ASS expression in gastric cancer was associated with a poor prognosis. Further study of mechanisms to silence the ASS gene or decrease the enzymatic activity of ASS protein has the potential to provide new treatments for patients with gastric cancer. PMID:25333458

  11. Aberrant activation of M phase proteins by cell proliferation-evoking carcinogens after 28-day administration in rats.

    PubMed

    Yafune, Atsunori; Taniai, Eriko; Morita, Reiko; Hayashi, Hitomi; Suzuki, Kazuhiko; Mitsumori, Kunitoshi; Shibutani, Makoto

    2013-06-07

    We have previously reported that hepatocarcinogens increase liver cells expressing p21(Cip1), a G1 checkpoint protein and M phase proteins after 28-day treatment in rats. This study aimed to identify early prediction markers of carcinogens available in many target organs after 28-day treatment in rats. Immunohistochemical analysis was performed on Ki-67, p21(Cip1) and M phase proteins [nuclear Cdc2, phospho-Histone H3 (p-Histone H3), Aurora B and heterochromatin protein 1α (HP1α)] with carcinogens targeting different organs. Carcinogens targeting thyroid (sulfadimethoxine; SDM), urinary bladder (phenylethyl isothiocyanate), forestomach (butylated hydroxyanisole; BHA), glandular stomach (catechol; CC), and colon (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and chenodeoxycholic acid) were examined using a non-carcinogenic toxicant (caprolactam) and carcinogens targeting other organs as negative controls. All carcinogens increased Ki-67(+), nuclear Cdc2(+), p-Histone H3(+) or Aurora B(+) carcinogenic target cells, except for both colon carcinogens, which did not increase cell proliferation. On the other hand, p21(Cip1+) cells increased with SDM and CC. HP1α responded only to BHA. Results revealed carcinogens evoking cell proliferation concurrently induced cell cycle arrest at M phase or showing chromosomal instability reflecting aberration in cell cycle regulation, irrespective of target organs, after 28-day treatment. Therefore, M phase proteins may be early prediction markers of carcinogens evoking cell proliferation in many target organs. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. Acute myeloid leukaemia: expression of MYC protein and its association with cytogenetic risk profile and overall survival.

    PubMed

    Mughal, Muhammad Kashif; Akhter, Ariz; Street, Lesley; Pournazari, Payam; Shabani-Rad, Meer-Taher; Mansoor, Adnan

    2017-09-01

    Acute myeloid leukaemia (AML) is a clinically aggressive disease with marked genetic heterogeneity. Cytogenetic abnormalities provide the basis for risk stratification into clinically favourable, intermediate, and unfavourable groups. There are additional genetic mutations, which further influence the prognosis of patients with AML. Most of these result in molecular aberrations whose downstream target is MYC. It is therefore logical to study the relationship between MYC protein expression and cytogenetic risk groups. We studied MYC expression by immunohistochemistry in a large cohort (n = 199) of AML patients and correlated these results with cytogenetic risk profile and overall survival (OS). We illustrated differential expression of MYC protein across various cytogenetic risk groups (p = 0.03). Highest expression of MYC was noted in AML patients with favourable cytogenetic risk group. In univariate analysis, MYC expression showed significant negative influence of OS in favourable and intermediate cytogenetic risk group (p = 0.001). Interestingly, MYC expression had a protective effect in the unfavourable cytogenetic risk group. In multivariate analysis, while age and cytogenetic risk group were significant factors influencing survival, MYC expression by immunohistochemistry methods also showed some marginal impact (p = 0.069). In conclusion, we have identified differential expression of MYC protein in relation to cytogenetic risk groups in AML patients and documented its possible impact on OS in favourable and intermediate cytogenetic risk groups. These preliminary observations mandate additional studies to further investigate the routine clinical use of MYC protein expression in AML risk stratification. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  13. A comparative study of cell cycle mediator protein expression patterns in anaplastic and papillary thyroid carcinoma.

    PubMed

    Evans, Juanita J; Crist, Henry S; Durvesh, Saima; Bruggeman, Richard D; Goldenberg, David

    2012-07-01

    Anaplastic thyroid carcinoma (ATC) is an extremely aggressive and rapidly fatal neoplasm. The aim of this study was to identify a limited cell cycle associated protein expression pattern unique to ATC and to correlate that pattern with clinical outcome. This represents one of the largest tissue micro-array projects comparing the cell cycle protein expression data of ATC to other well-differentiated tumors in the literature. Tissue microarrays were created from 21 patients with ATC and an age and gender matched cohort of patients with papillary thyroid carcinoma (PTC). Expression of epidermal growth factor receptor, cyclin D1, cyclin E, p53, p21, p16, aurora kinase A, opioid growth factor (OGF), OGF-receptor, thyroglobulin and Ki-67 was evaluated in a semi-quantitative fashion. Differences in protein expression between the cohorts were evaluated using chi-square tests with Bonferroni adjustments. Survival time and presence of metastasis at presentation were collected. The ATC cohort showed a statistically significant decrease (p < 0.05) in thyroglobulin expression and statistically significant increases (p < 0.05) in Ki-67 and p53 expression as compared with the PTC cohort. A trend toward loss of p16 and p21 expression was noted in the ATC cohort. A trend toward decreased survival was noted with p21 expression. These data indicate disruption of the normal cell cycle with aberrant expression of multiple protein markers suggesting increased proliferative activity and loss of control of cell cycle progression to G₁ phase. These findings support the assertion that ATC may represent the furthest end of a continuum of thyroid carcinoma dedifferentiation.

  14. Aberrant ligand-induced activation of G protein-coupled estrogen receptor 1 (GPER) results in developmental malformations during vertebrate embryogenesis.

    PubMed

    Jayasinghe, B Sumith; Volz, David C

    2012-01-01

    G protein-coupled estrogen receptor 1 (GPER) is a G protein-coupled receptor (GPCR) unrelated to nuclear estrogen receptors but strongly activated by 17β-estradiol in both mammals and fish. To date, the distribution and functional characterization of GPER within reproductive and nonreproductive vertebrate organs have been restricted to juvenile and adult animals. In contrast, virtually nothing is known about the spatiotemporal distribution and function of GPER during vertebrate embryogenesis. Using zebrafish as an animal model, we investigated the potential functional role and expression of GPER during embryogenesis. Based on real-time PCR and whole-mount in situ hybridization, gper was expressed as early as 1 h postfertilization (hpf) and exhibited strong stage-dependent expression patterns during embryogenesis. At 26 and 38 hpf, gper mRNA was broadly distributed throughout the body, whereas from 50 to 98 hpf, gper expression was increasingly localized to the heart, brain, neuromasts, craniofacial region, and somite boundaries of developing zebrafish. Continuous exposure to a selective GPER agonist (G-1)-but not continuous exposure to a selective GPER antagonist (G-15)-from 5 to 96 hpf, or within three developmental windows ranging from 10 to 72 hpf, resulted in adverse concentration-dependent effects on survival, gross morphology, and somite formation within the trunk of developing zebrafish embryos. Importantly, based on co-exposure studies, G-15 blocked severe G-1-induced developmental toxicity, suggesting that G-1 toxicity is mediated via aberrant activation of GPER. Overall, our findings suggest that xenobiotic-induced GPER activation represents a potentially novel and understudied mechanism of toxicity for environmentally relevant chemicals that affect vertebrate embryogenesis.

  15. Identification of aberrantly expressed long non-coding RNAs in stomach adenocarcinoma.

    PubMed

    Gu, Jianbin; Li, Yong; Fan, Liqiao; Zhao, Qun; Tan, Bibo; Hua, Kelei; Wu, Guobin

    2017-07-25

    Stomach adenocarcinoma (STAD) is a common malignancy worldwide. This study aimed to identify the aberrantly expressed long non-coding RNAs (lncRNAs) in STAD. Total of 74 DElncRNAs and 449 DEmRNAs were identified in STAD compared with paired non-tumor tissues. The DElncRNA/DEmRNA co-expression network was constructed, which covered 519 nodes and 2993 edges. The qRT-PCR validation results of DElncRNAs were consistent with our bioinformatics analysis based on RNA-sequencing. The DEmRNAs co-expressed with DElncRNAs were significantly enriched in gastric acid secretion, complement and coagulation cascades, pancreatic secretion, cytokine-cytokine receptor interaction and Jak-STAT signaling pathway. The expression levels of the nine candidate DElncRNAs in TCGA database were compatible with our RNA-sequencing. FEZF1-AS1, HOTAIR and LINC01234 had the potential diagnosis value for STAD. The lncRNA and mRNA expression profile of 3 STAD tissues and 3 matched adjacent non-tumor tissues was obtained through high-throughput RNA-sequencing. Differentially expressed lncRNAs/mRNAs (DElncRNAs/DEmRNAs) were identified in STAD. DElncRNA/DEmRNA co-expression network construction, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predict the biological functions of DElncRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was subjected to validate the expression levels of DEmRNAs and DElncRNAs. Moreover, the expression of DElncRNAs was validated through The Cancer Genome Atlas (TCGA) database. The diagnosis value of candidate DElncRNAs was accessed by receiver operating characteristic (ROC) analysis. Our work might provide useful information for exploring the tumorigenesis mechanism of STAD and pave the road for identification of diagnostic biomarkers in STAD.

  16. Expression of three topologically distinct membrane proteins elicits unique stress response pathways in the yeast Saccharomyces cerevisiae.

    PubMed

    Buck, Teresa M; Jordan, Rick; Lyons-Weiler, James; Adelman, Joshua L; Needham, Patrick G; Kleyman, Thomas R; Brodsky, Jeffrey L

    2015-06-01

    Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The α-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.1, which vary in their mass, membrane topologies, and quaternary structures. To examine transcriptional profiles in a null background, the proteins were expressed in yeast, which was previously used to examine the degradation requirements for each substrate. Surprisingly, the proteins failed to induce a canonical unfolded protein response or heat shock response, although messages encoding several cytosolic and ER lumenal protein folding factors rose when αENaC or CFTR was expressed. In contrast, the levels of these genes were unaltered by Kir2.1 expression; instead, the yeast iron regulon was activated. Nevertheless, a significant number of genes that respond to various environmental stressors were upregulated by all three substrates, and compared with previous microarray data we deduced the existence of a group of genes that reflect a novel misfolded membrane protein response. These data indicate that aberrant proteins in the ER elicit profound yet unique cellular responses. Copyright © 2015 the American Physiological Society.

  17. Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

    PubMed Central

    Hewitt, Stephen N.; Choi, Ryan; Kelley, Angela; Crowther, Gregory J.; Napuli, Alberto J.; Van Voorhis, Wesley C.

    2011-01-01

    Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies. PMID:21904041

  18. Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC).

    PubMed

    Niere, Farr; Namjoshi, Sanjeev; Song, Ehwang; Dilly, Geoffrey A; Schoenhard, Grant; Zemelman, Boris V; Mechref, Yehia; Raab-Graham, Kimberly F

    2016-02-01

    Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder-neurological disorders that exhibit elevated mTORC1 activity. Through a protein-protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes

  19. Aberrant c-erbB2 expression in cell clusters overlying focally disrupted breast myoepithelial cell layers: a trigger or sign for emergence of more aggressive cell clones?

    PubMed Central

    Zhang, Xichen; Hashemi, Shahreyar Shar; Yousefi, Morvarid; Ni, Jinsong; Wang, Qiuyue; Gao, Ling; Gong, Pengtao; Gao, Chunling; Sheng, Joy; Mason, Jeffrey; Man, Yan-gao

    2008-01-01

    Our recent studies revealed that cell clusters overlying focal myoepithelial cell layer disruption (FMCLD) had a significantly higher frequency of genetic instabilities and expression of invasion-related genes than their adjacent counterparts within the same duct. Our current study attempted to assess whether these cell clusters would also have elevated c-erbB2 expression. Human breast tumors (n=50) with a high frequency of FMCLD were analyzed with double immunohistochemistry, real-time RT-PCR, and chromogenic in situ hybridization for c-erbB2 protein and gene expression. Of 448 FMCLD detected, 404 (90.2%) were associated with cell clusters that had intense c-erbB2 immunoreactivities primarily in their cytoplasm, in contrast to their adjacent counterparts within the same duct, which had no or barely detectable c-erbB2 expression. These c-erbB2 positive cells were arranged as tongue-like projections, “puncturing” into the stroma, and about 20% of them were in direct continuity with tube-like structures that resembled blood vessels. Aberrant c-erbB2 expression was also seen in clusters of architecturally normal-appearing ducts that had distinct cytological abnormalities in both ME and epithelial cells, whereas not in their clear-cut normal counterparts. Molecular assays detected markedly higher c-erbB2 mRNA and gene amplification in cell clusters associated with FMCLD than in those associated with non-disrupted ME cell layers. Our findings suggest that cell clusters overlying FMCLD may represent the precursors of pending invasive lesions, and that aberrant cerbB2 expression may trigger or signify the emergence of biologically more aggressive cell clones. PMID:18726004

  20. Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)*

    PubMed Central

    Niere, Farr; Namjoshi, Sanjeev; Song, Ehwang; Dilly, Geoffrey A.; Schoenhard, Grant; Zemelman, Boris V.; Mechref, Yehia; Raab-Graham, Kimberly F.

    2016-01-01

    Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and

  1. Aberrant and alternative splicing in skeletal system disease.

    PubMed

    Fan, Xin; Tang, Liling

    2013-10-01

    The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. © 2013 Elsevier B.V. All rights reserved.

  2. Aberrant Expression of Xist in Aborted Porcine Fetuses Derived from Somatic Cell Nuclear Transfer Embryos

    PubMed Central

    Yuan, Lin; Wang, Anfeng; Yao, Chaogang; Huang, Yongye; Duan, Feifei; Lv, Qinyan; Wang, Dongxu; Ouyang, Hongsheng; Li, Zhanjun; Lai, Liangxue

    2014-01-01

    Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos. PMID:25429426

  3. MeCP2 deficiency in Rett syndrome causes epigenetic aberrations at the PWS/AS imprinting center that affects UBE3A expression.

    PubMed

    Makedonski, Kirill; Abuhatzira, Liron; Kaufman, Yotam; Razin, Aharon; Shemer, Ruth

    2005-04-15

    Rett syndrome (RS) is a severe and progressive neurodevelopmental disorder caused by heterozygous mutations in the X-linked methyl CpG binding protein 2 (MeCP2) gene. MeCP2 is a nuclear protein that binds specifically to methylated DNA and functions as a general transcription repressor in the context of chromatin remodeling complexes. RS shares clinical features with those of Angelman syndrome (AS), an imprinting neurodevelopmental disorder. In AS patients, the maternally expressed copy of UBE3A that codes for the ubiquitin protein ligase 3A (E6-AP) is repressed. The similar phenotype of these two syndromes led us to hypothesize that part of the RS phenotype is due to MeCP2-associated silencing of UBE3A. Indeed, UBE3A mRNA and protein are shown here to be significantly reduced in human and mouse MECP2 deficient brains. This reduced UBE3A level was associated with biallelic production of the UBE3A antisense RNA. In addition, MeCP2 deficiency resulted in elevated histone H3 acetylation and H3(K4) methylation and reduced H3(K9) methylation at the PWS/AS imprinting center, with no effect on DNA methylation or SNRPN expression. We conclude, therefore, that MeCP2 deficiency causes epigenetic aberrations at the PWS imprinting center. These changes in histone modifications result in loss of imprinting of the UBE3A antisense gene in the brain, increase in UBE3A antisense RNA level and, consequently reduction in UBE3A production.

  4. Expressing freedom and taking liberties: the paradoxes of aberrant science.

    PubMed

    Little, M

    2006-06-01

    Complete freedom does not exist, despite people's preparedness to die for it. Scientific freedom is much defended and yet much misunderstood. Scientists have limits imposed on their freedom by the disciplines and discourse communities in which they place themselves. Freedom within these socially constructed constraints needs to be distinguished from taking liberties with the rules and practices that make up these constraints, and validate the activities of special groups within society. Scientists (and the public) perceive taking liberties with science's rules and practices as aberrant science, and they often react punitively. Aberrant science can be broadly examined under four headings: wicked science, naughty science, dysfunctional science, and ideologically unacceptable science. When we examine examples of perceived aberrant science, we find that these categories of "misconduct" are connected and often confused. Scientific freedom needs to be redefined with due regard to current understandings of scientists as human beings facing powerful social pressures to deliver results of a particular kind.

  5. Nodal aberration theory for wild-filed asymmetric optical systems

    NASA Astrophysics Data System (ADS)

    Chen, Yang; Cheng, Xuemin; Hao, Qun

    2016-10-01

    Nodal Aberration Theory (NAT) was used to calculate the zero field position in Full Field Display (FFD) for the given aberration term. Aiming at wide-filed non-rotational symmetric decentered optical systems, we have presented the nodal geography behavior of the family of third-order and fifth-order aberrations. Meanwhile, we have calculated the wavefront aberration expressions when one optical element in the system is tilted, which was not at the entrance pupil. By using a three-piece-cellphone lens example in optical design software CodeV, the nodal geography is testified under several situations; and the wavefront aberrations are calculated when the optical element is tilted. The properties of the nodal aberrations are analyzed by using Fringe Zernike coefficients, which are directly related with the wavefront aberration terms and usually obtained by real ray trace and wavefront surface fitting.

  6. Phenotypic characterization of aberrant stem and progenitor cell populations in myelodysplastic syndromes.

    PubMed

    Ostendorf, Benjamin N; Flenner, Eva; Flörcken, Anne; Westermann, Jörg

    2018-01-01

    Recent reports have revealed myelodysplastic syndromes (MDS) to arise from cancer stem cells phenotypically similar to physiological hematopoietic stem cells. Myelodysplastic hematopoiesis maintains a hierarchical organization, but the proportion of several hematopoietic compartments is skewed and multiple surface markers are aberrantly expressed. These aberrant antigen expression patterns hold diagnostic and therapeutic promise. However, eradication of MDS requires targeting of early myelodysplasia propagating stem cells. This warrants an exact assessment of the differentiation stage at which aberrant expression occurs in transformed hematopoiesis. Here, we report results on the prospective and extensive dissection of the hematopoietic hierarchy in 20 patients with either low-risk MDS or MDS with excess blasts and compare it to hematopoiesis in patients with non-malignancy-associated cytopenia or B cell lymphoma without bone marrow infiltration. We found patients with MDS with excess blasts to exhibit characteristic expansions of specific immature progenitor compartments. We also identified the aberrant expression of several markers including ALDH, CLL-1, CD44, and CD47 to be specific features of hematopoiesis in MDS with excess blasts. We show that amongst these, aberrant CLL-1 expression manifested at the early uncommitted hematopoietic stem cell level, suggesting a potential role as a therapeutic target.

  7. Onco-GPCR signaling and dysregulated expression of microRNAs in human cancer.

    PubMed

    Nohata, Nijiro; Goto, Yusuke; Gutkind, J Silvio

    2017-01-01

    The G-protein-coupled receptor (GPCR) family is the largest family of cell-surface receptors involved in signal transduction. Aberrant expression of GPCRs and G proteins are frequently associated with prevalent human diseases, including cancer. In fact, GPCRs represent the therapeutic targets of more than a quarter of the clinical drugs currently on the market. MiRNAs (miRNAs) are also aberrantly expressed in many human cancers, and they have significant roles in the initiation, development and metastasis of human malignancies. Recent studies have revealed that dysregulation of miRNAs and their target genes expression are associated with cancer progression. The emerging information suggests that miRNAs play an important role in the fine tuning of many signaling pathways, including GPCR signaling. We summarize our current knowledge of the individual functions of miRNAs regulated by GPCRs and GPCR signaling-associated molecules, and miRNAs that regulate the expression and activity of GPCRs, their endogenous ligands and their coupled heterotrimeric G proteins in human cancer.

  8. Protein Translation in the Nucleus Accumbens Is Dysregulated during Cocaine Withdrawal and Required for Expression of Incubation of Cocaine Craving.

    PubMed

    Werner, Craig T; Stefanik, Michael T; Milovanovic, Mike; Caccamise, Aaron; Wolf, Marina E

    2018-03-14

    Exposure to drug-associated cues can induce drug craving and relapse in abstinent addicts. Cue-induced craving that progressively intensifies ("incubates") during withdrawal from cocaine has been observed in both rats and humans. Building on recent evidence that aberrant protein translation underlies incubation-related adaptations in the NAc, we used male rats to test the hypothesis that translation is dysregulated during cocaine withdrawal and/or when rats express incubated cocaine craving. We found that intra-NAc infusion of anisomycin, a general protein translation inhibitor, or rapamycin, an inhibitor of mammalian target of rapamycin, reduced the expression of incubated cocaine craving, consistent with previous results showing that inhibition of translation in slices normalized the adaptations that maintain incubation. We then examined signaling pathways involved in protein translation using NAc synaptoneurosomes prepared after >47 d of withdrawal from cocaine or saline self-administration, or after withdrawal plus a cue-induced seeking test. The most robust changes were observed following seeking tests. Most notably, we found that eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 2α (eIF2α) are dephosphorylated when cocaine rats undergo a cue-induced seeking test; both effects are consistent with increased translation during the test. Blocking eIF2α dephosphorylation and thereby restoring its inhibitory influence on translation, via intra-NAc injection of Sal003 just before the test, substantially reduced cocaine seeking. These results are consistent with dysregulation of protein translation in the NAc during cocaine withdrawal, enabling cocaine cues to elicit an aberrant increase in translation that is required for the expression of incubated cocaine craving. SIGNIFICANCE STATEMENT Cue-induced cocaine craving progressively intensifies (incubates) during withdrawal in both humans and rats. This may contribute to persistent vulnerability

  9. Aberrant chimeric RNA GOLM1-MAK10 encoding a secreted fusion protein as a molecular signature for human esophageal squamous cell carcinoma

    PubMed Central

    Zhang, Hao; Lin, Wan; Kannan, Kalpana; Luo, Liming; Li, Jing; Chao, Pei-Wen; Wang, Yan; Chen, Yu-Ping; Gu, Jiang; Yen, Laising

    2013-01-01

    It is increasingly recognized that chimeric RNAs may exert a novel layer of cellular complexity that contributes to oncogenesis and cancer progression, and could be utilized as molecular biomarkers and therapeutic targets. To date yet no fusion chimeric RNAs have been identified in esophageal cancer, the 6th most frequent cause of cancer death in the world. While analyzing the expression of 32 recurrent cancer chimeric RNAs in esophageal squamous cell carcinoma (ESCC) from patients and cancer cell lines, we identified GOLM1-MAK10, as a highly cancer-enriched chimeric RNA in ESCC. In situ hybridization revealed that the expression of the chimera is largely restricted to cancer cells in patient tumors, and nearly undetectable in non-neoplastic esophageal tissue from normal subjects. The aberrant chimera closely correlated with histologic differentiation and lymph node metastasis. Furthermore, we demonstrate that chimera GOLM1-MAK10 encodes a secreted fusion protein. Mechanistic studies reveal that GOLM1-MAK10 is likely derived from transcription read-through/splicing rather than being generated from a fusion gene. Collectively, these findings provide novel insights into the molecular mechanism involved in ESCC and provide a novel potential target for future therapies. The secreted fusion protein translated from GOLM1-MAK10 could also serve as a unique protein signature detectable by standard non-invasive assays. These observations are critical as there is no clinically useful molecular signature available for detecting this deadly disease or monitoring the treatment response. PMID:24243830

  10. Aberrant cognitive phenotypes and altered hippocampal BDNF expression related to epigenetic modifications in mice lacking the post-synaptic scaffolding protein SHANK1: Implications for autism spectrum disorder.

    PubMed

    Sungur, A Özge; Jochner, Magdalena C E; Harb, Hani; Kılıç, Ayşe; Garn, Holger; Schwarting, Rainer K W; Wöhr, Markus

    2017-08-01

    Autism spectrum disorder (ASD) is a class of neurodevelopmental disorders characterized by persistent deficits in social communication/interaction, together with restricted/repetitive patterns of behavior. ASD is among the most heritable neuropsychiatric conditions, and while available evidence points to a complex set of genetic factors, the SHANK gene family has emerged as one of the most promising candidates. Here, we assessed ASD-related phenotypes with particular emphasis on social behavior and cognition in Shank1 mouse mutants in comparison to heterozygous and wildtype littermate controls across development in both sexes. While social approach behavior was evident in all experimental conditions and social recognition was only mildly affected by genotype, Shank1 -/- null mutant mice were severely impaired in object recognition memory. This effect was particularly prominent in juveniles, not due to impairments in object discrimination, and replicated in independent mouse cohorts. At the neurobiological level, object recognition deficits were paralleled by increased brain-derived neurotrophic factor (BDNF) protein expression in the hippocampus of Shank1 -/- mice; yet BDNF levels did not differ under baseline conditions. We therefore investigated changes in the epigenetic regulation of hippocampal BDNF expression and detected an enrichment of histone H3 acetylation at the Bdnf promoter1 in Shank1 -/- mice, consistent with increased learning-associated BDNF. Together, our findings indicate that Shank1 deletions lead to an aberrant cognitive phenotype characterized by severe impairments in object recognition memory and increased hippocampal BDNF levels, possibly due to epigenetic modifications. This result supports the link between ASD and intellectual disability, and suggests epigenetic regulation as a potential therapeutic target. © 2017 Wiley Periodicals, Inc.

  11. Cooperative STAT/NF-κB signaling regulates lymphoma metabolic reprogramming and aberrant GOT2 expression.

    PubMed

    Feist, Maren; Schwarzfischer, Philipp; Heinrich, Paul; Sun, Xueni; Kemper, Judith; von Bonin, Frederike; Perez-Rubio, Paula; Taruttis, Franziska; Rehberg, Thorsten; Dettmer, Katja; Gronwald, Wolfram; Reinders, Jörg; Engelmann, Julia C; Dudek, Jan; Klapper, Wolfram; Trümper, Lorenz; Spang, Rainer; Oefner, Peter J; Kube, Dieter

    2018-04-17

    Knowledge of stromal factors that have a role in the transcriptional regulation of metabolic pathways aside from c-Myc is fundamental to improvements in lymphoma therapy. Using a MYC-inducible human B-cell line, we observed the cooperative activation of STAT3 and NF-κB by IL10 and CpG stimulation. We show that IL10 + CpG-mediated cell proliferation of MYC low cells depends on glutaminolysis. By 13 C- and 15 N-tracing of glutamine metabolism and metabolite rescue experiments, we demonstrate that GOT2 provides aspartate and nucleotides to cells with activated or aberrant Jak/STAT and NF-κB signaling. A model of GOT2 transcriptional regulation is proposed, in which the cooperative phosphorylation of STAT3 and direct joint binding of STAT3 and p65/NF-κB to the proximal GOT2 promoter are important. Furthermore, high aberrant GOT2 expression is prognostic in diffuse large B-cell lymphoma underscoring the current findings and importance of stromal factors in lymphoma biology.

  12. Correlations between corneal and total wavefront aberrations

    NASA Astrophysics Data System (ADS)

    Mrochen, Michael; Jankov, Mirko; Bueeler, Michael; Seiler, Theo

    2002-06-01

    Purpose: Corneal topography data expressed as corneal aberrations are frequently used to report corneal laser surgery results. However, the optical image quality at the retina depends on all optical elements of the eye such as the human lens. Thus, the aim of this study was to investigate the correlations between the corneal and total wavefront aberrations and to discuss the importance of corneal aberrations for representing corneal laser surgery results. Methods: Thirty three eyes of 22 myopic subjects were measured with a corneal topography system and a Tschernig-type wavefront analyzer after the pupils were dilated to at least 6 mm in diameter. All measurements were centered with respect to the line of sight. Corneal and total wavefront aberrations were calculated up to the 6th Zernike order in the same reference plane. Results: Statistically significant correlations (p < 0.05) between the corneal and total wavefront aberrations were found for the astigmatism (C3,C5) and all 3rd Zernike order coefficients such as coma (C7,C8). No statistically significant correlations were found for all 4th to 6th order Zernike coefficients except for the 5th order horizontal coma C18 (p equals 0.003). On average, all Zernike coefficients for the corneal aberrations were found to be larger compared to Zernike coefficients for the total wavefront aberrations. Conclusions: Corneal aberrations are only of limited use for representing the optical quality of the human eye after corneal laser surgery. This is due to the lack of correlation between corneal and total wavefront aberrations in most of the higher order aberrations. Besides this, the data present in this study yield towards an aberration balancing between corneal aberrations and the optical elements within the eye that reduces the aberration from the cornea by a certain degree. Consequently, ideal customized ablations have to take both, corneal and total wavefront aberrations, into consideration.

  13. Machine Learning-based Classification of Diffuse Large B-cell Lymphoma Patients by Their Protein Expression Profiles.

    PubMed

    Deeb, Sally J; Tyanova, Stefka; Hummel, Michael; Schmidt-Supprian, Marc; Cox, Juergen; Mann, Matthias

    2015-11-01

    Characterization of tumors at the molecular level has improved our knowledge of cancer causation and progression. Proteomic analysis of their signaling pathways promises to enhance our understanding of cancer aberrations at the functional level, but this requires accurate and robust tools. Here, we develop a state of the art quantitative mass spectrometric pipeline to characterize formalin-fixed paraffin-embedded tissues of patients with closely related subtypes of diffuse large B-cell lymphoma. We combined a super-SILAC approach with label-free quantification (hybrid LFQ) to address situations where the protein is absent in the super-SILAC standard but present in the patient samples. Shotgun proteomic analysis on a quadrupole Orbitrap quantified almost 9,000 tumor proteins in 20 patients. The quantitative accuracy of our approach allowed the segregation of diffuse large B-cell lymphoma patients according to their cell of origin using both their global protein expression patterns and the 55-protein signature obtained previously from patient-derived cell lines (Deeb, S. J., D'Souza, R. C., Cox, J., Schmidt-Supprian, M., and Mann, M. (2012) Mol. Cell. Proteomics 11, 77-89). Expression levels of individual segregation-driving proteins as well as categories such as extracellular matrix proteins behaved consistently with known trends between the subtypes. We used machine learning (support vector machines) to extract candidate proteins with the highest segregating power. A panel of four proteins (PALD1, MME, TNFAIP8, and TBC1D4) is predicted to classify patients with low error rates. Highly ranked proteins from the support vector analysis revealed differential expression of core signaling molecules between the subtypes, elucidating aspects of their pathobiology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Method for Expressing Clinical and Statistical Significance of Ocular and Corneal Wavefront Error Aberrations

    PubMed Central

    Smolek, Michael K.

    2011-01-01

    Purpose The significance of ocular or corneal aberrations may be subject to misinterpretation whenever eyes with different pupil sizes or the application of different Zernike expansion orders are compared. A method is shown that uses simple mathematical interpolation techniques based on normal data to rapidly determine the clinical significance of aberrations, without concern for pupil and expansion order. Methods Corneal topography (Tomey, Inc.; Nagoya, Japan) from 30 normal corneas was collected and the corneal wavefront error analyzed by Zernike polynomial decomposition into specific aberration types for pupil diameters of 3, 5, 7, and 10 mm and Zernike expansion orders of 6, 8, 10 and 12. Using this 4×4 matrix of pupil sizes and fitting orders, best-fitting 3-dimensional functions were determined for the mean and standard deviation of the RMS error for specific aberrations. The functions were encoded into software to determine the significance of data acquired from non-normal cases. Results The best-fitting functions for 6 types of aberrations were determined: defocus, astigmatism, prism, coma, spherical aberration, and all higher-order aberrations. A clinical screening method of color-coding the significance of aberrations in normal, postoperative LASIK, and keratoconus cases having different pupil sizes and different expansion orders is demonstrated. Conclusions A method to calibrate wavefront aberrometry devices by using a standard sample of normal cases was devised. This method could be potentially useful in clinical studies involving patients with uncontrolled pupil sizes or in studies that compare data from aberrometers that use different Zernike fitting-order algorithms. PMID:22157570

  15. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

    PubMed

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

    2016-05-12

    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations.

  16. On the Definition of Aberration

    NASA Astrophysics Data System (ADS)

    Xu, Minghui; Wang, Guangli

    2014-12-01

    There was a groundbreaking step in the history of astronomy in 1728 when the effect of aberration was discovered by James Bradley (1693-1762). Recently, the solar acceleration, due to the variations in the aberrational effect of extragalactic sources caused by it, has been determined from VLBI observations with an uncertainty of about 0.5 mm{\\cdot}{s^{-1}}{\\cdot}{yr^{-1}} level. As a basic concept in astrometry with a nearly 300-year history, the definition of aberration, however, is still equivocal and discordant in the literature. It has been under continuing debate whether it depends on the relative motion between the observer and the observed source or only on the motion of the observer with respect to the frame of reference. In this paper, we will review the debate and the inconsistency in the definition of the aberration since the last century, and then discuss its definition in detail, which involves the discussions on the planetary aberration, the stellar aberration, the proper motion of an object during the travel time of light from the object to the observer, and the way of selecting the reference frame to express and distinguish the motions of the source and the observer. The aberration is essentially caused by the transformation between coordinate systems, and consequently quantified by the velocity of the observer with respect to the selected reference frame, independent of the motion of the source. Obviously, this nature is totally different from that of the definition given by the IAU WG NFA (Capitaine, 2007) in 2006, which is stated as, ``the apparent angular displacement of the observed position of a celestial object from its geometric position, caused by the finite velocity of light in combination with the motions of the observer and of the observed object.''

  17. Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

    PubMed

    Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

    2016-01-01

    BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

  18. Mechanisms underlying aberrant expression of miR-29c in uterine leiomyoma.

    PubMed

    Chuang, Tsai-Der; Khorram, Omid

    2016-01-01

    To determine the expression of miR-29c and its target genes in leiomyoma and the role of NF-κB, specific protein 1 (SP1), and DNA methylation in its regulation. Experimental study. Academic research laboratory. Women undergoing hysterectomy for leiomyoma. Over- and underexpression of miR-29c; blockade of transcription factors. MiR-29c and its target gene levels in leiomyoma and the effects of blockade of transcription factors on miR-29c expression. Leiomyoma as compared with myometrium expressed significantly lower levels of miR-29c, with an inverse relationship with expression of its targets, COL3A1 and DNMT3A. Gain of function of miR-29c inhibited the expression of COL3A1 and DNMT3A at protein and mRNA levels, secreted COL3A1, and rate of cell proliferation. Loss of function of miR-29c had the opposite effect. E2, P, and their combination inhibited miR-29c in leiomyoma smooth muscle cells (LSMC). Phosphorylated NF-κB (p65) and SP1 protein expression were significantly increased in leiomyoma. SiRNA knockdown of SP1 and DNMT3A or their specific inhibitors significantly increased the expression of miR-29c, accompanied by the inhibition of cellular and secreted COL3A1 in siRNA-treated cells. Knockdown of p65 also induced miR-29c expression but had no effect on COL3A1 expression. MiR-29c expression is suppressed in leiomyoma, resulting in an increase in expression of its targets COL3A1 and DNMT3A. The suppression of miR-29c in LSMC is primarily mediated by SP1, NF-κB signaling, and epigenetic modification. Collectively, these results indicate a significant role for miR-29c in leiomyoma pathogenesis. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  19. Aberrant light directly impairs mood and learning through melanopsin-expressing neurons.

    PubMed

    LeGates, Tara A; Altimus, Cara M; Wang, Hui; Lee, Hey-Kyoung; Yang, Sunggu; Zhao, Haiqing; Kirkwood, Alfredo; Weber, E Todd; Hattar, Samer

    2012-11-22

    The daily solar cycle allows organisms to synchronize their circadian rhythms and sleep-wake cycles to the correct temporal niche. Changes in day-length, shift-work, and transmeridian travel lead to mood alterations and cognitive function deficits. Sleep deprivation and circadian disruption underlie mood and cognitive disorders associated with irregular light schedules. Whether irregular light schedules directly affect mood and cognitive functions in the context of normal sleep and circadian rhythms remains unclear. Here we show, using an aberrant light cycle that neither changes the amount and architecture of sleep nor causes changes in the circadian timing system, that light directly regulates mood-related behaviours and cognitive functions in mice. Animals exposed to the aberrant light cycle maintain daily corticosterone rhythms, but the overall levels of corticosterone are increased. Despite normal circadian and sleep structures, these animals show increased depression-like behaviours and impaired hippocampal long-term potentiation and learning. Administration of the antidepressant drugs fluoxetine or desipramine restores learning in mice exposed to the aberrant light cycle, suggesting that the mood deficit precedes the learning impairments. To determine the retinal circuits underlying this impairment of mood and learning, we examined the behavioural consequences of this light cycle in animals that lack intrinsically photosensitive retinal ganglion cells. In these animals, the aberrant light cycle does not impair mood and learning, despite the presence of the conventional retinal ganglion cells and the ability of these animals to detect light for image formation. These findings demonstrate the ability of light to influence cognitive and mood functions directly through intrinsically photosensitive retinal ganglion cells.

  20. Aberrant p63 and WT-1 expression in myoepithelial cells of pregnancy-associated breast cancer: implications for tumor aggressiveness and invasiveness

    PubMed Central

    Xu, Zheli; Wang, Wan; Deng, Chu-Xia; Man, Yan-gao

    2009-01-01

    Our recent studies revealed that focal alterations in breast myoepithelial cell layers significantly impact the biological presentation of associated epithelial cells. As pregnancy-associated breast cancer (PABC) has a significantly more aggressive clinical course and mortality rate than other forms of breast malignancies, our current study compared tumor suppressor expression in myoepithelial cells of PABC and non-PABC, to determine whether myoepithelial cells of PABC may have aberrant expression of tumor suppressors. Tissue sections from 20 cases of PABC and 20 cases of stage, grade, and age matched non-PABC were subjected to immunohistochemistry, and the expression of tumor suppressor maspin, p63, and Wilms' tumor 1 (WT-1) in calponin positive myoepithelial cells were statistically compared. The expression profiles of maspin, p63, and WT-1 in myoepithelial cells of all ducts encountered were similar between PABC and non-PABC. PABC, however, displayed several unique alterations in terminal duct and lobular units (TDLU), acini, and associated tumor tissues that were not seen in those of non-PABC, which included the absence of p63 and WT-1 expression in a vast majority of the myoepithelial cells, cytoplasmic localization of p63 in the entire epithelial cell population of some lobules, and substantially increasing WT-1 expression in vascular structures of the invasive cancer component. All or nearly all epithelial cells with aberrant p63 and WT-1 expression lacked the expression of estrogen receptor and progesterone receptor, whereas they had a substantially higher proliferation index than their counterparts with p63 and WT-1 expression. Hyperplastic cells with cytoplasmic p63 expression often adjacent to, and share a similar immunohistochemical and cytological profile with, invasive cancer cells. To our best knowledge, our main finings have not been previously reported. Our findings suggest that the functional status of myoepithelial cells may be significantly

  1. Aberrant adhesion impacts early development in a Dictyostelium model for juvenile neuronal ceroid lipofuscinosis

    PubMed Central

    Huber, Robert J.; Myre, Michael A.; Cotman, Susan L.

    2017-01-01

    ABSTRACT Neuronal ceroid lipofuscinosis (NCL), also known as Batten disease, refers to a group of severe neurodegenerative disorders that primarily affect children. The most common subtype of the disease is caused by loss-of-function mutations in CLN3, which is conserved across model species from yeast to human. The precise function of the CLN3 protein is not known, which has made targeted therapy development challenging. In the social amoeba Dictyostelium discoideum, loss of Cln3 causes aberrant mid-to-late stage multicellular development. In this study, we show that Cln3-deficiency causes aberrant adhesion and aggregation during the early stages of Dictyostelium development. cln3− cells form ∼30% more multicellular aggregates that are comparatively smaller than those formed by wild-type cells. Loss of Cln3 delays aggregation, but has no significant effect on cell speed or cAMP-mediated chemotaxis. The aberrant aggregation of cln3− cells cannot be corrected by manually pulsing cells with cAMP. Moreover, there are no significant differences between wild-type and cln3− cells in the expression of genes linked to cAMP chemotaxis (e.g., adenylyl cyclase, acaA; the cAMP receptor, carA; cAMP phosphodiesterase, pdsA; g-protein α 9 subunit, gpaI). However, during this time in development, cln3− cells show reduced cell-substrate and cell-cell adhesion, which correlate with changes in the levels of the cell adhesion proteins CadA and CsaA. Specifically, loss of Cln3 decreases the intracellular level of CsaA and increases the amount of soluble CadA in conditioned media. Together, these results suggest that the aberrant aggregation of cln3− cells is due to reduced adhesion during the early stages of development. Revealing the molecular basis underlying this phenotype may provide fresh new insight into CLN3 function. PMID:27669405

  2. Formation of Hirano Bodies Induced by Expression of an Actin Cross-Linking Protein with a Gain-of-Function Mutation

    PubMed Central

    Maselli, Andrew; Furukawa, Ruth; Thomson, Susanne A. M.; Davis, Richard C.; Fechheimer, Marcus

    2003-01-01

    Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa ΔEF1. The 34-kDa ΔEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa ΔEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa ΔEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and α-actinin. Finally, the expression of the 34-kDa ΔEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton. PMID:12912897

  3. Expression of Par3 polarity protein correlates with poor prognosis in ovarian cancer.

    PubMed

    Nakamura, Hiroe; Nagasaka, Kazunori; Kawana, Kei; Taguchi, Ayumi; Uehara, Yuriko; Yoshida, Mitsuyo; Sato, Masakazu; Nishida, Haruka; Fujimoto, Asaha; Inoue, Tomoko; Adachi, Katsuyuki; Nagamatsu, Takeshi; Arimoto, Takahide; Oda, Katsutoshi; Osuga, Yutaka; Fujii, Tomoyuki

    2016-11-17

    Previous studies have shown that the cell polarity protein partitioning defective 3 (Par3) plays an essential role in the formation of tight junctions and definition of apical-basal polarity. Aberrant function of this protein has been reported to be involved in epithelial-mesenchymal transition (EMT) and cancer invasion. The aim of this study was to examine the functional mechanism of Par3 in ovarian cancer. First, we investigated the association between Par3 expression level and survival of 50 ovarian cancer patients. Next, we conducted an in vitro analysis of ovarian cancer cell lines, focusing on the cell line JHOC5, to investigate Par3 function. To investigate the function of Par3 in invasion, the IL-6/STAT3 pathway was analyzed upon Par3 knockdown with siRNA. The effect of siRNA treatment was assessed by qPCR, ELISA, and western blotting. Invasiveness and cell proliferation following treatment with siRNA against Par3 were investigated using Matrigel chamber, wound healing, and cell proliferation assays. Expression array data for ovarian cancer patient samples revealed low Par3 expression was significantly associated with good prognosis. Univariate analysis of clinicopathological factors revealed significant association between high Par3 levels and peritoneal dissemination at the time of diagnosis. Knockdown of Par3 in JHOC5 cells suppressed cell invasiveness, migration, and cell proliferation with deregulation of IL-6/STAT3 activity. Taken together, these results suggest that Par3 expression is likely involved in ovarian cancer progression, especially in peritoneal metastasis. The underlying mechanism may be that Par3 modulates IL-6 /STAT3 signaling. Here, we propose that the expression of Par3 in ovarian cancer may control disease outcome.

  4. Multiplexed aberration measurement for deep tissue imaging in vivo

    PubMed Central

    Wang, Chen; Liu, Rui; Milkie, Daniel E.; Sun, Wenzhi; Tan, Zhongchao; Kerlin, Aaron; Chen, Tsai-Wen; Kim, Douglas S.; Ji, Na

    2014-01-01

    We describe a multiplexed aberration measurement method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine their phase gradients. Applicable to fluorescent-protein-labeled structures of arbitrary complexity, it allows us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improves structural and functional imaging of fine neuronal processes over a large imaging volume. PMID:25128976

  5. Bombyx mori nucleopolyhedrovirus BM5 protein regulates progeny virus production and viral gene expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kokusho, Ryuhei, E-mail: kokusho@ss.ab.a.u-tokyo.a

    2016-11-15

    Bombyx mori nucleopolyhedrovirus (BmNPV) orf5 (Bm5) is a core gene of lepidopteran baculoviruses and encodes the protein with the conserved amino acid residues (DUF3627) in its C-terminus. Here, we found that Bm5 disruption resulted in lower titers of budded viruses and fewer numbers of occlusion bodies (OBs) in B. mori cultured cells and larvae, although viral genome replication was not affected. Bm5 disruption also caused aberrant expression of various viral genes at the very late stage of infection. Immunocytochemical analysis revealed that BM5 localized to the nuclear membrane. We also found that DUF3627 is important for OB production, transcriptional regulationmore » of viral genes, and subcellular localization of BM5. Compared with wild-type BmNPV infection, larval death was delayed when B. mori larvae were infected with Bm5 mutants. These results suggest that BM5 is involved in progeny virus production and regulation of viral gene expression at the very late stage of infection. -- Highlights: •The role of BmNPV BM5 protein was examined in B. mori cultured cells and larvae. •BM5 contributes to efficient production of budded viruses and occlusion bodies. •BM5 regulates viral gene expression at the very late stage of infection. •BM5 dominantly localizes to the nuclear membrane. •Bm5 mutant showed v-cath down-regulation and resulting delay of larval death.« less

  6. MicroRNA-15b regulates reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression in human uterine leiomyoma.

    PubMed

    Guan, Yichun; Guo, Lankai; Zukerberg, Lawrence; Rueda, Bo R; Styer, Aaron K

    2016-08-17

    Human uterine leiomyoma (fibroids; LYO) are the most common benign neoplasms in reproductive-aged women. Dysregulated extracellular matrix and irregular LYO reversion-inducing cysteine-rich protein with Kazal motifs (RECK) expression are thought to be mediated by aberrant microRNA (miR) expression. The relationship of miR-15b and RECK expression in LYO has not been studied. The expression levels of miR-15b and RECK were determined by quantitative RT-PCR, Western blot, and immunohistochemistry in cultures derived from commercial primary leiomyoma (cpLYO) and myometrial (cpMYO) cell lines and leiomyoma (pLYO) and myometrium (pMYO) tissue from surgical samples respectively. The relationship between miR-15b and RECK expression in cpLYO and pLYO (compared to their respective myometrial controls) was evaluated following transfection of cell cultures with either miR-15b mimic or inhibitor. Elevated levels of miR-15b were observed in cpLYO (2.82-fold; p = 0.04) and pLYO cell (1.30-fold; p = 0.0001) cultures respectively compared to corresponding MYO cell controls. Following transfection with miR-15b mimic, cpLYO cells (0.62-fold; p < 0.0001) and pLYO cells (0.68-fold; p < 0.0001) demonstrated reduced RECK protein expression. Following transfection with miR-15b inhibitor, cpLYO cells (1.20-fold; p < 0.0001) and pLYO cells (1.31-fold; p = 0.0007) demonstrated elevated RECK protein expression. RECK protein expression was reduced in pLYO tissues (0.73-fold; p < 0.0001) and pLYO (0.47-fold; p = 0.047) cells when compared to the corresponding MYO tissue controls. Our findings suggest that miR-15b negatively regulates RECK expression in LYO, and increased miR-15b and decreased RECK expression may contribute to the pathobiology of LYO. The functional significance of miR-15b and RECK expression warrants further investigation as potential therapeutic targets for the treatment of human LYO.

  7. Aberrant expression of erythropoietin in uterine leiomyoma: implications in tumor growth.

    PubMed

    Asano, Ryoko; Asai-Sato, Mikiko; Miyagi, Yohei; Mizushima, Taichi; Koyama-Sato, Makiko; Nagashima, Yoji; Taguri, Masataka; Sakakibara, Hideya; Hirahara, Fumiki; Miyagi, Etsuko

    2015-08-01

    Myomatous erythrocytosis syndrome is a rare complication of uterine leiomyoma caused by erythropoietin (EPO) that is produced by tumor cells. We assessed the EPO expression in leiomyomas and investigated the effects of EPO on the tumor growth. Tissue samples were collected from 114 patients with uterine leiomyomas who underwent myomectomy or hysterectomy in Yokohama City University Hospital. From 17 patients, the corresponding normal myometrium was also collected. All samples were analyzed for EPO messenger RNA (mRNA) expression by real-time reverse transcription-polymerase chain reaction. EPO protein expression was determined by an enzyme-linked immunosorbent assay. The relationships between EPO expression and clinicopathological features were retrospectively analyzed using the patients' charts. Blood vessel density and maturity were assessed using hematoxylin-eosin staining and CD34 immunohistochemistry. EPO mRNA expression was detected in 108 of 114, or 95%, of the leiomyomas. The mean EPO mRNA expression in the leiomyoma was higher than the corresponding normal myometrium (3836 ± 4122 vs 1455 ± 2141; P = .025 by Wilcoxon rank test). The EPO mRNA expression in the leiomyomas varied extensively among samples, ranging from undetectable levels to 18-fold above the mean EPO mRNA of normal myometrium. EPO protein production was observed concomitant with mRNA expression. A positive correlation of leiomyoma size and EPO mRNA expression was shown by Spearman rank correlation coefficient (ρ = 0.294; P = .001), suggesting the involvement of EPO in leiomyoma growth. The blood vessel maturity was also significantly increased in EPO-producing leiomyomas (high vessel maturity in high vs low EPO group: 67% vs 20%; P = .013 by Fisher exact test). This report demonstrates that EPO is produced in most of conventional leiomyomas and supports a model in which EPO accelerates tumor growth, possibly by inducing vessel maturity. Our study suggests one possible mechanism by which

  8. Improving membrane protein expression and function using genomic edits

    DOE PAGES

    Jensen, Heather M.; Eng, Thomas; Chubukov, Victor; ...

    2017-10-12

    Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

  9. Improving membrane protein expression and function using genomic edits

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jensen, Heather M.; Eng, Thomas; Chubukov, Victor

    Expression of membrane proteins often leads to growth inhibition and perturbs central metabolism and this burden varies with the protein being overexpressed. There are also known strain backgrounds that allow greater expression of membrane proteins but that differ in efficacy across proteins. Here, we hypothesized that for any membrane protein, it may be possible to identify a modified strain background where its expression can be accommodated with less burden. To directly test this hypothesis, we used a bar-coded transposon insertion library in tandem with cell sorting to assess genome-wide impact of gene deletions on membrane protein expression. The expression ofmore » five membrane proteins (CyoB, CydB, MdlB, YidC, and LepI) and one soluble protein (GST), each fused to GFP, was examined. We identified Escherichia coli mutants that demonstrated increased membrane protein expression relative to that in wild type. For two of the proteins (CyoB and CydB), we conducted functional assays to confirm that the increase in protein expression also led to phenotypic improvement in function. This study represents a systematic approach to broadly identify genetic loci that can be used to improve membrane protein expression, and our method can be used to improve expression of any protein that poses a cellular burden.« less

  10. Clinical omics analysis of colorectal cancer incorporating copy number aberrations and gene expression data.

    PubMed

    Yoshida, Tsuyoshi; Kobayashi, Takumi; Itoda, Masaya; Muto, Taika; Miyaguchi, Ken; Mogushi, Kaoru; Shoji, Satoshi; Shimokawa, Kazuro; Iida, Satoru; Uetake, Hiroyuki; Ishikawa, Toshiaki; Sugihara, Kenichi; Mizushima, Hiroshi; Tanaka, Hiroshi

    2010-07-29

    Colorectal cancer (CRC) is one of the most frequently occurring cancers in Japan, and thus a wide range of methods have been deployed to study the molecular mechanisms of CRC. In this study, we performed a comprehensive analysis of CRC, incorporating copy number aberration (CRC) and gene expression data. For the last four years, we have been collecting data from CRC cases and organizing the information as an "omics" study by integrating many kinds of analysis into a single comprehensive investigation. In our previous studies, we had experienced difficulty in finding genes related to CRC, as we observed higher noise levels in the expression data than in the data for other cancers. Because chromosomal aberrations are often observed in CRC, here, we have performed a combination of CNA analysis and expression analysis in order to identify some new genes responsible for CRC. This study was performed as part of the Clinical Omics Database Project at Tokyo Medical and Dental University. The purpose of this study was to investigate the mechanism of genetic instability in CRC by this combination of expression analysis and CNA, and to establish a new method for the diagnosis and treatment of CRC. Comprehensive gene expression analysis was performed on 79 CRC cases using an Affymetrix Gene Chip, and comprehensive CNA analysis was performed using an Affymetrix DNA Sty array. To avoid the contamination of cancer tissue with normal cells, laser micro-dissection was performed before DNA/RNA extraction. Data analysis was performed using original software written in the R language. We observed a high percentage of CNA in colorectal cancer, including copy number gains at 7, 8q, 13 and 20q, and copy number losses at 8p, 17p and 18. Gene expression analysis provided many candidates for CRC-related genes, but their association with CRC did not reach the level of statistical significance. The combination of CNA and gene expression analysis, together with the clinical information

  11. Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

    PubMed Central

    Nagel, Stefan; Ehrentraut, Stefan; Tomasch, Jürgen; Quentmeier, Hilmar; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G.; MacLeod, Roderick A. F.

    2013-01-01

    Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies. PMID:23637834

  12. Ectopic expression of homeobox gene NKX2-1 in diffuse large B-cell lymphoma is mediated by aberrant chromatin modifications.

    PubMed

    Nagel, Stefan; Ehrentraut, Stefan; Tomasch, Jürgen; Quentmeier, Hilmar; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

    2013-01-01

    Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies.

  13. Expression of placental protein 14 by the new endometrial cancer cell line MFE-280 in vitro and by endometrial carcinomas in vivo.

    PubMed

    Hackenberg, R; Loos, S; Nia, A H; Kunzmann, R; Schulz, K D

    1998-01-01

    MFE-280 endometrial cancer cells express PP14 (placental protein 14) in vitro. PP14 is normally found in the secretory endometrium and in placental tissue. MFE-280 cells, which are tumorigenic in nude mice, were derived from a recurrent, poorly differentiated endometrial carcinoma. The cells were initially grown in suspension culture and later transferred to monolayer cultures. Karyotyping revealed near-diploidy with a complex heterogeneous aberration pattern. MFE-280 cells were positive for the cytokeratins 7, 8, 18 and 19 as well as for vimentin. The expression of PP14 in MFE-280 cells was demonstrated by immunochemistry and reverse transcriptase--polymerase chain reaction. PP14-mRNA was also detected in one out of five endometrial cancer specimen. In tumor tissue the expression of PP14 was not dependent on progestins.

  14. A genome-wide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets.

    PubMed

    Adamia, Sophia; Haibe-Kains, Benjamin; Pilarski, Patrick M; Bar-Natan, Michal; Pevzner, Samuel; Avet-Loiseau, Herve; Lode, Laurence; Verselis, Sigitas; Fox, Edward A; Burke, John; Galinsky, Ilene; Dagogo-Jack, Ibiayi; Wadleigh, Martha; Steensma, David P; Motyckova, Gabriela; Deangelo, Daniel J; Quackenbush, John; Stone, Richard; Griffin, James D

    2014-03-01

    Despite new treatments, acute myeloid leukemia (AML) remains an incurable disease. More effective drug design requires an expanded view of the molecular complexity that underlies AML. Alternative splicing of RNA is used by normal cells to generate protein diversity. Growing evidence indicates that aberrant splicing of genes plays a key role in cancer. We investigated genome-wide splicing abnormalities in AML and based on these abnormalities, we aimed to identify novel potential biomarkers and therapeutic targets. We used genome-wide alternative splicing screening to investigate alternative splicing abnormalities in two independent AML patient cohorts [Dana-Farber Cancer Institute (DFCI) (Boston, MA) and University Hospital de Nantes (UHN) (Nantes, France)] and normal donors. Selected splicing events were confirmed through cloning and sequencing analysis, and than validated in 193 patients with AML. Our results show that approximately 29% of expressed genes genome-wide were differentially and recurrently spliced in patients with AML compared with normal donors bone marrow CD34(+) cells. Results were reproducible in two independent AML cohorts. In both cohorts, annotation analyses indicated similar proportions of differentially spliced genes encoding several oncogenes, tumor suppressor proteins, splicing factors, and heterogeneous-nuclear-ribonucleoproteins, proteins involved in apoptosis, cell proliferation, and spliceosome assembly. Our findings are consistent with reports for other malignances and indicate that AML-specific aberrations in splicing mechanisms are a hallmark of AML pathogenesis. Overall, our results suggest that aberrant splicing is a common characteristic for AML. Our findings also suggest that splice variant transcripts that are the result of splicing aberrations create novel disease markers and provide potential targets for small molecules or antibody therapeutics for this disease. ©2013 AACR

  15. Aberrant Expression of Calretinin, D2-40 and Mesothelin in Mucinous and Non-Mucinous Colorectal Carcinomas and Relation to Clinicopathological Features and Prognosis.

    PubMed

    Foda, Abd AlRahman Mohammad; El-Hawary, Amira Kamal; Hamed, Hazem

    2016-10-01

    CRC is a heterogeneous disease in terms of morphology, invasive behavior, metastatic capacity, and clinical outcome. Recently, many so-called mesothelial markers, including calretinin, D2-40, WT1, thrombomodulin, mesothelin, and others, have been certified. The aim of this study was to assess the immunohistochemical expression of calretinin and other mesothelial markers (D2-40 and mesothelin) in colorectal mucinous adenocarcinoma (MA) and non mucinous adenocarcinoma (NMA) specimens and relation to clinicopathological features and prognosis using manual tissue microarray technique. We studied tumor tissue specimens from 150 patients with colorectal MA and NMA who underwent radical surgery from January 2007 to January 2012. High-density manual tissue microarrays were constructed using a modified mechanical pencil tip technique, and paraffin sections were submitted for immunohistochemistry using Calretinin, D2-40 and mesothelin expressions. We found that NMA showed significantly more calretinin and D2-40 expression than MA In contrast, no statistically significant difference between NMA and MA was detected in mesothelin expression. There were no statistically significant relations between any of the clinicopathological or histological parameters and any of the three markers. In a univariate analysis, neither calretinin nor D2-40 expressions showed any significant relations to DFS or OS. However, mesothelin luminal expression was significantly associated with worse DFS. Multivariate Cox regression analysis proved that luminal mesothelin expression was an independent negative prognostic factor in NMA. In conclusion, Calretinin, D2-40 and mesothelin are aberrantly expressed in a proportion of CRC cases with more expression in NMA than MA. Aberrant expression of these mesothelial markers was not associated with clinicopathological or histological features of CRCs. Only mesothelin expression appears to be a strong predictor of adverse prognosis.

  16. Analysis of genomic aberrations and gene expression profiling identifies novel lesions and pathways in myeloproliferative neoplasms

    PubMed Central

    Rice, K L; Lin, X; Wolniak, K; Ebert, B L; Berkofsky-Fessler, W; Buzzai, M; Sun, Y; Xi, C; Elkin, P; Levine, R; Golub, T; Gilliland, D G; Crispino, J D; Licht, J D; Zhang, W

    2011-01-01

    Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis, are myeloproliferative neoplasms (MPNs) with distinct clinical features and are associated with the JAK2V617F mutation. To identify genomic anomalies involved in the pathogenesis of these disorders, we profiled 87 MPN patients using Affymetrix 250K single-nucleotide polymorphism (SNP) arrays. Aberrations affecting chr9 were the most frequently observed and included 9pLOH (n=16), trisomy 9 (n=6) and amplifications of 9p13.3–23.3 (n=1), 9q33.1–34.13 (n=1) and 9q34.13 (n=6). Patients with trisomy 9 were associated with elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage. Gene expression profiling of patients with and without chr9 abnormalities (+9, 9pLOH), identified genes potentially involved in disease pathogenesis including JAK2, STAT5B and MAPK14. We also observed recurrent gains of 1p36.31–36.33 (n=6), 17q21.2–q21.31 (n=5) and 17q25.1–25.3 (n=5) and deletions affecting 18p11.31–11.32 (n=8). Combined SNP and gene expression analysis identified aberrations affecting components of a non-canonical PRC2 complex (EZH1, SUZ12 and JARID2) and genes comprising a ‘HSC signature' (MLLT3, SMARCA2 and PBX1). We show that NFIB, which is amplified in 7/87 MPN patients and upregulated in PV CD34+ cells, protects cells from apoptosis induced by cytokine withdrawal. PMID:22829077

  17. Indispensable roles of mammalian Cbl family proteins as negative regulators of protein tyrosine kinase signaling

    PubMed Central

    Band, Vimla

    2011-01-01

    All higher eukaryotes utilize protein tyrosine kinases (PTKs) as molecular switches to control a variety of cellular signals. Notably, many PTKs have been identified as proto-oncogenes whose aberrant expression, mutations or co-option by pathogens can lead to human malignancies. Thus, it is obvious that PTK functions must be precisely regulated in order to maintain homeostasis of an organism. Investigations over the past fifteen years have revealed that members of the Cbl family proteins can serve as negative regulators of PTK signaling, and biochemical and cell biological studies have unraveled the mechanistic basis of this regulation. Yet, it is only recently that the field has begun to appreciate the real significance of this novel regulatory apparatus in shaping PTK-mediated signaling in organismic contexts and in human diseases. Here, we discuss recent progress in murine models that are beginning to provide insights into the critical roles of Cbl proteins in physiological pathways, with important implications in understanding how aberrations of Cbl proteins contribute to oncogenesis. PMID:21655429

  18. The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns.

    PubMed

    Zee, Barry M; Dibona, Amy B; Alekseyenko, Artyom A; French, Christopher A; Kuroda, Mitzi I

    2016-01-01

    Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state.

  19. The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns

    PubMed Central

    Zee, Barry M.; Dibona, Amy B.; Alekseyenko, Artyom A.; French, Christopher A.; Kuroda, Mitzi I.

    2016-01-01

    Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state. PMID:27698495

  20. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  1. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses.

    PubMed

    Kroemer, Jeremy A; Bonning, Bryony C; Harrison, Robert L

    2015-01-21

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification.

  2. Peripheral T-Cell Lymphoma with Aberrant Expression of CD19, CD20, and CD79a: Case Report and Literature Review

    PubMed Central

    Matnani, Rahul G.; Stewart, Rachel L.; Pulliam, Joseph; Jennings, Chester D.; Kesler, Melissa

    2013-01-01

    A case of lymphoma of T-cell derivation with aberrant expression of three B-cell lineage markers (CD19, CD20, and CD79a), which was diagnosed on a left axillary excision, is described. Immunohistochemical studies and flow cytometry analysis demonstrated neoplastic cells expressing CD3, CD19, CD20, and CD79a with absence of CD4, CD8, CD10, CD30, CD34, CD56, CD68, TDT, MPO, PAX-5, and surface immunoglobulin. Gene rearrangement studies performed on paraffin blocks demonstrated monoclonal T-cell receptor gamma chain rearrangement with no evidence of clonal heavy chain rearrangement. The neoplastic cells were negative for Epstein-Barr virus (EBV) or Human Herpes Virus 8 (HHV-8). At the time of diagnosis, the PET scan demonstrated hypermetabolic neoplastic cells involving the left axilla, bilateral internal jugular areas, mediastinum, right hilum, bilateral lungs, and spleen. However, bone marrow biopsy performed for hemolytic anemia revealed normocellular bone marrow with trilineage maturation. The patient had no evidence of immunodeficiency or infection with EBV or HHV-8. This is the first reported case of a mature T-cell lymphoma with aberrant expression of three B-cell lineage markers. The current report also highlights the need for molecular gene rearrangement studies to determine the precise lineage of ambiguous neoplastic clones. PMID:24066244

  3. Exocyst Complex Protein Expression in the Human Placenta

    PubMed Central

    Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

    2014-01-01

    Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

  4. Reduced cortical BDNF expression and aberrant memory in Carf knockout mice

    PubMed Central

    McDowell, Kelli A.; Hutchinson, Ashley N.; Wong-Goodrich, Sarah J.E.; Presby, Matthew M.; Su, Dan; Rodriguiz, Ramona M.; Law, Krystal C.; Williams, Christina L.; Wetsel, William C.; West, Anne E.

    2010-01-01

    Transcription factors are a key point of convergence between the cell-intrinsic and extracellular signals that guide synaptic development and brain plasticity. Calcium-Response Factor (CaRF) is a unique transcription factor first identified as a binding protein for a calcium-response element in the gene encoding Brain-Derived Neurotrophic Factor (Bdnf). We have now generated Carf knockout (KO) mice to characterize the function of this factor in vivo. Intriguingly, Carf KO mice have selectively reduced expression of Bdnf exon IV-containing mRNA transcripts and BDNF protein in the cerebral cortex while BDNF levels in the hippocampus and striatum remain unchanged, implicating CaRF as a brain region-selective regulator of BDNF expression. At the cellular level, Carf KO mice show altered expression of GABAergic proteins at striatal synapses, raising the possibility that CaRF may contribute to aspects of inhibitory synapse development. Carf KO mice show normal spatial learning in the Morris water maze and normal context-dependent fear conditioning. However they have an enhanced ability to find a new platform location on the first day of reversal training in the water maze and they extinguish conditioned fear more slowly than their wildtype (WT) littermates. Finally, Carf KO mice show normal short-term and long-term memory in a novel object recognition task, but exhibit impairments during the remote memory phase of testing. Taken together these data reveal novel roles for CaRF in the organization and/or function of neural circuits that underlie essential aspects of learning and memory. PMID:20519520

  5. Improving membrane protein expression by optimizing integration efficiency

    PubMed Central

    2017-01-01

    The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

  6. Human papilloma virus, DNA methylation and microRNA expression in cervical cancer (Review).

    PubMed

    Jiménez-Wences, Hilda; Peralta-Zaragoza, Oscar; Fernández-Tilapa, Gloria

    2014-06-01

    Cancer is a complex disease caused by genetic and epigenetic abnormalities that affect gene expression. The progression from precursor lesions to invasive cervical cancer is influenced by persistent human papilloma virus (HPV) infection, which induces changes in the host genome and epigenome. Epigenetic alterations, such as aberrant miRNA expression and changes in DNA methylation status, favor the expression of oncogenes and the silencing of tumor-suppressor genes. Given that some miRNA genes can be regulated through epigenetic mechanisms, it has been proposed that alterations in the methylation status of miRNA promoters could be the driving mechanism behind their aberrant expression in cervical cancer. For these reasons, we assessed the relationship among HPV infection, cellular DNA methylation and miRNA expression. We conclude that alterations in the methylation status of protein-coding genes and various miRNA genes are influenced by HPV infection, the viral genotype, the physical state of the viral DNA, and viral oncogenic risk. Furthermore, HPV induces deregulation of miRNA expression, particularly at loci near fragile sites. This deregulation occurs through the E6 and E7 proteins, which target miRNA transcription factors such as p53.

  7. E-cadherin and β-catenin adhesion proteins correlate positively with connexins in colorectal cancer

    PubMed Central

    KANCZUGA-KODA, LUIZA; WINCEWICZ, ANDRZEJ; FUDALA, ANDRZEJ; ABRYCKI, TOMASZ; FAMULSKI, WALDEMAR; BALTAZIAK, MAREK; SULKOWSKI, STANISLAW; KODA, MARIUSZ

    2014-01-01

    The majority of solid cancers present with qualitative and quantitative aberrations of adhesion proteins, including E-cadherin and β-catenin, and connexin (Cx) gap junction proteins, which is consistent with alterations in the expression and location of such proteins in neoplastic cells. Since there are no data on the correlation between adhesion proteins and Cxs in human colorectal cancer (CRC), the aim of the present study was to evaluate the expression and correlation between these proteins. Tissue specimens were obtained from 151 cases of surgically removed colorectal adenocarcinomas. The samples were examined by immunohistochemistry with the use of antibodies against E-cadherin, β-catenin and the three Cxs: Cx26, Cx32 and Cx43. The aberrant expression of the studied adhesion proteins (primarily cytoplasmic for E-cadherin and cytoplasmic and/or nuclear for β-catenin) was observed, whereas only a minority of cases revealed normal membranous distribution of the labeling. The present study is the first in the literature to reveal a correlation between the expression of E-cadherin and β-catenin and the examined Cxs in CRC in humans. The positive correlation between the Cxs, particularly Cx26 and Cx32, and the adhesive proteins occurred in patients without lymph node metastases and in the moderately differentiated tumors (G2). Such a dependency was not observed in the analysis of the correlation between Cx43 and E-cadherin. However, a positive correlation between these proteins was observed in patients with lymph nodes metastases. Additionally, a link between the expression of these adhesion proteins was observed. The present study indicates, for the first time, that the expression of adhesion proteins, E-cadherin and β-catenin, is closely associated with the expression of three studied Cxs in CRC, and that this correlation may improve an understanding of the carcinogenic process in this cancer. PMID:24932249

  8. Vanadium inhibits DNA-protein cross-links and ameliorates surface level changes of aberrant crypt foci during 1,2-dimethylhydrazine induced rat colon carcinogenesis.

    PubMed

    Kanna, P Suresh; Saralaya, M G; Samanta, K; Chatterjee, M

    2005-01-01

    The trace mineral vanadium inhibits cancer development in a variety of experimental animal models. The present study was to gain insight into a putative anticancer effect of vanadium in a rat model of colon carcinogenesis. The in vivo study was intended to clarify the effect of vanadium on DNA-protein cross-links (DPC), surface level changes of aberrant crypt foci (ACF) and biotransformation status during 1,2-dimethylhydrazine (1,2-DMH) induced preneoplastic rat colon carcinogenesis. The comet assay showed statistically higher mean base values of DNA-protein mass (p<0.01) and mean frequencies of tailed cells (p<0.001) in the carcinogen-induced group after treatment with proteinase K. Treatment with vanadium in the form of ammonium monovanadate supplemented ad libitum in drinking water for the entire experimental period caused a significant (p<0.02) reduction (40%) in DNA-protein cross-links in colon cells. Further, the biotransformation status of vanadium was ascertained measuring the drug metabolising enzymes, glutathione S-transferase (GST) and cytochrome P-450 (Cyt P-450). Significantly, there was an increase in glutathione S-transferase and cytochrome P-450 levels (p<0.01 and p<0.02, respectively) in rats supplemented with vanadium as compared to their carcinogen controls. As an endpoint marker, we also evaluated the effect of vanadium on surface level changes of aberrant crypt foci induced by 1,2-DMH by scanning electron microscopy. Animals induced with 1,2-DMH and supplemented with vanadium showed a marked improvement in colonic architecture with less number of aberrant crypt foci in contrast to the animals induced with 1,2-DMH alone, thereby exhibiting its anti-carcinogenicity by modulating the markers studied herein.

  9. Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers.

    PubMed

    Kataoka, Keisuke; Shiraishi, Yuichi; Takeda, Yohei; Sakata, Seiji; Matsumoto, Misako; Nagano, Seiji; Maeda, Takuya; Nagata, Yasunobu; Kitanaka, Akira; Mizuno, Seiya; Tanaka, Hiroko; Chiba, Kenichi; Ito, Satoshi; Watatani, Yosaku; Kakiuchi, Nobuyuki; Suzuki, Hiromichi; Yoshizato, Tetsuichi; Yoshida, Kenichi; Sanada, Masashi; Itonaga, Hidehiro; Imaizumi, Yoshitaka; Totoki, Yasushi; Munakata, Wataru; Nakamura, Hiromi; Hama, Natsuko; Shide, Kotaro; Kubuki, Yoko; Hidaka, Tomonori; Kameda, Takuro; Masuda, Kyoko; Minato, Nagahiro; Kashiwase, Koichi; Izutsu, Koji; Takaori-Kondo, Akifumi; Miyazaki, Yasushi; Takahashi, Satoru; Shibata, Tatsuhiro; Kawamoto, Hiroshi; Akatsuka, Yoshiki; Shimoda, Kazuya; Takeuchi, Kengo; Seya, Tsukasa; Miyano, Satoru; Ogawa, Seishi

    2016-06-16

    Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3' region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3'-untranslated region (UTR). Disruption of the Pd-l1 3'-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3'-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.

  10. Glycosylation Changes in Serum Proteins Identify Patients with Pancreatic Cancer.

    PubMed

    Drabik, Anna; Bodzon-Kulakowska, Anna; Suder, Piotr; Silberring, Jerzy; Kulig, Jan; Sierzega, Marek

    2017-04-07

    After more than a decade of biomarker discovery using advanced proteomic and genomic approaches, very few biomarkers have been involved in clinical diagnostics. Most candidate biomarkers are focused on the protein component. Targeting post-translational modifications (PTMs) in combination with protein sequences will provide superior diagnostic information with regards to sensitivity and specificity. Glycosylation is one of the most common and functionally important PTMs. It plays a central role in many biological processes, including protein folding, host-pathogen interactions, immune response, and inflammation. Cancer-associated aberrant glycosylation has been identified in various types of cancer. Expression of cancer-specific glycan epitopes represents an excellent opportunity for diagnostics and potentially specific detection of tumors. Here, we report four proteins (LIFR, CE350, VP13A, HPT) found in sera from pancreatic cancer patients carrying aberrant glycan structures as compared to those of controls.

  11. Epstein-Barr virus infection induces aberrant TLR activation pathway and fibroblast-myofibroblast conversion in scleroderma.

    PubMed

    Farina, Antonella; Cirone, Mara; York, Michael; Lenna, Stefania; Padilla, Cristina; Mclaughlin, Sarah; Faggioni, Alberto; Lafyatis, Robert; Trojanowska, Maria; Farina, Giuseppina A

    2014-04-01

    Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein-Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV-Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-β1 (TGFβ1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation.

  12. Long non-coding RNA expression patterns in lung tissues of chronic cigarette smoke induced COPD mouse model.

    PubMed

    Zhang, Haiyun; Sun, Dejun; Li, Defu; Zheng, Zeguang; Xu, Jingyi; Liang, Xue; Zhang, Chenting; Wang, Sheng; Wang, Jian; Lu, Wenju

    2018-05-15

    Long non-coding RNAs (lncRNAs) have critical regulatory roles in protein-coding gene expression. Aberrant expression profiles of lncRNAs have been observed in various human diseases. In this study, we investigated transcriptome profiles in lung tissues of chronic cigarette smoke (CS)-induced COPD mouse model. We found that 109 lncRNAs and 260 mRNAs were significantly differential expressed in lungs of chronic CS-induced COPD mouse model compared with control animals. GO and KEGG analyses indicated that differentially expressed lncRNAs associated protein-coding genes were mainly involved in protein processing of endoplasmic reticulum pathway, and taurine and hypotaurine metabolism pathway. The combination of high throughput data analysis and the results of qRT-PCR validation in lungs of chronic CS-induced COPD mouse model, 16HBE cells with CSE treatment and PBMC from patients with COPD revealed that NR_102714 and its associated protein-coding gene UCHL1 might be involved in the development of COPD both in mouse and human. In conclusion, our study demonstrated that aberrant expression profiles of lncRNAs and mRNAs existed in lungs of chronic CS-induced COPD mouse model. From animal models perspective, these results might provide further clues to investigate biological functions of lncRNAs and their potential target protein-coding genes in the pathogenesis of COPD.

  13. Rice ABERRANT PANICLE ORGANIZATION 1, encoding an F-box protein, regulates meristem fate.

    PubMed

    Ikeda, Kyoko; Ito, Momoyo; Nagasawa, Nobuhiro; Kyozuka, Junko; Nagato, Yasuo

    2007-09-01

    Inflorescence architecture is one of the most important agronomical traits. Characterization of rice aberrant panicle organization 1 (apo1) mutants revealed that APO1 positively controls spikelet number by suppressing the precocious conversion of inflorescence meristems to spikelet meristems. In addition, APO1 is associated with the regulation of the plastchron, floral organ identity, and floral determinacy. Phenotypic analyses of apo1 and floral homeotic double mutants demonstrate that APO1 positively regulates class-C floral homeotic genes, but not class-B genes. Molecular studies revealed that APO1 encodes an F-box protein, an ortholog of Arabidopsis UNUSUAL FLORAL ORGAN (UFO), which is a positive regulator of class-B genes. Overexpression of APO1 caused an increase in inflorescence branches and spikelets. As the mutant inflorescences and flowers differed considerably between apo1 and ufo, the functions of APO1 and UFO appear to have diverged during evolution.

  14. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  15. Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

    PubMed

    Fu, Ssu-Ju; Jeng, Chung-Jiuan; Ma, Chia-Hao; Peng, Yi-Jheng; Lee, Chi-Ming; Fang, Ya-Ching; Lee, Yi-Ching; Tang, Sung-Chun; Hu, Meng-Chun; Tang, Chih-Yung

    2017-03-01

    Voltage-gated Ca V 2.1 channels comprise a pore-forming α 1A subunit with auxiliary α 2 δ and β subunits. Ca V 2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the Ca V 2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of Ca V 2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human Ca V 2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel Ca V 2.1-binding partner. In neurons, RNF138 and Ca V 2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of Ca V 2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the Ca V 2.1 protein level and enhances Ca V 2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of Ca V 2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on Ca V 2.1 WT functional expression, which can be attributed to defective membrane trafficking of Ca V 2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of Ca V 2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human Ca V 2.1 subunits. SIGNIFICANCE STATEMENT Loss-of-function mutations in the human Ca V 2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by

  16. Determination of aberration center of Ronchigram for automated aberration correctors in scanning transmission electron microscopy.

    PubMed

    Sannomiya, Takumi; Sawada, Hidetaka; Nakamichi, Tomohiro; Hosokawa, Fumio; Nakamura, Yoshio; Tanishiro, Yasumasa; Takayanagi, Kunio

    2013-12-01

    A generic method to determine the aberration center is established, which can be utilized for aberration calculation and axis alignment for aberration corrected electron microscopes. In this method, decentering induced secondary aberrations from inherent primary aberrations are minimized to find the appropriate axis center. The fitness function to find the optimal decentering vector for the axis was defined as a sum of decentering induced secondary aberrations with properly distributed weight values according to the aberration order. Since the appropriate decentering vector is determined from the aberration values calculated at an arbitrary center axis, only one aberration measurement is in principle required to find the center, resulting in /very fast center search. This approach was tested for the Ronchigram based aberration calculation method for aberration corrected scanning transmission electron microscopy. Both in simulation and in experiments, the center search was confirmed to work well although the convergence to find the best axis becomes slower with larger primary aberrations. Such aberration center determination is expected to fully automatize the aberration correction procedures, which used to require pre-alignment of experienced users. This approach is also applicable to automated aperture positioning. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Structural centrosome aberrations sensitize polarized epithelia to basal cell extrusion.

    PubMed

    Ganier, Olivier; Schnerch, Dominik; Nigg, Erich A

    2018-06-01

    Centrosome aberrations disrupt tissue architecture and may confer invasive properties to cancer cells. Here we show that structural centrosome aberrations, induced by overexpression of either Ninein-like protein (NLP) or CEP131/AZI1, sensitize polarized mammalian epithelia to basal cell extrusion. While unperturbed epithelia typically dispose of damaged cells through apical dissemination into luminal cavities, certain oncogenic mutations cause a switch in directionality towards basal cell extrusion, raising the potential for metastatic cell dissemination. Here we report that NLP-induced centrosome aberrations trigger the preferential extrusion of damaged cells towards the basal surface of epithelial monolayers. This switch in directionality from apical to basal dissemination coincides with a profound reorganization of the microtubule cytoskeleton, which in turn prevents the contractile ring repositioning that is required to support extrusion towards the apical surface. While the basal extrusion of cells harbouring NLP-induced centrosome aberrations requires exogenously induced cell damage, structural centrosome aberrations induced by excess CEP131 trigger the spontaneous dissemination of dying cells towards the basal surface from MDCK cysts. Thus, similar to oncogenic mutations, structural centrosome aberrations can favour basal extrusion of damaged cells from polarized epithelia. Assuming that additional mutations may promote cell survival, this process could sensitize epithelia to disseminate potentially metastatic cells. © 2018 The Authors.

  18. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  19. Immunostimulatory oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in 80% of CLL patients: A study of 132 CLL cases with correlation to FISH, IgVH status, and CD38 expression.

    PubMed

    Dicker, Frank; Schnittger, Susanne; Haferlach, Torsten; Kern, Wolfgang; Schoch, Claudia

    2006-11-01

    Compared with fluorescence in situ hybridization (FISH), conventional metaphase cytogenetics play only a minor prognostic role in chronic lymphocytic leukemia (CLL) so far, due to technical problems resulting from limited proliferation of CLL cells in vitro. Here, we present a simple method for in vitro stimulation of CLL cells that overcomes this limitation. In our unselected patient population, 125 of 132 cases could be successfully stimulated for metaphase generation by culture with the immunostimulatory CpG-oligonucleotide DSP30 plus interleukin 2. Of 125 cases, 101 showed chromosomal aberrations. The aberration rate is comparable to the rate detected by parallel interphase FISH. In 47 patients, conventional cytogenetics detected additional aberrations not detected by FISH analysis. A complex aberrant karyotype, defined as one having at least 3 aberrations, was detected in 30 of 125 patients, compared with only one such case as defined by FISH. Conventional cytogenetics frequently detected balanced and unbalanced translocations. A significant correlation of the poor-prognosis unmutated IgV(H) status with unbalanced translocations and of the likewise poor-prognosis CD38 expression to balanced translocations and complex aberrant karyotype was found. We demonstrate that FISH analysis underestimates the complexity of chromosomal aberrations in CLL. Therefore, conventional cytogenetics may define subgroups of patients with high risk of progression.

  20. Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis

    PubMed Central

    Deplus, Rachel; Lampe, Xavier; Krusy, Nathalie; Calonne, Emilie; Delbecque, Katty; Kridelka, Frederic; Fuks, François; Ennaji, My Mustapha; Delvenne, Philippe

    2012-01-01

    Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. PMID:22880087

  1. Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

    PubMed Central

    2010-01-01

    Background Transmembrane proteins (TM proteins) make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY). All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%). In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical characteristics. Surveys like this

  2. Dark proteins: effect of inclusion body formation on quantification of protein expression.

    PubMed

    Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R

    2008-09-01

    Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used

  3. Transplacental exposure to inorganic arsenic at a hepatocarcinogenic dose induces fetal gene expression changes in mice indicative of aberrant estrogen signaling and disrupted steroid metabolism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu Jie; Xie Yaxiong; Cooper, Ryan

    Exposure to inorganic arsenic in utero in C3H mice produces hepatocellular carcinoma in male offspring when they reach adulthood. To help define the molecular events associated with the fetal onset of arsenic hepatocarcinogenesis, pregnant C3H mice were given drinking water containing 0 (control) or 85 ppm arsenic from day 8 to 18 of gestation. At the end of the arsenic exposure period, male fetal livers were removed and RNA isolated for microarray analysis using 22K oligo chips. Arsenic exposure in utero produced significant (p < 0.001) alterations in expression of 187 genes, with approximately 25% of aberrantly expressed genes relatedmore » to either estrogen signaling or steroid metabolism. Real-time RT-PCR on selected genes confirmed these changes. Various genes controlled by estrogen, including X-inactive-specific transcript, anterior gradient-2, trefoil factor-1, CRP-ductin, ghrelin, and small proline-rich protein-2A, were dramatically over-expressed. Estrogen-regulated genes including cytokeratin 1-19 and Cyp2a4 were over-expressed, although Cyp3a25 was suppressed. Several genes involved with steroid metabolism also showed remarkable expression changes, including increased expression of 17{beta}-hydroxysteroid dehydrogenase-7 (HSD17{beta}7; involved in estradiol production) and decreased expression of HSD17{beta}5 (involved in testosterone production). The expression of key genes important in methionine metabolism, such as methionine adenosyltransferase-1a, betaine-homocysteine methyltransferase and thioether S-methyltransferase, were suppressed. Thus, exposure of mouse fetus to inorganic arsenic during a critical period in development significantly alters the expression of various genes encoding estrogen signaling and steroid or methionine metabolism. These alterations could disrupt genetic programming at the very early life stage, which could impact tumor formation much later in adulthood.« less

  4. TET1 Depletion Induces Aberrant CpG Methylation in Colorectal Cancer Cells

    PubMed Central

    Yamamoto, Eiichiro; Harada, Taku; Aoki, Hironori; Maruyama, Reo; Toyota, Mutsumi; Sasaki, Yasushi; Sugai, Tamotsu; Tokino, Takashi; Nakase, Hiroshi

    2016-01-01

    Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mechanism is not fully understood. 5-hydroxymethylcytosine levels and TET1 expression are both reduced in CRC, while epigenetic silencing of TET1 is reportedly associated with the CpG island methylator phenotype. In the present study, we aimed to clarify the relationship between loss of TET1 and aberrant DNA methylation in CRC. Stable TET1 knockdown clones were established using Colo320DM cells, which express high levels of TET1, and HCT116 cells, which express TET1 at a level similar to that in normal colonic tissue. Infinium HumanMethylation450 BeadChip assays revealed increased levels of 5-methylcytosine at more than 10,000 CpG sites in TET1-depleted Colo320DM cells. Changes in DNA methylation were observed at various positions within the genome, including promoters, gene bodies and intergenic regions, and the altered methylation affected expression of a subset of genes. By contrast, TET1 knockdown did not significantly affect DNA methylation in HCT116 cells. However, TET1 depletion was associated with attenuated effects of 5-aza-2’-deoxycytidine on gene expression profiles in both cell lines. These results suggest that loss of TET1 may induce aberrant DNA methylation and may attenuate the effect of 5-aza-2’-deoxycytidine in CRC cells. PMID:27977763

  5. Effects of immunosuppressive treatment on protein expression in rat kidney

    PubMed Central

    Kędzierska, Karolina; Sporniak-Tutak, Katarzyna; Sindrewicz, Krzysztof; Bober, Joanna; Domański, Leszek; Parafiniuk, Mirosław; Urasińska, Elżbieta; Ciechanowicz, Andrzej; Domański, Maciej; Smektała, Tomasz; Masiuk, Marek; Skrzypczak, Wiesław; Ożgo, Małgorzata; Kabat-Koperska, Joanna; Ciechanowski, Kazimierz

    2014-01-01

    The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents’ toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins’ synthesis. Very slight differences were observed between the group receiving cyclosporine, mycophenolate mofetil, and glucocorticoids (CMG) and the control group. In contrast, compared to the control group, animals receiving tacrolimus, mycophenolate mofetil, and glucocorticoids (TMG) exhibited higher expression of proteins responsible for renal drug metabolism and lower expression levels of cytoplasmic actin and the major urinary protein. In the TMG group, we observed higher expression of proteins responsible for drug metabolism and a decrease in the expression of respiratory chain enzymes (thioredoxin-2) and markers of distal renal tubular damage (heart fatty acid-binding protein) compared to expression in the CMG

  6. Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

    PubMed

    He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

    2017-04-01

    Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

  7. Aberrant Cortical Activity in Multiple GCaMP6-Expressing Transgenic Mouse Lines

    PubMed Central

    Buetfering, Christina; Groblewski, Peter A.; Manavi, Sahar; Miles, Jesse; White, Casey; Griffin, Fiona; Roll, Kate; Cross, Sissy; Nguyen, Thuyanh V.; Larsen, Rachael; Daigle, Tanya; Thompson, Carol L.; Olsen, Shawn; Hausser, Michael

    2017-01-01

    Abstract Transgenic mouse lines are invaluable tools for neuroscience but, as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, of either sex, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, although rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study. PMID:28932809

  8. Aberrant Cx26 hemichannels and keratitis-ichthyosis-deafness syndrome: insights into syndromic hearing loss

    PubMed Central

    Sanchez, Helmuth A.; Verselis, Vytas K.

    2014-01-01

    Mutation of the GJB2 gene, which encodes the connexin 26 (Cx26) gap junction (GJ) protein, is the most common cause of hereditary, sensorineural hearing loss. Cx26 is not expressed in hair cells, but is widely expressed throughout the non-sensory epithelial cells of the cochlea. Most GJB2 mutations produce non-syndromic deafness, but a subset produces syndromic deafness in which profound hearing loss is accompanied by a diverse array of infectious and neoplastic cutaneous disorders that can be fatal. Although GJ channels, which are assembled by the docking of two, so-called hemichannels (HCs), have been the main focus of deafness-associated disease models, it is now evident that the HCs themselves can function in the absence of docking and contribute to signaling across the cell membrane as a novel class of ion channel. A notable feature of syndromic deafness mutants is that the HCs exhibit aberrant behaviors providing a plausible basis for disease that is associated with excessive or altered contributions of Cx26 HCs that, in turn, lead to compromised cell integrity. Here we discuss some of the aberrant Cx26 HC properties that have been described for mutants associated with keratitis-ichthyosis-deafness (KID) syndrome, a particularly severe Cx26-associated syndrome, which shed light on genotype-phenotype relationships and causes underlying cochlear dysfunction. PMID:25386120

  9. Aberrant neuronal activity-induced signaling and gene expression in a mouse model of RASopathy

    PubMed Central

    Nakhaei-Rad, Saeideh; Montenegro-Venegas, Carolina; Pina-Fernández, Eneko; Marini, Claudia; Santos, Monica; Ahmadian, Mohammad R.; Stork, Oliver; Zenker, Martin

    2017-01-01

    Noonan syndrome (NS) is characterized by reduced growth, craniofacial abnormalities, congenital heart defects, and variable cognitive deficits. NS belongs to the RASopathies, genetic conditions linked to mutations in components and regulators of the Ras signaling pathway. Approximately 50% of NS cases are caused by mutations in PTPN11. However, the molecular mechanisms underlying cognitive impairments in NS patients are still poorly understood. Here, we report the generation and characterization of a new conditional mouse strain that expresses the overactive Ptpn11D61Y allele only in the forebrain. Unlike mice with a global expression of this mutation, this strain is viable and without severe systemic phenotype, but shows lower exploratory activity and reduced memory specificity, which is in line with a causal role of disturbed neuronal Ptpn11 signaling in the development of NS-linked cognitive deficits. To explore the underlying mechanisms we investigated the neuronal activity-regulated Ras signaling in brains and neuronal cultures derived from this model. We observed an altered surface expression and trafficking of synaptic glutamate receptors, which are crucial for hippocampal neuronal plasticity. Furthermore, we show that the neuronal activity-induced ERK signaling, as well as the consecutive regulation of gene expression are strongly perturbed. Microarray-based hippocampal gene expression profiling revealed profound differences in the basal state and upon stimulation of neuronal activity. The neuronal activity-dependent gene regulation was strongly attenuated in Ptpn11D61Y neurons. In silico analysis of functional networks revealed changes in the cellular signaling beyond the dysregulation of Ras/MAPK signaling that is nearly exclusively discussed in the context of NS at present. Importantly, changes in PI3K/AKT/mTOR and JAK/STAT signaling were experimentally confirmed. In summary, this study uncovers aberrant neuronal activity-induced signaling and regulation

  10. Interpreting Chromosome Aberration Spectra

    NASA Technical Reports Server (NTRS)

    Levy, Dan; Reeder, Christopher; Loucas, Bradford; Hlatky, Lynn; Chen, Allen; Cornforth, Michael; Sachs, Rainer

    2007-01-01

    Ionizing radiation can damage cells by breaking both strands of DNA in multiple locations, essentially cutting chromosomes into pieces. The cell has enzymatic mechanisms to repair such breaks; however, these mechanisms are imperfect and, in an exchange process, may produce a large-scale rearrangement of the genome, called a chromosome aberration. Chromosome aberrations are important in killing cells, during carcinogenesis, in characterizing repair/misrepair pathways, in retrospective radiation biodosimetry, and in a number of other ways. DNA staining techniques such as mFISH ( multicolor fluorescent in situ hybridization) provide a means for analyzing aberration spectra by examining observed final patterns. Unfortunately, an mFISH observed final pattern often does not uniquely determine the underlying exchange process. Further, resolution limitations in the painting protocol sometimes lead to apparently incomplete final patterns. We here describe an algorithm for systematically finding exchange processes consistent with any observed final pattern. This algorithm uses aberration multigraphs, a mathematical formalism that links the various aspects of aberration formation. By applying a measure to the space of consistent multigraphs, we will show how to generate model-specific distributions of aberration processes from mFISH experimental data. The approach is implemented by software freely available over the internet. As a sample application, we apply these algorithms to an aberration data set, obtaining a distribution of exchange cycle sizes, which serves to measure aberration complexity. Estimating complexity, in turn, helps indicate how damaging the aberrations are and may facilitate identification of radiation type in retrospective biodosimetry.

  11. Expression of multidrug resistance proteins in retinoblastoma

    PubMed Central

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307

  12. Different HER2 protein expression profiles aid in the histologic differential diagnosis between urothelial carcinoma in situ (CIS) and non-CIS conditions (dysplasia and reactive atypia) of the urinary bladder mucosa.

    PubMed

    Gunia, Sven; Koch, Stefan; Hakenberg, Oliver W; May, Matthias; Kakies, Christoph; Erbersdobler, Andreas

    2011-12-01

    We evaluated HER2 expression profiles in 32 carcinoma in situ (CIS) and 31 non-CIS conditions (5 dysplasia and 26 reactive atypia) of the urinary bladder mucosa by applying breast cancer scoring rules. In situ hybridization was performed on tissue microarrays to assess HER2 gene amplification status. Our immunoprofiling data disclosed moderate to strong HER2 expression in CIS, including the basal layer of the urothelium, and absent to weak HER2 expression in non-CIS conditions. From the histologic differential diagnostic standpoint, immunostaining for HER2 protein represents a useful adjunct to aid in the delineation between CIS and non-CIS conditions of the bladder mucosa. Pathogenically, aberrant HER2 protein expression in CIS seems to be more commonly associated with polysomy than with gene amplification. From a therapeutic viewpoint, prospective clinical studies should investigate the potential benefit of HER2-targeted therapies in CIS, particularly in cases unresponsive to conventional therapeutic regimens.

  13. The homeobox gene CDX2 is aberrantly expressed in most cases of acute myeloid leukemia and promotes leukemogenesis

    PubMed Central

    Scholl, Claudia; Bansal, Dimple; Döhner, Konstanze; Eiwen, Karina; Huntly, Brian J.P.; Lee, Benjamin H.; Rücker, Frank G.; Schlenk, Richard F.; Bullinger, Lars; Döhner, Hartmut; Gilliland, D. Gary; Fröhling, Stefan

    2007-01-01

    The homeobox transcription factor CDX2 plays an important role in embryonic development and regulates the proliferation and differentiation of intestinal epithelial cells in the adult. We have found that CDX2 is expressed in leukemic cells of 90% of patients with acute myeloid leukemia (AML) but not in hematopoietic stem and progenitor cells derived from normal individuals. Stable knockdown of CDX2 expression by RNA interference inhibited the proliferation of various human AML cell lines and strongly reduced their clonogenic potential in vitro. Primary murine hematopoietic progenitor cells transduced with Cdx2 acquired serial replating activity, were able to be continuously propagated in liquid culture, generated fully penetrant and transplantable AML in BM transplant recipients, and displayed dysregulated expression of Hox family members in vitro and in vivo. These results demonstrate that aberrant expression of the developmental regulatory gene CDX2 in the adult hematopoietic compartment is a frequent event in the pathogenesis of AML; suggest a role for CDX2 as part of a common effector pathway that promotes the proliferative capacity and self-renewal potential of myeloid progenitor cells; and support the hypothesis that CDX2 is responsible, in part, for the altered HOX gene expression that is observed in most cases of AML. PMID:17347684

  14. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    PubMed

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Analysis of nodal aberration properties in off-axis freeform system design.

    PubMed

    Shi, Haodong; Jiang, Huilin; Zhang, Xin; Wang, Chao; Liu, Tao

    2016-08-20

    Freeform surfaces have the advantage of balancing off-axis aberration. In this paper, based on the framework of nodal aberration theory (NAT) applied to the coaxial system, the third-order astigmatism and coma wave aberration expressions of an off-axis system with Zernike polynomial surfaces are derived. The relationship between the off-axis and surface shape acting on the nodal distributions is revealed. The nodal aberration properties of the off-axis freeform system are analyzed and validated by using full-field displays (FFDs). It has been demonstrated that adding Zernike terms, up to nine, to the off-axis system modifies the nodal locations, but the field dependence of the third-order aberration does not change. On this basis, an off-axis two-mirror freeform system with 500 mm effective focal length (EFL) and 300 mm entrance pupil diameter (EPD) working in long-wave infrared is designed. The field constant aberrations induced by surface tilting are corrected by selecting specific Zernike terms. The design results show that the nodes of third-order astigmatism and coma move back into the field of view (FOV). The modulation transfer function (MTF) curves are above 0.4 at 20 line pairs per millimeter (lp/mm) which meets the infrared reconnaissance requirement. This work provides essential insight and guidance for aberration correction in off-axis freeform system design.

  16. RNA-Binding Proteins in Female Reproductive Pathologies.

    PubMed

    Khalaj, Kasra; Miller, Jessica E; Fenn, Christian R; Ahn, SooHyun; Luna, Rayana L; Symons, Lindsey; Monsanto, Stephany P; Koti, Madhuri; Tayade, Chandrakant

    2017-06-01

    RNA-binding proteins are key regulatory molecules involved primarily in post-transcriptional gene regulation of RNAs. Post-transcriptional gene regulation is critical for adequate cellular growth and survival. Recent reports have shown key interactions between these RNA-binding proteins and other regulatory elements, such as miRNAs and long noncoding RNAs, either enhancing or diminishing their response to RNA stabilization. Many RNA-binding proteins have been reported to play a functional role in mediation of cytokines involved in inflammation and immune dysfunction, and some have been classified as global post-transcriptional regulators of inflammation. The ubiquitous expression of RNA-binding proteins in a wide variety of cell types and their unique mechanisms of degradative action provide evidence that they are involved in reproductive tract pathologies. Aberrant inflammation and immune dysfunction are major contributors to the pathogenesis and disease pathophysiology of many reproductive pathologies, including ovarian and endometrial cancers in the female reproductive tract. Herein, we discuss various RNA-binding proteins and their unique contributions to female reproductive pathologies with a focus on those mediated by aberrant inflammation and immune dysfunction. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  17. In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

    PubMed Central

    Cicek, Kader; Gulec, Burcu; Ungor, Rifat; Hasanova, Gulnara

    2017-01-01

    A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes. PMID:28827815

  18. The significance of PTEN and AKT aberrations in pediatric T-cell acute lymphoblastic leukemia

    PubMed Central

    Zuurbier, Linda; Petricoin, Emanuel F.; Vuerhard, Maartje J.; Calvert, Valerie; Kooi, Clarissa; Buijs-Gladdines, Jessica G.C.A.M.; Smits, Willem K.; Sonneveld, Edwin; Veerman, Anjo J.P.; Kamps, Willem A.; Horstmann, Martin; Pieters, Rob; Meijerink, Jules P.P.

    2012-01-01

    Background PI3K/AKT pathway mutations are found in T-cell acute lymphoblastic leukemia, but their overall impact and associations with other genetic aberrations is unknown. PTEN mutations have been proposed as secondary mutations that follow NOTCH1-activating mutations and cause cellular resistance to γ-secretase inhibitors. Design and Methods The impact of PTEN, PI3K and AKT aberrations was studied in a genetically well-characterized pediatric T-cell leukemia patient cohort (n=146) treated on DCOG or COALL protocols. Results PTEN and AKT E17K aberrations were detected in 13% and 2% of patients, respectively. Defective PTEN-splicing was identified in incidental cases. Patients without PTEN protein but lacking exon-, splice-, promoter mutations or promoter hypermethylation were present. PTEN/AKT mutations were especially abundant in TAL- or LMO-rearranged leukemia but nearly absent in TLX3-rearranged patients (P=0.03), the opposite to that observed for NOTCH1-activating mutations. Most PTEN/AKT mutant patients either lacked NOTCH1-activating mutations (P=0.006) or had weak NOTCH1-activating mutations (P=0.011), and consequently expressed low intracellular NOTCH1, cMYC and MUSASHI levels. T-cell leukemia patients without PTEN/AKT and NOTCH1-activating mutations fared well, with a cumulative incidence of relapse of only 8% versus 35% for PTEN/AKT and/or NOTCH1-activated patients (P=0.005). Conclusions PI3K/AKT pathway aberrations are present in 18% of pediatric T-cell acute lymphoblastic leukemia patients. Absence of strong NOTCH1-activating mutations in these cases may explain cellular insensitivity to γ-secretase inhibitors. PMID:22491738

  19. DNA Repair Defects and Chromosomal Aberrations

    NASA Technical Reports Server (NTRS)

    Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

    2009-01-01

    Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

  20. Advances in recombinant protein expression for use in pharmaceutical research.

    PubMed

    Assenberg, Rene; Wan, Paul T; Geisse, Sabine; Mayr, Lorenz M

    2013-06-01

    Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells. Copyright © 2013. Published by Elsevier Ltd.

  1. Using Peptide-Level Proteomics Data for Detecting Differentially Expressed Proteins.

    PubMed

    Suomi, Tomi; Corthals, Garry L; Nevalainen, Olli S; Elo, Laura L

    2015-11-06

    The expression of proteins can be quantified in high-throughput means using different types of mass spectrometers. In recent years, there have emerged label-free methods for determining protein abundance. Although the expression is initially measured at the peptide level, a common approach is to combine the peptide-level measurements into protein-level values before differential expression analysis. However, this simple combination is prone to inconsistencies between peptides and may lose valuable information. To this end, we introduce here a method for detecting differentially expressed proteins by combining peptide-level expression-change statistics. Using controlled spike-in experiments, we show that the approach of averaging peptide-level expression changes yields more accurate lists of differentially expressed proteins than does the conventional protein-level approach. This is particularly true when there are only few replicate samples or the differences between the sample groups are small. The proposed technique is implemented in the Bioconductor package PECA, and it can be downloaded from http://www.bioconductor.org.

  2. SBDS Protein Expression Patterns in the Bone Marrow

    PubMed Central

    Wong, Trisha E.; Calicchio, Monica L.; Fleming, Mark D.; Shimamura, Akiko; Harris, Marian H.

    2010-01-01

    Shwachman Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome caused by biallelic SBDS gene mutations. Here we examined SBDS protein levels in human bone marrow. SBDS protein expression was high in neutrophil progenitors, megakaryocytes, plasma cells and osteoblasts. In contrast, SBDS protein levels were low in all hematopoietic cell lineages from patients harboring the common SBDS mutations. We conclude that SBDS protein levels vary widely between specific marrow lineages. Uniformly low SBDS protein expression levels distinguish the majority of SDS patients from controls or other marrow failure syndromes. PMID:20658628

  3. Vectors for co-expression of an unrestricted number of proteins

    PubMed Central

    Scheich, Christoph; Kümmel, Daniel; Soumailakakis, Dimitri; Heinemann, Udo; Büssow, Konrad

    2007-01-01

    A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells. PMID:17311810

  4. [Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].

    PubMed

    Yin, Xiaotao; Wang, Wei; Tian, Renli; Xu, Yuanji; Yan, Jinqi; Zhang, Wei; Gao, Jiangping; Yu, Jiyun

    2013-08-01

    To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity. Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA. The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA. The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.

  5. The growing impact of lyophilized cell-free protein expression systems

    PubMed Central

    Hunt, J. Porter; Yang, Seung Ook; Wilding, Kristen M.; Bundy, Bradley C.

    2017-01-01

    ABSTRACT Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis. PMID:27791452

  6. Aberrant Gene Expression Profiles in Pluripotent Stem Cells Induced from Fibroblasts of a Klinefelter Syndrome Patient*

    PubMed Central

    Ma, Yu; Li, Chunliang; Gu, Junjie; Tang, Fan; Li, Chun; Li, Peng; Ping, Ping; Yang, Shi; Li, Zheng; Jin, Ying

    2012-01-01

    Klinefelter syndrome (KS) is the most common male chromosome aneuploidy. Its pathophysiology is largely unexplained due to the lack of adequate models. Here, we report the derivation of induced pluripotent stem cell (iPSCs) lines from a KS patient with a karyotype of 47, XXY. Derived KS-iPSCs meet all criteria of normal iPSCs with the potential for germ cell differentiation. Although X chromosome inactivation occurs in all KS-iPSCs, genome-wide transcriptome analysis identifies aberrantly expressed genes associated with the clinical features of KS. Our KS-iPSCs can serve as a cellular model for KS research. Identified genes may become biomarkers for early diagnosis or potential therapeutic targets for KS and significantly accelerate the understanding, diagnosis, and treatment of Klinefelter syndrome. PMID:23019320

  7. Aberrant expression of HMB-45 in traumatized melanocytic nevi.

    PubMed

    Leleux, Todd M; Prieto, Victor G; Diwan, A Hafeez

    2012-09-01

    Assessment of histologic and immunohistochemical maturation (with HMB-45 and anti-Ki-67) may be helpful in differentiating benign melanocytic nevi (BMN) from malignant melanoma. Recently, we reported loss of maturation and aberrant immunohistochemical findings in melanocytic nevi after liquid nitrogen cryotherapy (Adeniran et al, J Am Acad Dermatol 2009;61:341-5). Herein we report a similar phenomenon identified in traumatized melanocytic nevi (TMN). We sought to evaluate the histologic and immunohistochemical findings in early and late stages of traumatized nevi. Twenty-four cases of TMN were retrieved from the pathology archives. These were then assessed by two pathologists (T.L. and A.H.D.) using HMB-45 and MIB-1 (for Ki-67) antibodies. TMN showed some of the following findings: epidermal changes (parakeratosis, ulceration, serum crust, flattening of the epidermis) and dermal changes including fibrosis and the presence of melanophages. In some cases, there was architectural disorder of the overlying melanocytes, with crowding in the basal layer, but without significant pagetoid spread. Occasionally, the dermal scar contained larger, more epithelioid-appearing melanocytes than those beneath the scar. Fifty-four percent of TMN lacked obvious immunohistochemical maturation with HMB-45, since nevus cells within the scar or directly beneath it were strongly labeled. None of the TMN showed appreciable labeling for Ki-67. The exact clinical duration between trauma and biopsy could not be determined. Loss of maturation with HMB-45 in TMN can be a diagnostic pitfall in challenging cases. Concurrent evaluation of MIB-1 expression, along with the characteristic histologic features of trauma, should allow the correct diagnosis to be reached. Copyright © 2011 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

  8. Aberrant phenotypes in peripheral T cell lymphomas.

    PubMed Central

    Hastrup, N; Ralfkiaer, E; Pallesen, G

    1989-01-01

    Seventy six peripheral T cell lymphomas were examined immunohistologically to test their reactivity with a panel of monoclonal antibodies against 11 T cell associated antigens (CD1-8, CD27, UCHL1, and the T cell antigen receptor). Sixty two (82%) lymphomas showed aberrant phenotypes, and four main categories were distinguished as follows: (i) lack of one or several pan-T cell antigens (49, 64% of the cases); (ii) loss of both the CD4 and CD8 antigens (11, 15% of the cases); (iii) coexpression of the CD4 and CD8 antigens (13, 17% of the cases); and (iv) expression of the CD1 antigen (eight, 11% of the cases). No correlation was seen between the occurrence of aberrant phenotypes and the histological subtype. It is concluded that the demonstration of an aberrant phenotype is a valuable supplement to histological assessment in the diagnosis of peripheral T cell lymphomas. It is recommended that the panel of monoclonal antibodies against T cell differentiation antigens should be fairly large, as apparently any antigen may be lost in the process of malignant transformation. Images Figure PMID:2469701

  9. Aberrant DNA Methylation as a Biomarker and a Therapeutic Target of Cholangiocarcinoma.

    PubMed

    Nakaoka, Toshiaki; Saito, Yoshimasa; Saito, Hidetsugu

    2017-05-23

    Cholangiocarcinoma is an epithelial malignancy arising in the region between the intrahepatic bile ducts and the ampulla of Vater at the distal end of the common bile duct. The effect of current chemotherapy regimens against cholangiocarcinoma is limited, and the prognosis of patients with cholangiocarcinoma is poor. Aberrant DNA methylation and histone modification induce silencing of tumor suppressor genes and chromosomal instability during carcinogenesis. Studies have shown that the tumor suppressor genes and microRNAs (miRNAs) including MLH1 , p14 , p16 , death-associated protein kinase ( DAPK ), miR-370 and miR-376c are frequently methylated in cholangiocarcinoma. Silencing of these tumor suppressor genes and miRNAs plays critical roles in the initiation and progression of cholangiocarcinoma. In addition, recent studies have demonstrated that DNA methylation inhibitors induce expression of endogenous retroviruses and exert the anti-tumor effect of via an anti-viral immune response. Aberrant DNA methylation of tumor suppressor genes and miRNAs could be a powerful biomarker for the diagnosis and treatment of cholangiocarcinoma. Epigenetic therapy with DNA methylation inhibitors holds considerable promise for the treatment of cholangiocarcinoma through the reactivation of tumor suppressor genes and miRNAs as well as the induction of an anti-viral immune response.

  10. Aberrant DNA Methylation as a Biomarker and a Therapeutic Target of Cholangiocarcinoma

    PubMed Central

    Nakaoka, Toshiaki; Saito, Yoshimasa; Saito, Hidetsugu

    2017-01-01

    Cholangiocarcinoma is an epithelial malignancy arising in the region between the intrahepatic bile ducts and the ampulla of Vater at the distal end of the common bile duct. The effect of current chemotherapy regimens against cholangiocarcinoma is limited, and the prognosis of patients with cholangiocarcinoma is poor. Aberrant DNA methylation and histone modification induce silencing of tumor suppressor genes and chromosomal instability during carcinogenesis. Studies have shown that the tumor suppressor genes and microRNAs (miRNAs) including MLH1, p14, p16, death-associated protein kinase (DAPK), miR-370 and miR-376c are frequently methylated in cholangiocarcinoma. Silencing of these tumor suppressor genes and miRNAs plays critical roles in the initiation and progression of cholangiocarcinoma. In addition, recent studies have demonstrated that DNA methylation inhibitors induce expression of endogenous retroviruses and exert the anti-tumor effect of via an anti-viral immune response. Aberrant DNA methylation of tumor suppressor genes and miRNAs could be a powerful biomarker for the diagnosis and treatment of cholangiocarcinoma. Epigenetic therapy with DNA methylation inhibitors holds considerable promise for the treatment of cholangiocarcinoma through the reactivation of tumor suppressor genes and miRNAs as well as the induction of an anti-viral immune response. PMID:28545228

  11. Reconstructive correction of aberrations in nuclear particle spectrographs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Berz, M.; Joh, K.; Nolen, J.A.

    A method is presented that allows the reconstruction of trajectories in particle spectrographs and the reconstructive correction of residual aberrations that otherwise limit the resolution. Using a computed or fitted high order transfer map that describes the uncorrected aberrations of the spectrograph, it is possible to calculate a map via an analytic recursion relation that allows the computation of the corrected data of interest such as reaction energy and scattering angle as well as the reconstructed trajectories in terms of position measurements in two planes near the focal plane. The technique is only limited by the accuracy of the positionmore » measurements, the incoherent spot sizes, and the accuracy of the transfer map. In practice the method can be expressed as an inversion of a nonlinear map and implemented in the differential algebraic framework. The method is applied to correct residual aberrations in the S800 spectrograph which is under construction at the National Superconducting Cyclotron Laboratory at Michigan State University and to two other high resolution spectrographs.« less

  12. Study in mice shows that an aggressive type of breast cancer is linked to an inflammatory protein

    Cancer.gov

    Aberrant expression of an inflammatory protein, nitric oxide synthase 2 (NOS2), may enhance the progression and metastasis of an aggressive and less common form of breast cancer, known as the estrogen receptor-negative type of disease.

  13. [Expression of c-jun protein after experimental rat brain concussion].

    PubMed

    Wang, Feng; Li, Yong-hong

    2010-02-01

    To observe e-jun protein expression after rat brain concussion and explore the forensic pathologic markers following brain concussion. Fifty-five rats were randomly divided into brain concussion group and control group. The expression of c-jun protein was observed by immunohistochemistry. There were weak positive expression of c-jun protein in control group. In brain concussion group, however, some neutrons showed positive expression of c-jun protein at 15 min after brain concussion, and reach to the peak at 3 h after brain concussion. The research results suggest that detection of c-jun protein could be a marker to determine brain concussion and estimate injury time after brain concussion.

  14. Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

    PubMed Central

    Rasala, Beth A; Muto, Machiko; Lee, Philip A; Jager, Michal; Cardoso, Rosa MF; Behnke, Craig A; Kirk, Peter; Hokanson, Craig A; Crea, Roberto; Mendez, Michael; Mayfield, Stephen P

    2010-01-01

    Summary Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear if this is due to few attempts or to limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, greater than 50% expressed at levels sufficient for commercial production. Three expressed at 2% to 3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well-expressed serum amyloid protein. All of the algal chloroplast-expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivty achieved demonstrate the utility of C. reinhardtii as a robust platform for human therapeutic protein production. PMID:20230484

  15. Trichostatin A specifically improves the aberrant expression of transcription factor genes in embryos produced by somatic cell nuclear transfer

    PubMed Central

    Inoue, Kimiko; Oikawa, Mami; Kamimura, Satoshi; Ogonuki, Narumi; Nakamura, Toshinobu; Nakano, Toru; Abe, Kuniya; Ogura, Atsuo

    2015-01-01

    Although mammalian cloning by somatic cell nuclear transfer (SCNT) has been established in various species, the low developmental efficiency has hampered its practical applications. Treatment of SCNT-derived embryos with histone deacetylase (HDAC) inhibitors can improve their development, but the underlying mechanism is still unclear. To address this question, we analysed gene expression profiles of SCNT-derived 2-cell mouse embryos treated with trichostatin A (TSA), a potent HDAC inhibitor that is best used for mouse cloning. Unexpectedly, TSA had no effect on the numbers of aberrantly expressed genes or the overall gene expression pattern in the embryos. However, in-depth investigation by gene ontology and functional analyses revealed that TSA treatment specifically improved the expression of a small subset of genes encoding transcription factors and their regulatory factors, suggesting their positive involvement in de novo RNA synthesis. Indeed, introduction of one of such transcription factors, Spi-C, into the embryos at least partially mimicked the TSA-induced improvement in embryonic development by activating gene networks associated with transcriptional regulation. Thus, the effects of TSA treatment on embryonic gene expression did not seem to be stochastic, but more specific than expected, targeting genes that direct development and trigger zygotic genome activation at the 2-cell stage. PMID:25974394

  16. Gene promoter methylation and protein expression of BRMS1 in uterine cervix in relation to high-risk human papilloma virus infection and cancer.

    PubMed

    Panagopoulou, Maria; Lambropoulou, Maria; Balgkouranidou, Ioanna; Nena, Evangelia; Karaglani, Makrina; Nicolaidou, Christina; Asimaki, Anthi; Konstantinidis, Theocharis; Constantinidis, Theodoros C; Kolios, George; Kakolyris, Stylianos; Agorastos, Theodoros; Chatzaki, Ekaterini

    2017-04-01

    Cervical cancer is strongly related to certain high-risk types of human papilloma virus infection. Breast cancer metastasis suppressor 1 (BRMS1) is a tumor suppressor gene, its expression being regulated by DNA promoter methylation in several types of cancers. This study aims to evaluate the methylation status of BRMS1 promoter in relation to high-risk types of human papilloma virus infection and the development of pre-cancerous lesions and describe the pattern of BRMS1 protein expression in normal, high-risk types of human papilloma virus-infected pre-cancerous and malignant cervical epithelium. We compared the methylation status of BRMS1 in cervical smears of 64 women with no infection by high-risk types of human papilloma virus to 70 women with proven high-risk types of human papilloma virus infection, using real-time methylation-specific polymerase chain reaction. The expression of BRMS1 protein was described by immunohistochemistry in biopsies from cervical cancer, pre-cancerous lesions, and normal cervices. Methylation of BRMS1 promoter was detected in 37.5% of women with no high-risk types of human papilloma virus infection and was less frequent in smears with high-risk types of human papilloma virus (11.4%) and in women with pathological histology (cervical intraepithelial neoplasia) (11.9%). Methylation was detected also in HeLa cervical cancer cells. Immunohistochemistry revealed nuclear BRMS1 protein staining in normal high-risk types of human papilloma virus-free cervix, in cervical intraepithelial neoplasias, and in malignant tissues, where staining was occasionally also cytoplasmic. In cancer, expression was stronger in the more differentiated cancer blasts. In conclusion, BRMS1 promoter methylation and aberrant protein expression seem to be related to high-risk types of human papilloma virus-induced carcinogenesis in uterine cervix and is worthy of further investigation.

  17. Low-Order Aberrations in Band-limited Lyot Coronagraphs

    NASA Astrophysics Data System (ADS)

    Sivaramakrishnan, Anand; Soummer, Rémi; Sivaramakrishnan, Allic V.; Lloyd, James P.; Oppenheimer, Ben R.; Makidon, Russell B.

    2005-12-01

    We study the way Lyot coronagraphs with unapodized entrance pupils respond to small, low-order phase aberrations. This study is applicable to ground-based adaptive optics coronagraphs operating at 90% and higher Strehl ratios, as well as to some space-based coronagraphs with intrinsically higher Strehl ratio imaging. We utilize a second-order expansion of the monochromatic point-spread function (written as a power spectrum of a power series in the phase aberration over clear aperture) to derive analytical expressions for the response of a ``band-limited'' Lyot coronagraph (BLC) to small, low-order, phase aberrations. The BLC possesses a focal plane mask with an occulting spot whose opacity profile is a spatially band-limited function rather than a hard-edged, opaque disk. The BLC is, to first order, insensitive to tilt and astigmatism. Undersizing the stop in the reimaged pupil plane (the Lyot plane) following the focal plane mask can alleviate second-order effects of astigmatism, at the expense of system throughput and angular resolution. The optimal degree of such undersizing depends on individual instrument designs and goals. Our analytical work engenders physical insight and complements existing numerical work on this subject. Our methods can be extended to treat the passage of higher order aberrations through band-limited Lyot coronagraphs by using our polynomial decomposition or an analogous Fourier approach.

  18. Aberrant expression of stress-induced phosphoprotein 1 in colorectal cancer and its clinicopathologic significance.

    PubMed

    Zhang, Zhimei; Ren, Hui; Yang, Liang; Zhang, Xinhua; Liang, Wei; Wu, Hui; Huang, Leilei; Kang, Jihui; Xu, Jianbo; Zhai, Ertao; Cai, Shirong; He, Yulong

    2018-06-04

    Stress-Inducible Phosphoprotein1 (STIP1) is an adaptor protein that bridges HSP70 and HSP90 folding and a secretory protein that regulates malignant tumor progression. The aim of the present study was to demonstrate the clinicopathological significance and prognostic role of STIP1 in colorectal cancer (CRC). We used data from The Cancer Genome Atlas (TCGA) to analyze STIP1 expression in CRC and utilized 8 pairs of fresh-frozen tissue samples to investigate STIP1 expression in CRC tissues and adjacent normal tissues using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays. We also used immunohistochemical staining to detect STIP1 expression in 144 formalin-fixed, paraffin-embedded (FFPE) CRC tissue samples and determine the clinical significance of STIP1 expression in CRC. The results of bioinformatics analysis, qRT-PCR, and western blot showed that STIP1 expression was higher in CRC tissues than in adjacent normal tissues. High STIP1 expression was significantly correlated with advanced T stage (P=.01), N stage (P=.001), M stage (P﹤0.001), and TNM stage (P﹤0.001). Moreover, Kaplan-Meier analyses indicated that higher STIP1 expression predicted a worse prognosis in patients with CRC, and Cox regression analysis revealed that STIP1 was an independent prognostic factor for overall survival and disease-free survival in patients with CRC. In conclusion, our results suggest that STIP1 acts as an oncogene in CRC and can therefore serve as a biomarker for the prognosis of patients with CRC. Copyright © 2018. Published by Elsevier Inc.

  19. Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

    PubMed Central

    Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

    2001-01-01

    Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

  20. High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    PubMed Central

    Dortay, Hakan; Akula, Usha Madhuri; Westphal, Christin; Sittig, Marie; Mueller-Roeber, Bernd

    2011-01-01

    Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here. PMID:21541323

  1. Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes.

    PubMed

    Mehlin, Christopher; Boni, Erica; Buckner, Frederick S; Engel, Linnea; Feist, Tiffany; Gelb, Michael H; Haji, Lutfiyah; Kim, David; Liu, Colleen; Mueller, Natascha; Myler, Peter J; Reddy, J T; Sampson, Joshua N; Subramanian, E; Van Voorhis, Wesley C; Worthey, Elizabeth; Zucker, Frank; Hol, Wim G J

    2006-08-01

    As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.

  2. Aberrant expression and DNA methylation of lipid metabolism genes in PCOS: a new insight into its pathogenesis.

    PubMed

    Pan, Jie-Xue; Tan, Ya-Jing; Wang, Fang-Fang; Hou, Ning-Ning; Xiang, Yu-Qian; Zhang, Jun-Yu; Liu, Ye; Qu, Fan; Meng, Qing; Xu, Jian; Sheng, Jian-Zhong; Huang, He-Feng

    2018-01-01

    Polycystic ovary syndrome (PCOS), whose etiology remains uncertain, is a highly heterogenous and genetically complex endocrine disorder. The aim of this study was to identify differentially expressed genes (DEGs) in granulosa cells (GCs) from PCOS patients and make epigenetic insights into the pathogenesis of PCOS. Included in this study were 110 women with PCOS and 119 women with normal ovulatory cycles undergoing in vitro fertilization acting as the control group. RNA-seq identified 92 DEGs unique to PCOS GCs in comparison with the control group. Bioinformatic analysis indicated that synthesis of lipids and steroids was activated in PCOS GCs. 5-Methylcytosine analysis demonstrated that there was an approximate 25% reduction in global DNA methylation of GCs in PCOS women (4.44 ± 0.65%) compared with the controls (6.07 ± 0.72%; P  < 0.05). Using MassArray EpiTYPER quantitative DNA methylation analysis, we also found hypomethylation of several gene promoters related to lipid and steroid synthesis, which might result in the aberrant expression of these genes. Our results suggest that hypomethylated genes related to the synthesis of lipid and steroid may dysregulate expression of these genes and promote synthesis of steroid hormones including androgen, which could partially explain mechanisms of hyperandrogenism in PCOS.

  3. [Pichia pastoris as an expression system for recombinant protein production].

    PubMed

    Ciarkowska, Anna; Jakubowska, Anna

    2013-01-01

    Pichia pastoris has become increasingly popular as a host for recombinant protein production in recent years. P. pastoris is more cost effective and allows achieving higher expression levels than insect and mammalian cells. It also offers some significant advantages over E. coli expression systems, such as avoiding problems with proper protein folding. Also, P. pastoris as an eukaryotic organism can carry out posttranslational modifications of produced proteins. Additionally, P. pastoris can produce high levels of recombinant proteins in extracellular medium which simplifies protein purification. Having many advantages over other expression systems makes P. pastoris an organism of choice for industrial protein production.

  4. [Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it].

    PubMed

    Weng, Daihui; Lei, Yingfeng; Dong, Yangchao; Han, Peijun; Ye, Chuantao; Yang, Jing; Wang, Yuan; Yin, Wen

    2015-12-01

    To construct the plasmid expressing the fusion protein of Dengue virus type 2 (DENV2) nonstructural protein 3 (NS3) with affinity tag, and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification (TAP) assay. Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments, after amplified by PCR with DENV2 cDNA as template, were digested and cloned into the mammalian eukaryotic expression vector pCI-SF with the tandem affinity tag (FLAG-StrepII). The recombinant pCI-NS3-SF was transiently transformed by Lipofectamine(TM) 2000 into HEK293T cells, and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay. The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system. TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3.

  5. Computational Model of the Modulation of Gene Expression Following DNA Damage

    NASA Technical Reports Server (NTRS)

    Cucinotta, F. A.; Dicello, J. F.; Nikjoo, H.; Cherubini, R.

    2002-01-01

    High linear energy transfer (LET) radiation, such as heavy ions or neutrons, has an increased biological effectiveness compared to X rays for gene mutation, genomic instability, and carcinogenesis. In the traditional paradigm, mutations or chromosomal aberrations are causative of late effects. However, in recent years experimental evidence has demonstrated the important role of the description of the modification of gene expression by radiation in understanding the mechanisms of radiation action. In this report, approaches are discussed to the mathematical description of mRNA and protein expression kinetics following DNA damage. Several hypotheses for models of radiation modulation of protein expression are discussed including possible non-linear processes that evolve from the linear dose responses that follow the initial DNA damage produced by radiation.

  6. Aberrant intracellular localization of Varicella-Zoster virus regulatory proteins during latency

    PubMed Central

    Lungu, Octavian; Panagiotidis, Christos A.; Annunziato, Paula W.; Gershon, Anne A.; Silverstein, Saul J.

    1998-01-01

    Varicella-Zoster virus (VZV) is a herpesvirus that becomes latent in sensory neurons after primary infection (chickenpox) and subsequently may reactivate to cause zoster. The mechanism by which this virus maintains latency, and the factors involved, are poorly understood. Here we demonstrate, by immunohistochemical analysis of ganglia obtained at autopsy from seropositive patients without clinical symptoms of VZV infection that viral regulatory proteins are present in latently infected neurons. These proteins, which localize to the nucleus of cells during lytic infection, predominantly are detected in the cytoplasm of latently infected neurons. The restriction of regulatory proteins from the nucleus of latently infected neurons might interrupt the cascade of virus gene expression that leads to a productive infection. Our findings raise the possibility that VZV has developed a novel mechanism for maintenance of latency that contrasts with the transcriptional repression that is associated with latency of herpes simplex virus, the prototypic alpha herpesvirus. PMID:9618542

  7. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  8. Performance benchmarking of four cell-free protein expression systems.

    PubMed

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins. © 2015 Wiley Periodicals, Inc.

  9. Micro-Scale Genomic DNA Copy Number Aberrations as Another Means of Mutagenesis in Breast Cancer

    PubMed Central

    Chao, Hann-Hsiang; He, Xiaping; Parker, Joel S.; Zhao, Wei; Perou, Charles M.

    2012-01-01

    Introduction In breast cancer, the basal-like subtype has high levels of genomic instability relative to other breast cancer subtypes with many basal-like-specific regions of aberration. There is evidence that this genomic instability extends to smaller scale genomic aberrations, as shown by a previously described micro-deletion event in the PTEN gene in the Basal-like SUM149 breast cancer cell line. Methods We sought to identify if small regions of genomic DNA copy number changes exist by using a high density, gene-centric Comparative Genomic Hybridizations (CGH) array on cell lines and primary tumors. A custom tiling array for CGH (244,000 probes, 200 bp tiling resolution) was created to identify small regions of genomic change, which was focused on previously identified basal-like-specific, and general cancer genes. Tumor genomic DNA from 94 patients and 2 breast cancer cell lines was labeled and hybridized to these arrays. Aberrations were called using SWITCHdna and the smallest 25% of SWITCHdna-defined genomic segments were called micro-aberrations (<64 contiguous probes, ∼ 15 kb). Results Our data showed that primary tumor breast cancer genomes frequently contained many small-scale copy number gains and losses, termed micro-aberrations, most of which are undetectable using typical-density genome-wide aCGH arrays. The basal-like subtype exhibited the highest incidence of these events. These micro-aberrations sometimes altered expression of the involved gene. We confirmed the presence of the PTEN micro-amplification in SUM149 and by mRNA-seq showed that this resulted in loss of expression of all exons downstream of this event. Micro-aberrations disproportionately affected the 5′ regions of the affected genes, including the promoter region, and high frequency of micro-aberrations was associated with poor survival. Conclusion Using a high-probe-density, gene-centric aCGH microarray, we present evidence of small-scale genomic aberrations that can contribute to

  10. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  11. [Construction and expression of a eukaryotic expression vector containing human CR2-Fc fusion protein].

    PubMed

    Li, Xinxin; Wu, Zhihao; Zhang, Chuanfu; Jia, Leili; Song, Hongbin; Xu, Yuanyong

    2014-01-01

    To construct a eukaryotic expression vector containing human complement receptor 2 (CR2)-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary (CHO) cells. The extracellular domain of human CR2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The ones with stable expression of the fusion protein were obtained by means of G418 selection. The expression of the CR2-Fc fusion protein was detected and confirmed by SDS-PAGE and Western blotting. Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. SDS-PAGE showed that relative molecular mass (Mr;) of the purified product was consistent with the expected value. Western blotting further proved the single band at the same position. We constructed the eukaryotic expression vector of CR2-Fc/PCI-neo successfully. The obtained fusion protein was active and can be used for the further study of the role in HIV control.

  12. Aberrant expression of interleukin-22 and its targeting microRNAs in oral lichen planus: a preliminary study.

    PubMed

    Shen, Zhengyu; Du, Guanhuan; Zhou, Zengtong; Liu, Wei; Shi, Linjun; Xu, Hui

    2016-08-01

    Oral lichen planus (OLP) is a T cell-mediated autoimmune disease involving oral mucosa. Interleukin-22 (IL-22) as the signature cytokine of T helper 22 cells is increasingly recognized as a key regulator in various autoimmune diseases. Our previous study reported that IL-22 immunoexpression in OLP was significantly increased compared with the normal controls. The objective of this preliminary study was to compare the IL-22 expression levels in oral biopsies from patients with OLP (n = 50) against normal oral mucosa (n = 19) using RT-qPCR and Western blot, identify the potential targeting miRNAs of IL-22, and examine the miRNA expression levels in OLP. Interleukin-22 expression level in OLP was significantly increased compared with the normal controls. The Dual-Luciferase reporter assay system in human embryonic kidney 293 (HEK293) cells demonstrated that miR-562 and miR-203 were the target miRNAs of IL-22, which was consistent with predictions from bioinformatics software analyses. Interestingly, miR-562 expression in OLP was significantly decreased, but miR-203 expression in OLP was significantly increased compared with the normal controls. This preliminary study for the first time reported that aberrant expression levels of miR-562 and miR-203 were associated with high expression of IL-22 and demonstrated the target relationship between miRNAs and IL-22 in HEK293 cells. Our data indicated that IL-22 and its targeting miRNAs contribute to the pathogenesis of OLP. Further studies are required to investigate the regulatory pathways of IL-22 and miR-562 and miR-203 in OLP. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Aberrant intermediate filament and synaptophysin expression is a frequent event in malignant melanoma: an immunohistochemical study of 73 cases.

    PubMed

    Romano, Ryan C; Carter, Jodi M; Folpe, Andrew L

    2015-08-01

    Malignant melanomas are known to express vimentin, among other intermediate filaments. Though anomalous keratin expression by malignant melanoma has been reported, its frequency is not well-established and this phenomenon is not well-known. We have seen in consultation a number of malignant melanomas with anomalous expression of keratin, other intermediate filaments, or synaptophysin, and therefore studied a large group of primary and metastatic melanomas to determine the frequency of these events. About 73 cases of malignant melanoma (22 primaries and 51 metastases) from 71 patients (51 male, 20 female; mean 59 years, range 17-87 years) were retrieved from our archives. Prior diagnoses were confirmed by re-review of hematoxylin and eosin sections and relevant (e.g., S100 protein, HMB45, Melan-A, and tyrosinase) immunohistochemical studies. Available sections were immunostained for keratin (OSCAR and AE1/AE3 antibodies), desmin, neurofilament protein, glial fibrillary acidic protein, synaptophysin, and chromogranin A. Not all cases could be tested for all markers. Cases were predominantly epithelioid (48/73, 66%) or spindle cell/desmoplastic (25/73, 34%). S100 protein, Melan-A, HMB45, and tyrosinase were positive in 60/65 (92%), 34/64 (53%), 30/60 (50%), 25/48 (52%) of cases, respectively. All five S100-protein-negative cases expressed at least one of the other melanocytic markers: Melan-A (two of four, 50%), HMB45 (two of three, 67%), and tyrosinase (one of two, 50%). All cases expressed at least one melanocytic marker. Cases were positive for keratin (OSCAR, 17/61, 28%; AE1/AE3, 16/40, 40%), desmin (11/47, 24%), neurofilament protein (5/31, 16%), glial fibrillary acidic protein (3/32, 9%), and synaptophysin (10/34, 29%), typically only in a minority of cells. Chromogranin was negative (0/32, 0%). Altogether 9/73 cases (12%) showed expression of >1 intermediate filament. All S100-protein-negative melanomas showed anomalous intermediate filament expression (keratin

  14. Endogenous oncogenic Nras mutation promotes aberrant GM-CSF signaling in granulocytic/monocytic precursors in a murine model of chronic myelomonocytic leukemia.

    PubMed

    Wang, Jinyong; Liu, Yangang; Li, Zeyang; Du, Juan; Ryu, Myung-Jeom; Taylor, Philip R; Fleming, Mark D; Young, Ken H; Pitot, Henry; Zhang, Jing

    2010-12-23

    Oncogenic NRAS mutations are frequently identified in myeloid diseases involving monocyte lineage. However, its role in the genesis of these diseases remains elusive. We report a mouse bone marrow transplantation model harboring an oncogenic G12D mutation in the Nras locus. Approximately 95% of recipient mice develop a myeloproliferative disease resembling the myeloproliferative variant of chronic myelomonocytic leukemia (CMML), with a prolonged latency and acquisition of multiple genetic alterations, including uniparental disomy of oncogenic Nras allele. Based on single-cell profiling of phospho-proteins, a novel population of CMML cells is identified to display aberrant granulocyte-macrophage colony stimulating factor (GM-CSF) signaling in both the extracellular signal-regulated kinase (ERK) 1/2 and signal transducer and activator of transcription 5 (Stat5) pathways. This abnormal signaling is acquired during CMML development. Further study suggests that aberrant Ras/ERK signaling leads to expansion of granulocytic/monocytic precursors, which are highly responsive to GM-CSF. Hyperactivation of Stat5 in CMML cells is mainly through expansion of these precursors rather than up-regulation of surface expression of GM-CSF receptors. Our results provide insights into the aberrant cytokine signaling in oncogenic NRAS-associated myeloid diseases.

  15. Epigenetic silencing of the NR4A3 tumor suppressor, by aberrant JAK/STAT signaling, predicts prognosis in gastric cancer

    NASA Astrophysics Data System (ADS)

    Yeh, Chung-Min; Chang, Liang-Yu; Lin, Shu-Hui; Chou, Jian-Liang; Hsieh, Hsiao-Yen; Zeng, Li-Han; Chuang, Sheng-Yu; Wang, Hsiao-Wen; Dittner, Claudia; Lin, Cheng-Yu; Lin, Jora M. J.; Huang, Yao-Ting; Ng, Enders K. W.; Cheng, Alfred S. L.; Wu, Shu-Fen; Lin, Jiayuh; Yeh, Kun-Tu; Chan, Michael W. Y.

    2016-08-01

    While aberrant JAK/STAT signaling is crucial to the development of gastric cancer (GC), its effects on epigenetic alterations of its transcriptional targets remains unclear. In this study, by expression microarrays coupled with bioinformatic analyses, we identified a putative STAT3 target gene, NR4A3 that was downregulated in MKN28 GC daughter cells overexpressing a constitutively activated STAT3 mutant (S16), as compared to an empty vector control (C9). Bisulphite pyrosequencing and demethylation treatment showed that NR4A3 was epigenetically silenced by promoter DNA methylation in S16 and other GC cell lines including AGS cells, showing constitutive activation of STAT3. Subsequent experiments revealed that NR4A3 promoter binding by STAT3 might repress its transcription. Long-term depletion of STAT3 derepressed NR4A3 expression, by promoter demethylation, in AGS GC cells. NR4A3 re-expression in GC cell lines sensitized the cells to cisplatin, and inhibited tumor growth in vitro and in vivo, in an animal model. Clinically, GC patients with high NR4A3 methylation, or lower NR4A3 protein expression, had significantly shorter overall survival. Intriguingly, STAT3 activation significantly associated only with NR4A3 methylation in low-stage patient samples. Taken together, aberrant JAK/STAT3 signaling epigenetically silences a potential tumor suppressor, NR4A3, in gastric cancer, plausibly representing a reliable biomarker for gastric cancer prognosis.

  16. Mask-induced aberration in EUV lithography

    NASA Astrophysics Data System (ADS)

    Nakajima, Yumi; Sato, Takashi; Inanami, Ryoichi; Nakasugi, Tetsuro; Higashiki, Tatsuhiko

    2009-04-01

    We estimated aberrations using Zernike sensitivity analysis. We found the difference of the tolerated aberration with line direction for illumination. The tolerated aberration of perpendicular line for illumination is much smaller than that of parallel line. We consider this difference to be attributable to the mask 3D effect. We call it mask-induced aberration. In the case of the perpendicular line for illumination, there was a difference in CD between right line and left line without aberration. In this report, we discuss the possibility of pattern formation in NA 0.25 generation EUV lithography tool. In perpendicular pattern for EUV light, the dominant part of aberration is mask-induced aberration. In EUV lithography, pattern correction based on the mask topography effect will be more important.

  17. AR-v7 protein expression is regulated by protein kinase and phosphatase

    PubMed Central

    Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

    2015-01-01

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

  18. Analysis of the expression level and methylation of tumor protein p53, phosphatase and tensin homolog and mutS homolog 2 in N-methyl-N-nitrosourea-induced thymic lymphoma in C57BL/6 mice.

    PubMed

    Huo, Xueyun; Li, Zhenkun; Zhang, Shuangyue; Li, Changlong; Guo, Meng; Lu, Jing; Lv, Jianyi; Du, Xiaoyan; Chen, Zhenwen

    2017-10-01

    Tumorigenesis is often caused by somatic mutation or epigenetic changes in genes that regulate aspects of cell death, proliferation and survival. Although the functions of multiple tumor suppressor genes have been well studied in isolation, how these genes cooperate during the progression of a single tumor remains unclear in numerous cases. The present study used N-methyl-N-nitrosourea (MNU), one of the most potent mutagenic nitrosourea compounds, to induce thymic lymphoma in C57BL/6J mice. Subsequently, the protein expression levels of phosphatase and tensin homolog (PTEN), transformation protein 53 and mutS homolog 2 (MSH2) were evaluated concomitantly in the thymus, liver, kidney and spleen of MNU-treated mice by western blotting. To determine whether changes in expression level were due to aberrant epigenetic regulation, the present study further examined the methylation status of each gene by MassARRAY analysis. During the tumorigenesis process of an MNU-induced single thymic lymphoma, the expression level of PTEN was revealed to be reduced in thymic lymphoma samples but not in normal or non-tumor thymus tissue samples. Furthermore, a marked reduction of P53 expression levels were demonstrated in thymic lymphomas and spleens with a metastatic tumor. Conversely, MSH2 upregulation was identified only in liver, kidney, and spleen samples that were infiltrated by thymic lymphoma cells. Furthermore, the present study revealed that a number of 5'-C-phosphate-G-3' sites located in the promoter of aberrantly expressed genes had significantly altered methylation statuses. These results improve the understanding of the course of mutagen-induced cancer, and highlight that epigenetic regulation may serve an important function in cancer.

  19. MiR-509-3-5p causes aberrant mitosis and anti-proliferative effect by suppression of PLK1 in human lung cancer A549 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Xian-Hui; Lu, Yao; Liang, Jing-Jing

    MicroRNAs (miRNAs) are potent post-transcriptional regulators of gene expression and play roles in DNA damage response (DDR). PLK1 is identified as a modulator of DNA damage checkpoint. Although down-regulation of PLK1 by certain microRNAs has been reported, little is known about the interplay between PLK1 and miR-509-3-5p in DDR. Here we have demonstrated that miR-509-3-5p repressed PLK1 expression by targeting PLK1 3′-UTR, thereby causing mitotic aberration and growth arrest of human lung cancer A549 cells. Repression of PLK1 by miR-509-3-5p was further evidenced by over-expression of miR-509-3-5p in A549, HepG2 and HCT116p53{sup −/−} cancer cells, in which PLK1 protein wasmore » suppressed. Consistently, miR-509-3-5p was stimulated, while PLK1 protein was down-regulated in A549 cells exposed to CIS and ADR, suggesting that suppression of PLK1 by miR-509-3-5p is a component of CIS/ADR-induced DDR pathway. Flow cytometry and immunofluorescence labeling showed that over-expression of miR-509-3-5p in A549 induced G2/M arrest and aberrant mitosis characterized by abnormal bipolar mitotic spindles, condensed chromosomes, lagging DNA and chromosome bridges. In addition, over-expression of miR-509-3-5p markedly blocked A549 cell proliferation and sensitized the cells to CIS and ADR treatment. Taken together, miR-509-3-5p is a feasible suppressor for cancer by targeting PLK1. Our data may provide aid in potential design of combined chemotherapy and in our better understanding of the roles of microRNAs in response to DNA damage. - Highlights: • MiR-509-3-5p represses PLK1 expression by targeting PLK1 3ГЉВ№-UTR. • Expression of miR-509-3-5p is induced and PLK1 repressed upon DNA damage. • Overexpression of miR-509-3-5p induces G2/M arrest and aberrant mitosis. • MiR-509-3-5p inhibits cell proliferation and sensitizes cells to DNA damage agents.« less

  20. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

    PubMed Central

    2012-01-01

    Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains. PMID:22569138

  1. Up-regulated EMMPRIN/CD147 protein expression might play a role in colorectal carcinogenesis and its subsequent progression without an alteration of its glycosylation and mRNA level.

    PubMed

    Zheng, Hua-chuan; Wang, Wei; Xu, Xiao-yan; Xia, Pu; Yu, Miao; Sugiyama, Toshiro; Takano, Yasuo

    2011-04-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) was reported to involve in the invasion and metastasis of malignancies by regulating the expression of vascular endothelial growth factor (VEGF) in stromal and cancer cells. The study aimed to clarify the role of EMMPRIN expression in tumorigenesis and progression of colorectal carcinomas (CRC). EMMPRIN expression was examined on tissue microarray containing colorectal carcinomas, adenoma and non-neoplastic mucosa (NNM) by immunohistochemistry and in situ hybridization (ISH). Colorectal carcinoma cell lines (DLD-1, HCT-15, SW480 and WiDr) and tissues were studied for EMMPRIN expression by Western blot or RT-PCR, followed by sequencing. All carcinoma cell lines showed EMMPRIN expression at both mRNA and protein levels. Two synonymous mutations were found in carcinoma cell lines at codon109 (GCT → GCC: Ala) or 179 (GAT → GAC: Asp). Frozen CRC tissues displayed higher EMMPRIN expression than paired NNM (P < 0.05). EMMPRIN expression was immunohistochemically stronger in colorectal high-grade adenoma, adenocarcinoma and metastatic carcinoma than non-neoplastic superficial epithelium and low-grade adenoma (P < 0.05). In contrast, its mRNA level was similar from colorectal NNM, adenoma to adenocarcinoma by ISH, in line with the findings of RT-PCR (P > 0.05). Immunohistochemically, EMMPRIN expression was positively correlated with tumor size, depth of invasion, vascular or lymphatic invasion, grade of infiltration (INF), ki-67 and VEGF expression of CRCs (P < 0.05). Among them, depth of invasion was an independent associated factor for EMMPRIN expression in CRCs (P < 0.05). Up-regulated EMMPRIN protein expression might contribute to colorectal carcinogenesis without the alteration of its glycosylation and mRNA level. Aberrant EMMPRIN protein expression might promote growth or invasion of CRCs possibly through increased ki-67 expression and inducible angiogenesis via up-regulating VEGF expression.

  2. Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

    PubMed Central

    Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

    2010-01-01

    Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

  3. Protein disorder is positively correlated with gene expression in E. coli

    PubMed Central

    Paliy, Oleg; Gargac, Shawn M.; Cheng, Yugong; Uversky, Vladimir N.; Dunker, A. Keith

    2009-01-01

    We considered on a global scale the relationship between the predicted fraction of protein disorder and RNA and protein expression in E. coli. Fraction of protein disorder correlated positively with both measured RNA expression levels of E. coli genes in three different growth media and with predicted abundance levels of E. coli proteins. Though weak, the correlation was highly significant. Correlation of protein disorder with RNA expression did not depend on the growth rate of E. coli cultures and was not caused by a small subset of genes showing exceptionally high concordance in their disorder and expression levels. Global analysis was complemented by detailed consideration of several groups of proteins. PMID:18465893

  4. Artificial neural network for the determination of Hubble Space Telescope aberration from stellar images

    NASA Technical Reports Server (NTRS)

    Barrett, Todd K.; Sandler, David G.

    1993-01-01

    An artificial-neural-network method, first developed for the measurement and control of atmospheric phase distortion, using stellar images, was used to estimate the optical aberration of the Hubble Space Telescope. A total of 26 estimates of distortion was obtained from 23 stellar images acquired at several secondary-mirror axial positions. The results were expressed as coefficients of eight orthogonal Zernike polynomials: focus through third-order spherical. For all modes other than spherical the measured aberration was small. The average spherical aberration of the estimates was -0.299 micron rms, which is in good agreement with predictions obtained when iterative phase-retrieval algorithms were used.

  5. [Tripartite-motif protein 25 and pyruvate kinase M2 protein expression in non-small cell lung cancer].

    PubMed

    Jing, Huai-Zhi; Qiu, Feng; Chen, Shi-Zhi; Su, Lin; Qu, Can

    2015-03-01

    To investigate the expression of tripartite-motif protein 25 (TRIM25) and pyruvate kinase M2 (PKM2) protein in non-small cell lung cancer (NSCLC) and explore their role in the occurrence and progression of NSCLC. The expressions of TRIM25 and PKM2 protein were detected in 60 NSCLC specimens and 20 adjacent normal lung tissue (>5 cm from the lesions) with immunofluorescence histochemical method and in 10 fresh specimens of NSCLC with Western blotting. The results were analyzed in relation with the clinicopathological features of the patients. The positivity rates of TRIM25 expression was 45% in the 60 lung carcinoma specimens, significantly higher than that in the 20 normal lung tissues (10%, P=0.005). TRIM25 protein was expressed in 28.6% of lung adenocarcinoma tissues and in 59.4% of squamous carcinoma tissues (P=0.017). TRIM25 protein expression was positively correlated with the TNM stages and lymph node metastasis of NSCLC (P<0.05). The expressions of PKM2 protein in 60 cases of lung carcinoma was 73.3%,while in 20 cases of normal lung tissues the expressions was 30%(P=0.001). The positivity rates of PKM2 expression differed significantly between lung adenocarcinoma and squamous carcinoma (57.1% vs 87.5%, P=0.008). An inverse correlation was noted between TRIM25 and PKM2 expressions (P=0.026). TRIM25 and PKM2 protein may participate in the occurrence and progression of NSCLC, and their expressions are inversely correlated.

  6. Distinct Protein Expression Profiles of Solid-Pseudopapillary Neoplasms of the Pancreas.

    PubMed

    Park, Minhee; Lim, Jong-Sun; Lee, Hyoung-Joo; Na, Keun; Lee, Min Jung; Kang, Chang Moo; Paik, Young-Ki; Kim, Hoguen

    2015-08-07

    Solid-pseudopapillary neoplasm (SPN) is an uncommon pancreatic tumor with mutation in CTNNB1 and distinct clinical and pathological features. We compared the proteomic profiles of SPN to mRNA expression. Pooled SPNs and pooled non-neoplastic pancreatic tissues were examined with high-resolution mass spectrometry. We identified 329 (150 up-regulated and 179 down-regulated) differentially expressed proteins in SPN. We identified 191 proteins (58.1% of the 329 dysregulated proteins) with the same expression tendencies in SPN based on mRNA data. Many overexpressed proteins were related to signaling pathways known to be activated in SPNs. We found that several proteins involved in Wnt signaling, including DKK4 and β-catenin, and proteins that bind β-catenin, such as FUS and NONO, were up-regulated in SPNs. Molecules involved in glycolysis, including PKM2, ENO2, and HK1, were overexpressed in accordance to their mRNA levels. In summary, SPN showed (1) distinct protein expression changes that correlated with mRNA expression, (2) overexpression of Wnt signaling proteins and proteins that bind directly to β-catenin, and (3) overexpression of proteins involved in metabolism. These findings may help develop early diagnostic biomarkers and molecular targets.

  7. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  8. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P

    2015-01-13

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

  9. Loss of the mitochondrial protein-only ribonuclease P complex causes aberrant tRNA processing and lethality in Drosophila.

    PubMed

    Sen, Aditya; Karasik, Agnes; Shanmuganathan, Aranganathan; Mirkovic, Elena; Koutmos, Markos; Cox, Rachel T

    2016-07-27

    Proteins encoded by mitochondrial DNA are translated using mitochondrially encoded tRNAs and rRNAs. As with nuclear encoded tRNAs, mitochondrial tRNAs must be processed to become fully functional. The mitochondrial form of ribonuclease P (mt:RNase P) is responsible for 5'-end maturation and is comprised of three proteins; mitochondrial RNase P protein (MRPP) 1 and 2 together with proteinaceous RNase P (PRORP). However, its mechanism and impact on development is not yet known. Using homology searches, we have identified the three proteins composing Drosophila mt:RNase P: Mulder (PRORP), Scully (MRPP2) and Roswell (MRPP1). Here, we show that each protein is essential and localizes with mitochondria. Furthermore, reducing levels of each causes mitochondrial deficits, which appear to be due at least in part to defective mitochondrial tRNA processing. Overexpressing two members of the complex, Mulder and Roswell, is also lethal, and in the case of Mulder, causes abnormal mitochondrial morphology. These data are the first evidence that defective mt:RNase P causes mitochondrial dysfunction, lethality and aberrant mitochondrial tRNA processing in vivo, underscoring its physiological importance. This in vivo mt:RNase P model will advance our understanding of how loss of mitochondrial tRNA processing causes tissue failure, an important aspect of human mitochondrial disease. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  10. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  11. Camera processing with chromatic aberration.

    PubMed

    Korneliussen, Jan Tore; Hirakawa, Keigo

    2014-10-01

    Since the refractive index of materials commonly used for lens depends on the wavelengths of light, practical camera optics fail to converge light to a single point on an image plane. Known as chromatic aberration, this phenomenon distorts image details by introducing magnification error, defocus blur, and color fringes. Though achromatic and apochromatic lens designs reduce chromatic aberration to a degree, they are complex and expensive and they do not offer a perfect correction. In this paper, we propose a new postcapture processing scheme designed to overcome these problems computationally. Specifically, the proposed solution is comprised of chromatic aberration-tolerant demosaicking algorithm and post-demosaicking chromatic aberration correction. Experiments with simulated and real sensor data verify that the chromatic aberration is effectively corrected.

  12. Methods and constructs for expression of foreign proteins in photosynthetic organisms

    DOEpatents

    Laible, Philip D.; Hanson, Deborah K.

    2002-01-01

    A method for expressing and purifying foreign proteins in photosynthetic organisms comprising the simultaneous expression of both the heterologous protein and a means for compartmentalizing or sequestering of the protein.

  13. An amelogenin mutation leads to disruption of the odontogenic apparatus and aberrant expression of Notch I

    PubMed Central

    Chen, Xu; Li, Yong; Alawi, Faizan; Bouchard, Jessica R.; Kulkarni, Ashok B.; Gibson, Carolyn W.

    2012-01-01

    BACKGROUND Amelogenins are highly conserved proteins secreted by ameloblasts in the dental organ of developing teeth. These proteins regulate dental enamel thickness and structure in humans and mice. Mice that express an amelogenin transgene with a P70T mutation (TgP70T) develop abnormal epithelial proliferation in an amelogenin null (KO) background. Some of these cellular masses have the appearance of proliferating stratum intermedium, which is the layer adjacent to the ameloblasts in unerupted teeth. As Notch proteins are thought to constitute the developmental switch that separates ameloblasts from stratum intermedium, these signaling proteins were evaluated in normal and proliferating tissues. METHODS Mandibles were dissected for histology and immunohistochemistry using Notch I antibodies. Molar teeth were dissected for western blotting and RT-PCR for evaluation of Notch levels through imaging and statistical analyses. RESULTS Notch I was immunolocalized to ameloblasts of TgP70TKO mice, KO ameloblasts stained, but less strongly, and wild-type teeth had minimal staining. Cells within the proliferating epithelial cell masses were positive for Notch I and had an appearance reminiscent of calcifying epithelial odontogenic tumor with amyloid-like deposits. Notch I protein and mRNA were elevated in molar teeth from TgP70TKO mice. CONCLUSION Expression of TgP70T leads to abnormal structures in mandibles and maxillae of mice with the KO genetic background and these mice have elevated levels of Notch I in developing molars. As cells within the masses also express transgenic amelogenins, development of the abnormal proliferations suggests communication between amelogenin producing cells and the proliferating cells, dependent on the presence of the mutated amelogenin protein. PMID:20923441

  14. Similar protein expression profiles of ovarian and endometrial high-grade serous carcinomas.

    PubMed

    Hiramatsu, Kosuke; Yoshino, Kiyoshi; Serada, Satoshi; Yoshihara, Kosuke; Hori, Yumiko; Fujimoto, Minoru; Matsuzaki, Shinya; Egawa-Takata, Tomomi; Kobayashi, Eiji; Ueda, Yutaka; Morii, Eiichi; Enomoto, Takayuki; Naka, Tetsuji; Kimura, Tadashi

    2016-03-01

    Ovarian and endometrial high-grade serous carcinomas (HGSCs) have similar clinical and pathological characteristics; however, exhaustive protein expression profiling of these cancers has yet to be reported. We performed protein expression profiling on 14 cases of HGSCs (7 ovarian and 7 endometrial) and 18 endometrioid carcinomas (9 ovarian and 9 endometrial) using iTRAQ-based exhaustive and quantitative protein analysis. We identified 828 tumour-expressed proteins and evaluated the statistical similarity of protein expression profiles between ovarian and endometrial HGSCs using unsupervised hierarchical cluster analysis (P<0.01). Using 45 statistically highly expressed proteins in HGSCs, protein ontology analysis detected two enriched terms and proteins composing each term: IMP2 and MCM2. Immunohistochemical analyses confirmed the higher expression of IMP2 and MCM2 in ovarian and endometrial HGSCs as well as in tubal and peritoneal HGSCs than in endometrioid carcinomas (P<0.01). The knockdown of either IMP2 or MCM2 by siRNA interference significantly decreased the proliferation rate of ovarian HGSC cell line (P<0.01). We demonstrated the statistical similarity of the protein expression profiles of ovarian and endometrial HGSC beyond the organs. We suggest that increased IMP2 and MCM2 expression may underlie some of the rapid HGSC growth observed clinically.

  15. The protein expression landscape of the Arabidopsis root

    PubMed Central

    Petricka, Jalean J.; Schauer, Monica A.; Megraw, Molly; Breakfield, Natalie W.; Thompson, J. Will; Georgiev, Stoyan; Soderblom, Erik J.; Ohler, Uwe; Moseley, Martin Arthur; Grossniklaus, Ueli; Benfey, Philip N.

    2012-01-01

    Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development. PMID:22447775

  16. Novel Partitivirus Enhances Virulence of and Causes Aberrant Gene Expression in Talaromyces marneffei.

    PubMed

    Lau, Susanna K P; Lo, George C S; Chow, Franklin W N; Fan, Rachel Y Y; Cai, James J; Yuen, Kwok-Yung; Woo, Patrick C Y

    2018-06-12

    Talaromyces marneffei is the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia. We report the discovery of a novel partitivirus, Talaromyces marneffei partitivirus -1 (TmPV1). TmPV1 was detected in 7 (12.7%) of 55 clinical T. marneffei isolates. Complete genome sequencing of the seven TmPV1 isolates revealed two double-stranded RNA (dsRNA) segments encoding RNA-dependent RNA polymerase (RdRp) and capsid protein, respectively. Phylogenetic analysis showed that TmPV1 occupied a distinct clade among the members of the genus Gammapartitivirus Transmission electron microscopy confirmed the presence of isometric, nonenveloped viral particles of 30 to 45 nm in diameter, compatible with partitiviruses, in TmPV1-infected T. marneffei Quantitative reverse transcription-PCR (qRT-PCR) demonstrated higher viral load of TmPV1 in the yeast phase than in the mycelial phase of T. marneffei Two virus-free isolates, PM1 and PM41, were successfully infected by purified TmPV1 using protoplast transfection. Mice challenged with TmPV1-infected T. marneffei isolates showed significantly shortened survival time ( P < 0.0001) and higher fungal burden in organs than mice challenged with isogenic TmPV1-free isolates. Transcriptomic analysis showed that TmPV1 causes aberrant expression of various genes in T. marneffei , with upregulation of potential virulence factors and suppression of RNA interference (RNAi)-related genes. This is the first report of a mycovirus in a thermally dimorphic fungus. Further studies are required to ascertain the mechanism whereby TmPV1 enhances the virulence of T. marneffei in mice and the potential role of RNAi-related genes in antiviral defense in T. marneffei IMPORTANCE Talaromyces marneffei (formerly Penicillium marneffei ) is the most important thermal dimorphic fungus in Southeast Asia, causing highly fatal systemic penicilliosis in HIV-infected and immunocompromised patients. We discovered a novel mycovirus, TmPV1

  17. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yamakoshi, Takako; Makino, Teruhiko, E-mail: tmakino@med.u-toyama.ac.jp; Ur Rehman, Mati

    2013-03-01

    Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain andmore » a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.« less

  18. The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity.

    PubMed

    Yi, Woelsung; Gupta, Sanjay; Ricker, Edd; Manni, Michela; Jessberger, Rolf; Chinenov, Yurii; Molina, Henrik; Pernis, Alessandra B

    2017-08-15

    Post-transcriptional modifications can control protein abundance, but the extent to which these alterations contribute to the expression of T helper (T H ) lineage-defining factors is unknown. Tight regulation of Bcl6 expression, an essential transcription factor for T follicular helper (T FH ) cells, is critical as aberrant T FH cell expansion is associated with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here we show that lack of the SLE risk variant Def6 results in deregulation of Bcl6 protein synthesis in T cells as a result of enhanced activation of the mTORC1-4E-BP-eIF4E axis, secondary to aberrant assembly of a raptor-p62-TRAF6 complex. Proteomic analysis reveals that this pathway selectively controls the abundance of a subset of proteins. Rapamycin or raptor deletion ameliorates the aberrant T FH cell expansion in mice lacking Def6. Thus deregulation of mTORC1-dependent pathways controlling protein synthesis can result in T-cell dysfunction, indicating a mechanism by which mTORC1 can promote autoimmunity.Excessive expansion of the T follicular helper (T FH ) cell pool is associated with autoimmune disease and Def6 has been identified as an SLE risk variant. Here the authors show that Def6 limits proliferation of T FH cells in mice via alteration of mTORC1 signaling and inhibition of Bcl6 expression.

  19. Transient Expression of Chimeric Fluorescent Reporter Proteins in Pollen Tubes to Study Protein Polar Secretion and Dynamics.

    PubMed

    Zhong, Guitao; Liu, Ronghe; Zhuang, Menglong; Wang, Hao

    2017-01-01

    Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting. A large amount of newly synthesized proteins are packed and polarly transported to the apical region to support the rapid and highly polarized tip growth. Here, we described a detailed step-by-step protocol for the transient expression of chimeric fluorescent reporter proteins in growing Arabidopsis and tobacco pollen tubes to study polar transportation logistics and mechanisms. In addition, we have optimized the Arabidopsis and tobacco in vitro pollen germination medium and the conditions to maximize the efficiency of protein expression. As a proof of concept, we have used this protocol to express actin microfilament and late endosomal fluorescent markers in Arabidopsis and tobacco pollen tubes.

  20. Selective clearance of aberrant tau proteins and rescue of neurotoxicity by transcription factor EB.

    PubMed

    Polito, Vinicia A; Li, Hongmei; Martini-Stoica, Heidi; Wang, Baiping; Yang, Li; Xu, Yin; Swartzlander, Daniel B; Palmieri, Michela; di Ronza, Alberto; Lee, Virginia M-Y; Sardiello, Marco; Ballabio, Andrea; Zheng, Hui

    2014-09-01

    Accumulating evidence implicates impairment of the autophagy-lysosome pathway in Alzheimer's disease (AD). Recently discovered, transcription factor EB (TFEB) is a molecule shown to play central roles in cellular degradative processes. Here we investigate the role of TFEB in AD mouse models. In this study, we demonstrate that TFEB effectively reduces neurofibrillary tangle pathology and rescues behavioral and synaptic deficits and neurodegeneration in the rTg4510 mouse model of tauopathy with no detectable adverse effects when expressed in wild-type mice. TFEB specifically targets hyperphosphorylated and misfolded Tau species present in both soluble and aggregated fractions while leaving normal Tau intact. We provide in vitro evidence that this effect requires lysosomal activity and we identify phosphatase and tensin homolog (PTEN) as a direct target of TFEB that is required for TFEB-dependent aberrant Tau clearance. The specificity and efficacy of TFEB in mediating the clearance of toxic Tau species makes it an attractive therapeutic target for treating diseases of tauopathy including AD. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  1. Impact of Chronic Alcohol Ingestion on Cardiac Muscle Protein Expression

    PubMed Central

    Fogle, Rachel L.; Lynch, Christopher J.; Palopoli, Mary; Deiter, Gina; Stanley, Bruce A.; Vary, Thomas C.

    2014-01-01

    Background Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved. Methods The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT™). Following the reaction with the ICAT™ reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches. Results Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system. Conclusions Based on the changes in proteins, we speculate modulation of

  2. High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

    PubMed

    Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

    2018-02-01

    Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. HABP1/p32/gC1qR induces aberrant growth and morphology in Schizosaccharomyces pombe through its N-terminal {alpha} helix

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mallick, Jaideep; Datta, Kasturi

    2005-10-01

    Hyaluronan binding protein (HABP1), located on human chromosome 17p13.3, was identified and characterized as being involved in cellular signaling from our laboratory. Here, we demonstrate that HABP1 expression in Schizosaccharomyces pombe induces growth inhibition, morphological abnormalities like elongation, multinucleation and aberrant cell septum formation in several strains of S. pombe, implicating its role in cell cycle progression and cytokinesis. This argument is further strengthened by an observed delay in the maximal expression of cell cycle regulatory proteins like CDC 2 and CDC 25 coupled to the direct interaction of HABP1 with CDC 25. In order to pinpoint the interacting domainmore » of HABP1, its N- and C-terminal truncated variants ({delta}N.HABP1 and {delta}C.HABP1, respectively) were utilized which revealed that while expression of the former did not alter the phenotype, the latter generated morphological changes similar to those imparted upon HABP1 expression. It was also noted that along with HABP1, {delta}C.HABP1 too directly interacts with CDC 25 while {delta}N.HABP1 does not. Taken together, these data suggest that HABP1 induces morphological changes and modulates the cell cycle by interacting with proteins like CDC 25 through its N-terminal {alpha}-helix.« less

  4. Assessing the utility of gene co-expression stability in combination with correlation in the analysis of protein-protein interaction networks

    PubMed Central

    2011-01-01

    Background Gene co-expression, in the form of a correlation coefficient, has been valuable in the analysis, classification and prediction of protein-protein interactions. However, it is susceptible to bias from a few samples having a large effect on the correlation coefficient. Gene co-expression stability is a means of quantifying this bias, with high stability indicating robust, unbiased co-expression correlation coefficients. We assess the utility of gene co-expression stability as an additional measure to support the co-expression correlation in the analysis of protein-protein interaction networks. Results We studied the patterns of co-expression correlation and stability in interacting proteins with respect to their interaction promiscuity, levels of intrinsic disorder, and essentiality or disease-relatedness. Co-expression stability, along with co-expression correlation, acts as a better classifier of hub proteins in interaction networks, than co-expression correlation alone, enabling the identification of a class of hubs that are functionally distinct from the widely accepted transient (date) and obligate (party) hubs. Proteins with high levels of intrinsic disorder have low co-expression correlation and high stability with their interaction partners suggesting their involvement in transient interactions, except for a small group that have high co-expression correlation and are typically subunits of stable complexes. Similar behavior was seen for disease-related and essential genes. Interacting proteins that are both disordered have higher co-expression stability than ordered protein pairs. Using co-expression correlation and stability, we found that transient interactions are more likely to occur between an ordered and a disordered protein while obligate interactions primarily occur between proteins that are either both ordered, or disordered. Conclusions We observe that co-expression stability shows distinct patterns in structurally and functionally

  5. Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense.

    PubMed

    Horiuchi, Yuki; Laskaratou, Danai; Sliwa, Michel; Ruckebusch, Cyril; Hatori, Kuniyuki; Mizuno, Hideaki; Hotta, Jun-Ichi

    2018-01-26

    Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT) 14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense . We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein "ember" from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 10⁵ M -1 ·cm -1 . The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

  6. Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

    PubMed

    Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

    2015-08-01

    Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

  7. Phytomonas: A non-pathogenic trypanosomatid model for functional expression of proteins.

    PubMed

    Miranda, Mariana R; Sayé, Melisa; Reigada, Chantal; Carrillo, Carolina; Pereira, Claudio A

    2015-10-01

    Phytomonas are protozoan parasites from the Trypanosomatidae family which infect a wide variety of plants. Herein, Phytomonas Jma was tested as a model for functional expression of heterologous proteins. Green fluorescent protein expression was evaluated in Phytomonas and compared with Trypanosoma cruzi, the etiological agent of Chagas' disease. Phytomonas was able to express GFP at levels similar to T. cruzi although the transgenic selection time was higher. It was possible to establish an efficient transfection and selection protocol for protein expression. These results demonstrate that Phytomonas can be a good model for functional expression of proteins from other trypanosomatids, presenting the advantage of being completely safe for humans. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Implantation failure in mice with a disruption in Phospholipase C beta 1 gene: lack of embryonic attachment, aberrant steroid hormone signalling and defective endocannabinoid metabolism

    PubMed Central

    Filis, Panayiotis; Kind, Peter C.; Spears, Norah

    2013-01-01

    Phospholipase C beta 1 (PLCβ1) is a downstream effector of G-protein-coupled receptor signalling and holds central roles in reproductive physiology. Mice with a disruption in the Plcβ1 gene are infertile with pleiotropic reproductive defects, the major reproductive block in females being implantation failure. Here, PLCβ1 was demonstrated at the luminal and glandular epithelia throughout the pre- and peri-implantation period, with transient stromal expression during 0.5–1.5 days post coitum (dpc). Examination of implantation sites at 4.5 dpc showed that in females lacking functional PLCβ1 (knock-out (KO) females), embryos failed to establish proper contact with the uterine epithelium. Proliferating luminal epithelial cells were evident in KO implantation sites, indicating failure to establish a receptive uterus. Real-time PCR demonstrated that KO implantation sites had aberrant ovarian steroid signalling, with high levels of estrogen receptor α, lactoferrin and amphiregulin mRNA, while immunohistochemistry revealed very low levels of estrogen receptor α protein, possibly due to rapid receptor turnover. KO implantation sites expressed markedly less fatty acid amide hydrolase and monoacylglycerol lipase, indicating that endocannabinoid metabolism was also affected. Collectively, our results show that PLCβ1 is essential for uterine preparation for implantation, and that defective PLCβ1-mediated signalling during implantation is associated with aberrant ovarian steroid signalling and endocannabinoid metabolism. PMID:23295235

  9. High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

    PubMed Central

    Bruni, Renato

    2014-01-01

    Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

  10. Expression of hypoxia-inducible 2 (HIG2) protein in uterine cancer.

    PubMed

    Nishimura, S; Tsuda, H; Nomura, H; Kataoka, F; Chiyoda, T; Tanaka, H; Tanaka, K; Susumu, N; Aoki, D

    2011-01-01

    For both cervical cancer (UCC) and endometrial cancer (EMC) there are no effective prognostic markers. In this study, we evaluated HIG2 protein expression in 332 uterine cancers (186 UCCs and 146 EMCs) and examined the relationship between HIG2 protein expression and clinical factors, including prognosis. Totally, HIG2 expression was detected in 58% of UCC and 66% of EMC. However, there was no significant relationship between HIG2 expression and age, clinical stage and histology in either UCC or EMC. In addition, HIG2 protein expression was not related to prognosis of UCC or EMC. The positivity rate of HIG2 protein was 56% and 61% in early-stage UCC and EMC, respectively and 67% in non-squamous cell carcinoma of UCC. The positivity rate of HIG2 protein was high even in early-stage UCC and EMC

  11. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

    PubMed

    Lynch, Jennifer R; Wang, Jenny Yingzi

    2016-05-11

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  12. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    PubMed Central

    Lynch, Jennifer R.; Wang, Jenny Yingzi

    2016-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies. PMID:27187360

  13. Ectopic Activation of Wnt/β-Catenin Signaling in Lens Fiber Cells Results in Cataract Formation and Aberrant Fiber Cell Differentiation

    PubMed Central

    Antosova, Barbora; Smolikova, Jana; Borkovcova, Romana; Strnad, Hynek; Lachova, Jitka; Machon, Ondrej; Kozmik, Zbynek

    2013-01-01

    The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency. PMID:24205179

  14. Transient expression of CCL21as recombinant protein in tomato.

    PubMed

    Beihaghi, Maria; Marashi, Hasan; Bagheri, Abdolreza; Sankian, Mojtaba

    2018-03-01

    The main goal of this study was to investigate the possibility of expressing recombinant protein of C-C chemokine ligand 21 (CCL21) in Solanum lycopersicum via agroinfiltration. CCL21 is a chemokine can be used for anti-metastatic of cancer cell lines. To examine the expression of CCL21 protein in S. lycopersicum , the construct of ccl21 was synthesized. This construct was cloned into pBI121 and the resulting CCL21 plasmid was agro-infiltrated into S. lycopersicum leaves. Within three days after infiltration, Expression of the foreign gene was confirmed by quantitative Real-time PCR. A recombinant CCL21 protein was immunogenically detected by western blot, dot blot and ELISA assay. And results showed that the foreign gene was expressed in the transformed leaves in high level. Also scratch assay was used to investigate the role of this protein in anti-metastatic function. The results demonstrated anti-metastatic of cancer cells in the presence of this protein.

  15. The Kunitz-protease inhibitor domain in amyloid precursor protein reduces cellular mitochondrial enzymes expression and function.

    PubMed

    Chua, Li-Min; Lim, Mei-Li; Wong, Boon-Seng

    2013-08-09

    Mitochondrial dysfunction is a prominent feature of Alzheimer's disease (AD) and this can be contributed by aberrant metabolic enzyme function. But, the mechanism causing this enzymatic impairment is unclear. Amyloid precursor protein (APP) is known to be alternatively spliced to produce three major isoforms in the brain (APP695, APP751, APP770). Both APP770 and APP751 contain the Kunitz Protease Inhibitory (KPI) domain, but the former also contain an extra OX-2 domain. APP695 on the other hand, lacks both domains. In AD, up-regulation of the KPI-containing APP isoforms has been reported. But the functional contribution of this elevation is unclear. In the present study, we have expressed and compared the effect of the non-KPI containing APP695 and the KPI-containing APP751 on mitochondrial function. We found that the KPI-containing APP751 significantly decreased the expression of three major mitochondrial metabolic enzymes; citrate synthase, succinate dehydrogenase and cytochrome c oxidase (COX IV). This reduction lowers the NAD(+)/NADH ratio, COX IV activity and mitochondrial membrane potential. Overall, this study demonstrated that up-regulation of the KPI-containing APP isoforms is likely to contribute to the impairment of metabolic enzymes and mitochondrial function in AD. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

    PubMed

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

    Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

  17. Aberrant activity of NKL homeobox gene NKX3-2 in a T-ALL subset

    PubMed Central

    Meyer, Corinna; Kaufmann, Maren; Zaborski, Margarete; MacLeod, Roderick A. F.; Drexler, Hans G.

    2018-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a hematopoietic malignancy originating from T-cell progenitors in which differentiation is blocked at early stages. Physiological expression of specific NKL homeobox genes obeys a hematopoietic NKL-code implicated in the process of lymphopoiesis while in differentiated T-cells these genes are silenced. We propose that this developmental expression pattern underlies the observation that NKL homeobox genes are the most ubiquitous group of transcription factors deregulated in T-ALL, including TLX1, TLX3, NKX2-5 and NKX3-1. Here, we describe a novel member of the NKL homeobox gene subclass, NKX3-2 (BAPX1), which is aberrantly activated in 18% of pediatric T-ALL patients analyzed while being normally expressed in developing spleen. Identification of NKX3-2 expression in T-ALL cell line CCRF-CEM qualified these cells to model its deregulation and function in a leukemic context. Genomic and chromosomal analyses demonstrated normal configuration of the NKX3-2 locus at chromosome 4p15, thus excluding cytogenetic dysregulation. Comparative expression profiling analysis of NKX3-2 patient data revealed deregulated activity of BMP- and MAPK-signalling. These candidate pathways were experimentally confirmed to mediate aberrant NKX3-2 expression. We also show that homeobox gene SIX6, plus MIR17HG and GATA3 are downstream targets of NKX3-2 and plausibly contribute to the pathogenesis of this malignancy by suppressing T-cell differentiation. Finally, NKL homeobox gene NKX2-5 was activated by NKX3-2 in CCRF-CEM and by FOXG1 in PEER, representing mutually inhibitory activators of this translocated oncogene. Together, our findings reveal a novel oncogenic NKL homeobox gene subclass member which is aberrantly expressed in a large subset of T-ALL patients and participates in a deregulated gene network likely to arise in developing spleen. PMID:29746601

  18. Aberrant activity of NKL homeobox gene NKX3-2 in a T-ALL subset.

    PubMed

    Nagel, Stefan; Meyer, Corinna; Kaufmann, Maren; Zaborski, Margarete; MacLeod, Roderick A F; Drexler, Hans G

    2018-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a hematopoietic malignancy originating from T-cell progenitors in which differentiation is blocked at early stages. Physiological expression of specific NKL homeobox genes obeys a hematopoietic NKL-code implicated in the process of lymphopoiesis while in differentiated T-cells these genes are silenced. We propose that this developmental expression pattern underlies the observation that NKL homeobox genes are the most ubiquitous group of transcription factors deregulated in T-ALL, including TLX1, TLX3, NKX2-5 and NKX3-1. Here, we describe a novel member of the NKL homeobox gene subclass, NKX3-2 (BAPX1), which is aberrantly activated in 18% of pediatric T-ALL patients analyzed while being normally expressed in developing spleen. Identification of NKX3-2 expression in T-ALL cell line CCRF-CEM qualified these cells to model its deregulation and function in a leukemic context. Genomic and chromosomal analyses demonstrated normal configuration of the NKX3-2 locus at chromosome 4p15, thus excluding cytogenetic dysregulation. Comparative expression profiling analysis of NKX3-2 patient data revealed deregulated activity of BMP- and MAPK-signalling. These candidate pathways were experimentally confirmed to mediate aberrant NKX3-2 expression. We also show that homeobox gene SIX6, plus MIR17HG and GATA3 are downstream targets of NKX3-2 and plausibly contribute to the pathogenesis of this malignancy by suppressing T-cell differentiation. Finally, NKL homeobox gene NKX2-5 was activated by NKX3-2 in CCRF-CEM and by FOXG1 in PEER, representing mutually inhibitory activators of this translocated oncogene. Together, our findings reveal a novel oncogenic NKL homeobox gene subclass member which is aberrantly expressed in a large subset of T-ALL patients and participates in a deregulated gene network likely to arise in developing spleen.

  19. Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

    PubMed

    Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

    2017-10-01

    Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant

  20. Thymidylate synthase (TS) protein expression as a prognostic factor in advanced colorectal cancer: a comparison with TS mRNA expression.

    PubMed

    Nakagawa, Tateo; Shimada, Mitsuo; Kurita, Nobuhiro; Iwata, Takashi; Nishioka, Masanori; Yoshikawa, Kozo; Higashijima, Jun; Utsunomiya, Tohru

    2012-06-01

    The role of intratumoral thymidylate synthase (TS) mRNA or protein expression is still controversial and little has been reported regarding relation of them in colorectal cancer. Forty-six patients with advanced colorectal cancer who underwent surgical resection were included. TS mRNA expression was determined by the Danenberg tumor profile method based on laser-captured micro-dissection of the tumor cells. TS protein expression was evaluated using immunohistochemical staining. TS mRNA expression tended to relate TS protein expression. Statistical significance was not found in overall survival between the TS mRNA high group and low group regardless of performing adjuvant chemotherapy. The overall survival in the TS protein negative group was significantly higher than that in positive group in all and the patients without adjuvant chemotherapy. Multivariate analysis showed TS protein expression was as an independent prognostic factor. TS protein expression tends to be related TS mRNA expression and is an independent prognostic factor in advanced colorectal cancer.

  1. Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

    PubMed

    Yue, Chang-Wu; Zhang, Yi-Zheng

    2009-03-01

    A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

  2. Aberrant EPHB4 gene methylation and childhood acute lymphoblastic leukemia

    PubMed Central

    Li, Yuhua; Wang, Huihui; Chen, Xiaowen; Mai, Huirong; Li, Changgang; Wen, Feiqiu

    2017-01-01

    The present study aimed to investigate the association between aberrant DNA methylation of the promoter region of the ephrin type-B receptor 4 (EPHB4) gene and the development of childhood acute lymphoblastic leukemia (ALL). Bisulfite sequencing polymerase chain reaction (BSP) was performed to determine the methylation density of cytosine-guanine pair islands in the promoter region of EPHB4, in bone marrow samples from 40 children with ALL. The mRNA and protein expression levels of EPHB4 were detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. A total of 10 children with idiopathic thrombocytopenic purpura (ITP) were recruited as controls. The results revealed that the average methylation density of the bone marrow samples from the patients with ALL was significantly higher, compared with the patients with ITP (P=0.046). The relative mRNA expression levels of EPHB4 in the patients with ITP (25.08±4.03) and the patients with ALL without methylation (12.33±2.16) were significantly higher, compared with that observed in the patients with ALL with methylation (6.48±2.73; P<0.01). Pearson analysis revealed a significant negative linear correlation between EPHB4 gene methylation and its expression levels (r=−0.957; P<0.01). Western blot analysis indicated that EPHB4 protein expression levels were low in the methylated ALL samples. An evaluation of the two-year disease-free survival (DFS) of the patients with ALL was performed, which revealed that the patients with unmethylated ALL exhibited a significantly higher two-year DFS rate, as compared with patients with methylated ALL (P=0.036). These results suggest that the methylation of the EPHB4 gene is prevalent in childhood ALL and may result in expressional inactivation, which consequently promotes ALL pathogenesis and is associated with an unfavorable prognosis. Therefore, the EPHB4 gene may function as a potential tumor suppressor in childhood ALL. PMID:29085439

  3. TNFRSF14 aberrations in follicular lymphoma increase clinically significant allogeneic T-cell responses.

    PubMed

    Kotsiou, Eleni; Okosun, Jessica; Besley, Caroline; Iqbal, Sameena; Matthews, Janet; Fitzgibbon, Jude; Gribben, John G; Davies, Jeffrey K

    2016-07-07

    Donor T-cell immune responses can eradicate lymphomas after allogeneic hematopoietic stem cell transplantation (AHSCT), but can also damage healthy tissues resulting in harmful graft-versus-host disease (GVHD). Next-generation sequencing has recently identified many new genetic lesions in follicular lymphoma (FL). One such gene, tumor necrosis factor receptor superfamily 14 (TNFRSF14), abnormal in 40% of FL patients, encodes the herpes virus entry mediator (HVEM) which limits T-cell activation via ligation of the B- and T-lymphocyte attenuator. As lymphoma B cells can act as antigen-presenting cells, we hypothesized that TNFRSF14 aberrations that reduce HVEM expression could alter the capacity of FL B cells to stimulate allogeneic T-cell responses and impact the outcome of AHSCT. In an in vitro model of alloreactivity, human lymphoma B cells with TNFRSF14 aberrations had reduced HVEM expression and greater alloantigen-presenting capacity than wild-type lymphoma B cells. The increased immune-stimulatory capacity of lymphoma B cells with TNFRSF14 aberrations had clinical relevance, associating with higher incidence of acute GVHD in patients undergoing AHSCT. FL patients with TNFRSF14 aberrations may benefit from more aggressive immunosuppression to reduce harmful GVHD after transplantation. Importantly, this study is the first to demonstrate the impact of an acquired genetic lesion on the capacity of tumor cells to stimulate allogeneic T-cell immune responses which may have wider consequences for adoptive immunotherapy strategies. © 2016 by The American Society of Hematology.

  4. Suppression of polyglutamine protein toxicity by co-expression of a heat-shock protein 40 and a heat-shock protein 110

    PubMed Central

    Kuo, Y; Ren, S; Lao, U; Edgar, B A; Wang, T

    2013-01-01

    A network of heat-shock proteins mediates cellular protein homeostasis, and has a fundamental role in preventing aggregation-associated neurodegenerative diseases. In a Drosophila model of polyglutamine (polyQ) disease, the HSP40 family protein, DNAJ-1, is a superior suppressor of toxicity caused by the aggregation of polyQ containing proteins. Here, we demonstrate that one specific HSP110 protein, 70 kDa heat-shock cognate protein cb (HSC70cb), interacts physically and genetically with DNAJ-1 in vivo, and that HSC70cb is necessary for DNAJ-1 to suppress polyglutamine-induced cell death in Drosophila. Expression of HSC70cb together with DNAJ-1 significantly enhanced the suppressive effects of DNAJ-1 on polyQ-induced neurodegeneration, whereas expression of HSC70cb alone did not suppress neurodegeneration in Drosophila models of either general polyQ disease or Huntington's disease. Furthermore, expression of a human HSP40, DNAJB1, together with a human HSP110, APG-1, protected cells from polyQ-induced neural degeneration in flies, whereas expression of either component alone had little effect. Our data provide a functional link between HSP40 and HSP110 in suppressing the cytotoxicity of aggregation-prone proteins, and suggest that HSP40 and HSP110 function together in protein homeostasis control. PMID:24091676

  5. ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro.

    PubMed

    Cherwa, James E; Tyson, Joshua; Bedwell, Gregory J; Brooke, Dewey; Edwards, Ashton G; Dokland, Terje; Prevelige, Peter E; Fane, Bentley A

    2017-01-01

    During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical

  6. Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3 nm precision using aberration-corrected scanning transmission electron microscopy

    PubMed Central

    Dukes, Madeline J.; Ramachandra, Ranjan; Baudoin, Jean-Pierre; Jerome, W. Gray; de Jonge, Niels

    2011-01-01

    Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3 nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under the electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells. PMID:21440635

  7. N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae.

    PubMed

    Ojima-Kato, Teruyo; Nagai, Satomi; Nakano, Hideo

    2017-05-01

    Despite advances in microbial protein expression systems, low production of proteins remains a great concern for some genes. Here we report that the insertion of a short peptide tag, consisting of Ser-Lys-Ile-Lys (SKIK), adjacent to the start codon of genes encoding difficult-to-express proteins can increase protein expression in Escherichia coli and Saccharomyces cerevisiae. Protein expression levels of a mouse monoclonal antibody (mAb), rabbit mAbs obtained from clonal B cells, and an artificially designed peptide were significantly increased simply by the addition of the SKIK tag in E. coli systems. In particular, a ∼30-fold increase in protein production was observed for the mouse mAb, and the artificially designed peptide band became detectable in sodium dodecyl sulfate-poly acrylamide gel electrophoresis after coomassie brilliant blue staining or western blotting on adding the SKIK tag. The tag also increased the expression of tagged proteins in S. cerevisiae and an E. coli cell-free protein synthesis system. Although the mechanism of high protein expression on addition of the tag is unclear, our findings offer great benefits to biotechnology research and industry. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. The N Terminus of Andes Virus L Protein Suppresses mRNA and Protein Expression in Mammalian Cells

    PubMed Central

    Heinemann, Patrick; Schmidt-Chanasit, Jonas

    2013-01-01

    Little is known about the structure and function of the 250-kDa L protein of hantaviruses, although it plays a central role in virus genome transcription and replication. When attempting to study Andes virus (ANDV) L protein in mammalian cells, we encountered difficulties. Even in a strong overexpression system, ANDV L protein could not be detected by immunoblotting. Deletion analysis revealed that the 534 N-terminal amino acid residues determine the low-expression phenotype. Inhibition of translation due to RNA secondary structures around the start codon, rapid proteasomal degradation, and reduced half-life time were excluded. However, ANDV L protein expression could be rescued upon mutation of the catalytic PD-E-K motif and further conserved residues of the putative endonuclease at the N terminus of the protein. In addition, wild-type ANDV L rather than expressible L mutants suppressed the level of L mRNA, as well as reporter mRNAs. Wild-type L protein also reduced the synthesis of cellular proteins in the high-molecular-weight range. Using expressible ANDV L mutants as a tool for localization studies, we show that L protein colocalizes with ANDV N and NSs but not Gc protein. A fraction of L protein also colocalized with the cellular processing (P) body component DCP1a. Overall, these data suggest that ANDV L protein possesses a highly active endonuclease at the N terminus suppressing the level of its own as well as heterologous mRNAs upon recombinant expression in mammalian cells. PMID:23576516

  9. Evolved Escherichia coli strains for amplified, functional expression of membrane proteins.

    PubMed

    Gul, Nadia; Linares, Daniel M; Ho, Franz Y; Poolman, Bert

    2014-01-09

    The major barrier to the physical characterization and structure determination of membrane proteins is low protein yield and/or low functionality in recombinant expression. The enteric bacterium Escherichia coli is the most widely employed organism for producing recombinant proteins. Beside several advantages of this expression host, one major drawback is that the protein of interest does not always adopt its native conformation and may end up in large insoluble aggregates. We describe a robust strategy to increase the likelihood of overexpressing membrane proteins in a functional state. The method involves fusion in tandem of green fluorescent protein and the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC) to the C-terminus of a target membrane protein. The fluorescence of green fluorescent protein is used to report the folding state of the target protein, whereas ErmC is used to select for increased expression. By gradually increasing the erythromycin concentration of the medium and testing different membrane protein targets, we obtained a number of evolved strains of which four (NG2, NG3, NG5 and NG6) were characterized and their genome was fully sequenced. Strikingly, each of the strains carried a mutation in the hns gene, whose product is involved in genome organization and transcriptional silencing. The degree of expression of (membrane) proteins correlates with the severity of the hns mutation, but cells in which hns was deleted showed an intermediate expression performance. We propose that (partial) removal of the transcriptional silencing mechanism changes the levels of proteins essential for the functional overexpression of membrane proteins. © 2013.

  10. Downregulation of ATM Gene and Protein Expression in Canine Mammary Tumors.

    PubMed

    Raposo-Ferreira, T M M; Bueno, R C; Terra, E M; Avante, M L; Tinucci-Costa, M; Carvalho, M; Cassali, G D; Linde, S D; Rogatto, S R; Laufer-Amorim, R

    2016-11-01

    The ataxia telangiectasia mutated (ATM) gene encodes a protein associated with DNA damage repair and maintenance of genomic integrity. In women, ATM transcript and protein downregulation have been reported in sporadic breast carcinomas, and the absence of ATM protein expression has been associated with poor prognosis. The aim of this study was to evaluate ATM gene and protein expression in canine mammary tumors and their association with clinical outcome. ATM gene and protein expression was evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in normal mammary gland samples (n = 10), benign mammary tumors (n = 11), nonmetastatic mammary carcinomas (n = 19), and metastatic mammary carcinomas (n = 11). Lower ATM transcript levels were detected in benign mammary tumors and carcinomas compared with normal mammary glands (P = .011). Similarly, lower ATM protein expression was observed in benign tumors (P = .0003), nonmetastatic mammary carcinomas (P < .0001), and the primary sites of metastatic carcinomas (P < .0001) compared with normal mammary glands. No significant differences in ATM gene or protein levels were detected among benign tumors and nonmetastatic and metastatic mammary carcinomas (P > .05). The levels of ATM gene or protein expression were not significantly associated with clinical and pathological features or with survival. Similar to human breast cancer, the data in this study suggest that ATM gene and protein downregulation is involved in canine mammary gland tumorigenesis. © The Author(s) 2016.

  11. Non-axisymmetric Aberration Patterns from Wide-field Telescopes Using Spin-weighted Zernike Polynomials

    NASA Astrophysics Data System (ADS)

    Kent, Stephen M.

    2018-04-01

    If the optical system of a telescope is perturbed from rotational symmetry, the Zernike wavefront aberration coefficients describing that system can be expressed as a function of position in the focal plane using spin-weighted Zernike polynomials. Methodologies are presented to derive these polynomials to arbitrary order. This methodology is applied to aberration patterns produced by a misaligned Ritchey–Chrétien telescope and to distortion patterns at the focal plane of the DESI optical corrector, where it is shown to provide a more efficient description of distortion than conventional expansions.

  12. Non-axisymmetric Aberration Patterns from Wide-field Telescopes Using Spin-weighted Zernike Polynomials

    DOE PAGES

    Kent, Stephen M.

    2018-02-15

    If the optical system of a telescope is perturbed from rotational symmetry, the Zernike wavefront aberration coefficients describing that system can be expressed as a function of position in the focal plane using spin-weighted Zernike polynomials. Methodologies are presented to derive these polynomials to arbitrary order. This methodology is applied to aberration patterns produced by a misaligned Ritchey Chretian telescope and to distortion patterns at the focal plane of the DESI optical corrector, where it is shown to provide a more efficient description of distortion than conventional expansions.

  13. Rhythmic expression of DEC2 protein in vitro and in vivo.

    PubMed

    Sato, Fuyuki; Muragaki, Yasuteru; Kawamoto, Takeshi; Fujimoto, Katsumi; Kato, Yukio; Zhang, Yanping

    2016-06-01

    Basic helix-loop-helix (bHLH) transcription factor DEC2 (bHLHE41/Sharp1) is one of the clock genes that show a circadian rhythm in various tissues. DEC2 regulates differentiation, sleep length, tumor cell invasion and apoptosis. Although studies have been conducted on the rhythmic expression of DEC2 mRNA in various tissues, the precise molecular mechanism of DEC2 expression is poorly understood. In the present study, we examined whether DEC2 protein had a rhythmic expression. Western blot analysis for DEC2 protein revealed a rhythmic expression in mouse liver, lung and muscle and in MCF-7 and U2OS cells. In addition, AMP-activated protein kinase (AMPK) activity (phosphorylation of AMPK) in mouse embryonic fibroblasts (MEFs) exhibited a rhythmic expression under the condition of medium change or glucose-depleted medium. However, the rhythmic expression of DEC2 in MEF gradually decreased in time under these conditions. The medium change affected the levels of DEC2 protein and phosphorylation of AMPK. In addition, the levels of DEC2 protein showed a rhythmic expression in vivo and in MCF-7 and U2OS cells. The results showed that the phosphorylation of AMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. These results may provide new insights into rhythmic expression and the regulation between DEC2 protein and AMPK activity.

  14. Novel isoprenylated proteins identified by an expression library screen.

    PubMed

    Biermann, B J; Morehead, T A; Tate, S E; Price, J R; Randall, S K; Crowell, D N

    1994-10-14

    Isoprenylated proteins are involved in eukaryotic cell growth and signal transduction. The protein determinant for prenylation is a short carboxyl-terminal motif containing a cysteine, to which the isoprenoid is covalently attached via thioether linkage. To date, isoprenylated proteins have almost all been identified by demonstrating the attachment of an isoprenoid to previously known proteins. Thus, many isoprenylated proteins probably remain undiscovered. To identify novel isoprenylated proteins for subsequent biochemical study, colony blots of a Glycine max cDNA expression library were [3H]farnesyl-labeled in vitro. Proteins identified by this screen contained several different carboxyl termini that conform to consensus farnesylation motifs. These proteins included known farnesylated proteins (DnaJ homologs) and several novel proteins, two of which contained six or more tandem repeats of a hexapeptide having the consensus sequence (E/G)(G/P)EK(P/K)K. Thus, plants contain a diverse array of genes encoding farnesylated proteins, and our results indicate that fundamental differences in the identities of farnesylated proteins may exist between plants and other eukaryotes. Expression library screening by direct labeling can be adapted to identify isoprenylated proteins from other organisms, as well as proteins with other post-translational modifications.

  15. Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen.

    PubMed

    Wang, Gangduo; Wang, Jianling; Khan, M Firoze

    2017-09-01

    Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1 mmol/kg/day by gavage) for 7 days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.

  16. Genome engineering for improved recombinant protein expression in Escherichia coli.

    PubMed

    Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

    2014-12-19

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

  17. Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein.

    PubMed

    Rosmorduc, O; Sirma, H; Soussan, P; Gordien, E; Lebon, P; Horisberger, M; Bréchot, C; Kremsdorf, D

    1999-05-01

    Chronic hepatitis B treatment has been significantly improved by interferon (IFN) treatment. However, some studies have suggested that hepatitis B virus (HBV) might have a direct effect on the resistance to IFN. Defective particles, generated by spliced HBV RNA and associated with chronic hepatitis B, have been previously characterized; expression of these particles leads to cytoplasmic accumulation of the capsid protein. The aim of this study was to investigate the role of these defective genomes in IFN resistance. The global antiviral activity of IFN was studied by virus yield reduction assays, the expression of three IFN-induced antiviral proteins was analysed by Western blotting and confocal microscopy, and the regulation of MxA gene expression was studied by Northern blotting and the luciferase assay, in Huh7 cells transfected with a complete or the defective HBV genome. Results showed that the expression of the defective genome reduces the antiviral activity of IFN and that this modulation involves a selective inhibition of MxA protein induction by the HBV capsid protein. Our results also show the trans-suppressive effect of the HBV capsid on the MxA promoter, which might participate in this phenomenon. In conclusion, this study shows a direct interplay between the IFN-sensitive pathway and the capsid protein and might implicate this defective HBV genome in virus persistence.

  18. [Suppression of COX-2 protein to cell apoptosis in non-small cell lung cancer].

    PubMed

    Sun, Limei; Zhao, Yue; Wang, Lujian; Song, Min; Song, Jiye

    2007-06-20

    One of mechanisms of carcinogenesis is suppression of cell apoptosis which leads to accumulation of aberrant cells. The aim of this study is to investigate cell apoptosis and COX-2 protein expression in non-small cell lung cancer (NSCLC). Cell apoptosis, expression of COX-2 and microvessel density (MVD) were detcted in 111 NSCLC samples by TdT-mediated dUTP nick end labeling (TUNEL) technique and immunohistochemical staining. The positive rate of COX-2 protein expression was 67.6% (75/111), and there were 53 patients with high level cell apoptosis (47.7%). Expression of COX-2 protien was significantly related to TNM stages (P=0.025) and lymph node metastasis (P=0.018). The MVD in NSCLC tissues with positive COX-2 expression was significantly higher than that in negative expression ones (P=0.000). COX model showed that lymph node metastasis (P=0.006) and positive expression of COX-2 protein (P=0.000) were independent prognostic factors of NSCLC. The expression of COX-2 protein may suppress cell apoptosis of tumor, and it may serve as a potential marker of prognosis for NSCLC.

  19. Aberrant expression of microRNAs and the miR-1/MET pathway in canine hepatocellular carcinoma.

    PubMed

    Lai, Y-C; Ushio, N; Rahman, M M; Katanoda, Y; Ogihara, K; Naya, Y; Moriyama, A; Iwanaga, T; Saitoh, Y; Sogawa, T; Sunaga, T; Momoi, Y; Izumi, H; Miyoshi, N; Endo, Y; Fujiki, M; Kawaguchi, H; Miura, N

    2018-06-01

    Canine hepatocellular carcinoma (HCC) is the most common primary hepatic tumour in dogs. MicroRNA (miRNA) dysregulation has been reported in human HCC and shown to have diagnostic and prognostic value; however, there are no data on miRNA expression in canine HCC. The aim of the present study was to investigate differentially expressed miRNAs in canine HCC. Analysis of miRNA expression in canine HCC tissues and cell lines by quantitative reverse transcription PCR showed that miR-1, miR-122, let-7a, and let-7g were downregulated, whereas miR-10b and miR-21 were upregulated in canine HCC. MET is one of the target genes of miR-1. MET was upregulated in canine HCC at the gene and protein levels, and a significant correlation between the concomitant downregulation of miR-1 and upregulation of MET was observed. Fast/intermediate-proliferating canine HCC cell lines had higher MET gene and protein expression levels than the slow-proliferating cell line. These findings suggest that miRNAs are differentially expressed in canine HCC, and that the miR-1/MET pathway may be associated with canine HCC cell proliferation. © 2018 John Wiley & Sons Ltd.

  20. Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

    USDA-ARS?s Scientific Manuscript database

    The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

  1. Ocular wavefront aberrations in the common marmoset Callithrix jacchus: effects of age and refractive error

    PubMed Central

    Coletta, Nancy J.; Marcos, Susana; Troilo, David

    2012-01-01

    The common marmoset, Callithrix jacchus, is a primate model for emmetropization studies. The refractive development of the marmoset eye depends on visual experience, so knowledge of the optical quality of the eye is valuable. We report on the wavefront aberrations of the marmoset eye, measured with a clinical Hartmann-Shack aberrometer (COAS, AMO Wavefront Sciences). Aberrations were measured on both eyes of 23 marmosets whose ages ranged from 18 to 452 days. Twenty-one of the subjects were members of studies of emmetropization and accommodation, and two were untreated normal subjects. Eleven of the 21 experimental subjects had worn monocular diffusers or occluders and ten had worn binocular spectacle lenses of equal power. Monocular deprivation or lens rearing began at about 45 days of age and ended at about 108 days of age. All refractions and aberration measures were performed while the eyes were cyclopleged; most aberration measures were made while subjects were awake, but some control measurements were performed under anesthesia. Wavefront error was expressed as a seventh-order Zernike polynomial expansion, using the Optical Society of America’s naming convention. Aberrations in young marmosets decreased up to about 100 days of age, after which the higher-order RMS aberration leveled off to about 0.10 micron over a 3 mm diameter pupil. Higher-order aberrations were 1.8 times greater when the subjects were under general anesthesia than when they were awake. Young marmoset eyes were characterized by negative spherical aberration. Visually deprived eyes of the monocular deprivation animals had greater wavefront aberrations than their fellow untreated eyes, particularly for asymmetric aberrations in the odd-numbered Zernike orders. Both lens-treated and deprived eyes showed similar significant increases in Z3-3 trefoil aberration, suggesting the increase in trefoil may be related to factors that do not involve visual feedback. PMID:20800078

  2. LS-CAP: an algorithm for identifying cytogenetic aberrations in hepatocellular carcinoma using microarray data.

    PubMed

    He, Xianmin; Wei, Qing; Sun, Meiqian; Fu, Xuping; Fan, Sichang; Li, Yao

    2006-05-01

    Biological techniques such as Array-Comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH) and affymetrix single nucleotide pleomorphism (SNP) array have been used to detect cytogenetic aberrations. However, on genomic scale, these techniques are labor intensive and time consuming. Comparative genomic microarray analysis (CGMA) has been used to identify cytogenetic changes in hepatocellular carcinoma (HCC) using gene expression microarray data. However, CGMA algorithm can not give precise localization of aberrations, fails to identify small cytogenetic changes, and exhibits false negatives and positives. Locally un-weighted smoothing cytogenetic aberrations prediction (LS-CAP) based on local smoothing and binomial distribution can be expected to address these problems. LS-CAP algorithm was built and used on HCC microarray profiles. Eighteen cytogenetic abnormalities were identified, among them 5 were reported previously, and 12 were proven by CGH studies. LS-CAP effectively reduced the false negatives and positives, and precisely located small fragments with cytogenetic aberrations.

  3. Functions of bromodomain-containing proteins and their roles in homeostasis and cancer.

    PubMed

    Fujisawa, Takao; Filippakopoulos, Panagis

    2017-04-01

    Bromodomains (BRDs) are evolutionarily conserved protein-protein interaction modules that are found in a wide range of proteins with diverse catalytic and scaffolding functions and are present in most tissues. BRDs selectively recognize and bind to acetylated Lys residues - particularly in histones - and thereby have important roles in the regulation of gene expression. BRD-containing proteins are frequently dysregulated in cancer, they participate in gene fusions that generate diverse, frequently oncogenic proteins, and many cancer-causing mutations have been mapped to the BRDs themselves. Importantly, BRDs can be targeted by small-molecule inhibitors, which has stimulated many translational research projects that seek to attenuate the aberrant functions of BRD-containing proteins in disease.

  4. Optimised 'on demand' protein arraying from DNA by cell free expression with the 'DNA to Protein Array' (DAPA) technology.

    PubMed

    Schmidt, Ronny; Cook, Elizabeth A; Kastelic, Damjana; Taussig, Michael J; Stoevesandt, Oda

    2013-08-02

    We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. [Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

    PubMed

    Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

    2016-01-01

    We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

  6. A Generic Protocol for Intracellular Expression of Recombinant Proteins in Bacillus subtilis.

    PubMed

    Phan, Trang; Huynh, Phuong; Truong, Tuom; Nguyen, Hoang

    2017-01-01

    Bacillus subtilis (B. subtilis) is a potential and attractive host for the production of recombinant proteins. Different expression systems for B. subtilis have been developed recently, and various target proteins have been recombinantly synthesized and purified using this host. In this chapter, we introduce a generic protocol to express a recombinant protein in B. subtilis. It includes protocols for (1) using our typical expression vector (plasmid pHT254) to introduce a target gene, (2) transformation of the target vector into B. subtilis, and (3) evaluation of the actual expression of a recombinant protein.

  7. Expression of membrane-associated proteins within single emulsion cell facsimiles.

    PubMed

    Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

    2012-07-07

    MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

  8. Expression and Localization of Plant Protein Disulfide Isomerase.

    PubMed Central

    Shorrosh, B. S.; Subramaniam, J.; Schubert, K. R.; Dixon, R. A.

    1993-01-01

    A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules. PMID:12231974

  9. Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

    PubMed

    Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A

    2013-03-01

    The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/β-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via

  10. OF TRYPANOSOMATIDS. ENDOTRANSFORMATIONS AND ABERRATIONS].

    PubMed

    Frolov, A O; Malysheva, M N; Kostygov, A Yu

    2016-01-01

    Endotransformations and aberrations of the life cycle in the evolutionary history of trypanosomatids (Kinetoplastea: Trypanosomatidae) are analyzed. We treat the term "endotransformations" as evolutionarily fixed changes of phases and/or developmental stages of parasites. By contrast, we treat aberrations as evolutionary unstable, periodically arising deformations of developmental phases of trypanosomatids, never leading to life cycle changes. Various examples of life cycle endotransformations and aberrations in representatives of the family Trypanosomatidae are discussed.

  11. Parasitization by Scleroderma guani influences protein expression in Tenebrio molitor pupae.

    PubMed

    Zhu, Jia-Ying; Wu, Guo-Xing; Ze, Sang-Zi; Stanley, David W; Yang, Bin

    2014-07-01

    Ectoparasitoid wasps deposit their eggs onto the surface and inject venom into their hosts. Venoms are chemically complex and they exert substantial impact on hosts, including permanent or temporary paralysis and developmental arrest. These visible venom effects are due to changes in expression of genes encoding physiologically relevant proteins. While the influence of parasitization on gene expression in several lepidopterans has been reported, the molecular details of parasitoid/beetle relationships remain mostly unknown. This shortcoming led us to pose the hypothesis that envenomation by the ectoparasitic ant-like bethylid wasp Scleroderma guani leads to changes in protein expression in the yellow mealworm beetle Tenebrio molitor. We tested our hypothesis by comparing the proteomes of non-parasitized and parasitized host pupae using iTRAQ-based proteomics. We identified 41 proteins that were differentially expressed (32↑- and 9↓-regulated) in parasitized pupae. We assigned these proteins to functional categories, including immunity, stress and detoxification, energy metabolism, development, cytoskeleton, signaling and others. We recorded parallel changes in mRNA levels and protein abundance in 14 selected proteins following parasitization. Our findings support our hypothesis by documenting changes in protein expression in parasitized hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Insect cells-baculovirus system for the production of difficult to express proteins.

    PubMed

    Osz-Papai, Judit; Radu, Laura; Abdulrahman, Wassim; Kolb-Cheynel, Isabelle; Troffer-Charlier, Nathalie; Birck, Catherine; Poterszman, Arnaud

    2015-01-01

    The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

  13. Fetal deficiency of Lin28 programs life-long aberrations in growth and glucose metabolism

    PubMed Central

    Shinoda, Gen; Shyh-Chang, Ng; de Soysa, T. Yvanka; Zhu, Hao; Seligson, Marc T.; Shah, Samar P.; Abo-Sido, Nora; Yabuuchi, Akiko; Hagan, John P.; Gregory, Richard I.; Asara, John M.; Cantley, Lewis C.; Moss, Eric G.; Daley, George Q.

    2013-01-01

    LIN28A/B are RNA binding proteins implicated by genetic association studies in human growth and glucose metabolism. Mice with ectopic over-expression of Lin28a have shown related phenotypes. Here we describe the first comprehensive analysis of the physiologic consequences of Lin28a and Lin28b deficiency in knockout (KO) mice. Lin28a/b-deficiency led to dwarfism starting at different ages, and compound gene deletions showed a cumulative dosage effect on organismal growth. Conditional gene deletion at specific developmental stages revealed that fetal but neither neonatal nor adult deficiency resulted in growth defects and aberrations in glucose metabolism. Tissue-specific KO mice implicated skeletal muscle-deficiency in the abnormal programming of adult growth and metabolism. The effects of Lin28b KO can be rescued by Tsc1 haplo-insufficiency in skeletal muscles. Our data implicate fetal expression of Lin28a/b in the regulation of life-long effects on metabolism and growth, and demonstrate that fetal Lin28b acts at least in part via mTORC1 signaling. PMID:23666760

  14. Forkhead box protein A2, a pioneer factor for hepatogenesis, is involved in the expression of hepatic phenotype of alpha-fetoprotein-producing adenocarcinoma.

    PubMed

    Yamamura, Nobuhisa; Fugo, Kazunori; Kishimoto, Takashi

    2017-09-01

    Alpha-fetoprotein (AFP)-producing adenocarcinoma is a high-malignant variant of adenocarcinoma with a hepatic or fetal-intestinal phenotype. The number of cases of AFP-producing adenocarcinomas is increasing, but the molecular mechanism underlying the aberrant production of AFP is unclear. Here we sought to assess the role of Forkhead box A (FoxA)2, which is a pioneer transcription factor in the differentiation of hepatoblasts. FoxA2 expression was investigated in five cases of AFP-producing gastric adenocarcinomas by immunohistochemistry, and all cases showed FoxA2 expression. Chromatin immunoprecipitation revealed the DNA binding of FoxA2 on the regulatory element of AFP gene in AFP-producing adenocarcinoma cells. The inhibition of FoxA2 expression with siRNA reduced the mRNA expression of liver-specific proteins, including AFP, albumin, and transferrin. The inhibition of FoxA2 also reduced the expressions of liver-enriched nuclear factors, i.e., hepatocyte nuclear factor (HNF) 4α and HNF6, although the expressions of HNF1α and HNF1β were not affected. The same effect as FoxA2 knockdown in AFP producing adenocarcinoma cells was also observed in hepatocellular carcinoma cells. Our results suggest that FoxA2 plays a key role in the expression of hepatic phenotype of AFP-producing adenocarcinomas. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Novel leukocyte protein, Trojan, differentially expressed during thymocyte development.

    PubMed

    Petrov, Petar; Motobu, Maki; Salmi, Jussi; Uchida, Tatsuya; Vainio, Olli

    2010-04-01

    "Trojan" is a novel cell surface protein, discovered from chicken embryonic thymocytes on the purpose to identify molecules involved in T cell differentiation. The molecule is predicted as a type I transmembrane protein having a Sushi and two fibronectin type III domains and a pair of intracellular phosphorylation sites. Its transcript expression is specific for lymphoid tissues and the presence of the protein on the surface of recirculating lymphocytes and macrophages was confirmed by immunofluorescence analysis. In thymus, about half of the double negative (CD4(-) CD8(-)) and CD8 single positive and the majority of CD4 single positive cells express Trojan with a relatively high intensity. However, only a minority of the double positive (CD4(+) CD8(+)) cells are positive for Trojan. This expression pattern, similar to that of some proteins with anti-apoptotic and function, like IL-7Ralpha, makes Trojan an attractive candidate of having an anti-apoptotic role. Copyright 2010 Elsevier Ltd. All rights reserved.

  16. Rift Valley Fever Virus Structural and Nonstructural Proteins: Recombinant Protein Expression and Immunoreactivity Against Antisera from Sheep

    PubMed Central

    Faburay, Bonto; Wilson, William; McVey, D. Scott; Drolet, Barbara S.; Weingartl, Hana; Madden, Daniel; Young, Alan; Ma, Wenjun

    2013-01-01

    Abstract The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA). PMID:23962238

  17. Protein-protein interactions and cancer: targeting the central dogma.

    PubMed

    Garner, Amanda L; Janda, Kim D

    2011-01-01

    Between 40,000 and 200,000 protein-protein interactions have been predicted to exist within the human interactome. As these interactions are of a critical nature in many important cellular functions and their dysregulation is causal of disease, the modulation of these binding events has emerged as a leading, yet difficult therapeutic arena. In particular, the targeting of protein-protein interactions relevant to cancer is of fundamental importance as the tumor-promoting function of several aberrantly expressed proteins in the cancerous state is directly resultant of its ability to interact with a protein-binding partner. Of significance, these protein complexes play a crucial role in each of the steps of the central dogma of molecular biology, the fundamental processes of genetic transmission. With the many important discoveries being made regarding the mechanisms of these genetic process, the identification of new chemical probes are needed to better understand and validate the druggability of protein-protein interactions related to the central dogma. In this review, we provide an overview of current small molecule-based protein-protein interaction inhibitors for each stage of the central dogma: transcription, mRNA splicing and translation. Importantly, through our analysis we have uncovered a lack of necessary probes targeting mRNA splicing and translation, thus, opening up the possibility for expansion of these fields.

  18. Monochromatic ocular wave aberrations in young monkeys

    PubMed Central

    Ramamirtham, Ramkumar; Kee, Chea-su; Hung, Li-Fang; Qiao-Grider, Ying; Roorda, Austin; Smith, Earl L.

    2006-01-01

    High-order monochromatic aberrations could potentially influence vision-dependent refractive development in a variety of ways. As a first step in understanding the effects of wave aberration on refractive development, we characterized the maturational changes that take place in the high-order aberrations of infant rhesus monkey eyes. Specifically, we compared the monochromatic wave aberrations of infant and adolescent animals and measured the longitudinal changes in the high-order aberrations of infant monkeys during the early period when emmetropization takes place. Our main findings were that (1) adolescent monkey eyes have excellent optical quality, exhibiting total RMS errors that were slightly better than those for adult human eyes that have the same numerical aperture and (2) shortly after birth, infant rhesus monkeys exhibited relatively larger magnitudes of high-order aberrations predominately spherical aberration, coma, and trefoil, which decreased rapidly to assume adolescent values by about 200 days of age. The results demonstrate that rhesus monkey eyes are a good model for studying the contribution of individual ocular components to the eye’s overall aberration structure, the mechanisms responsible for the improvements in optical quality that occur during early ocular development, and the effects of high-order aberrations on ocular growth and emmetropization. PMID:16750549

  19. Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System.

    PubMed

    Steinmetz, Eric J; Auldridge, Michele E

    2017-11-01

    The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process. This unit presents a strategy for parallel screening of fusion partners for enhanced expression or solubility. The Expresso® Solubility and Expression Screening System includes a panel of seven distinct fusion partners and utilizes an extremely simple cloning strategy to enable rapid screening and identification of the most effective fusion partner. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  20. Expression of APG-2 protein, a member of the heat shock protein 110 family, in developing rat brain.

    PubMed

    Okui, M; Ito, F; Ogita, K; Kuramoto, N; Kudoh, J; Shimizu, N; Ide, T

    2000-01-01

    APG-2 protein is a member of the heat shock protein 110 family, and it is thought to play an important role in the maintenance of neuronal functions under physiological and stress conditions. However, neither the tissue-distribution of APG-2 protein nor developmental change of its expression has been studied at the protein level. Therefore, we generated an antiserum against APG-2 protein and studied expression of this protein in rat brain and other tissues by use of the Western blot method. The results showed a high expression of APG-2 protein in various regions of the central nervous system (cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, cerebellum, medulla pons, and spinal cord) throughout the entire postnatal stage. Similarly, a high level of APG-2 protein was detected in the whole brain of rat embryos and in adult rat tissues such as liver, lung, spleen, and kidney. In contrast, its expression in heart was high at postnatal days 1 and 3, but thereafter drastically decreased to a low level. Furthermore, APG-2 protein was detected in neuronal primary cultures prepared from rat cerebral cortex, and its level did not change notably during neuronal differentiation. These results show that APG-2 protein is constitutively expressed in various tissues and also in neuronal cells throughout the entire embryonic and postnatal period. suggesting that it might play an important role in these tissues under non-stress conditions.

  1. Marker Protein Expression Combined With Expression Heterogeneity is a Powerful Indicator of Malignancy in Acral Lentiginous Melanomas.

    PubMed

    Cintra Lopes Carapeto, Fernando; Neves Comodo, Andréia; Germano, Andressa; Pereira Guimarães, Daiane; Barcelos, Denise; Fernandes, Mariana; Landman, Gilles

    2017-02-01

    Samples of acral lentiginous melanomas (ALMs) were obtained from the Department of Pathology at Escola Paulista de Medicina-Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil. Demographic, clinical, and follow-up data were obtained from the charts of Hospital São Paulo. From 2 tissue microarrays containing 60 nevi and quadruplicate samples of ≥1.0-mm of 49 ALM, sections were stained to evaluate SCF, KIT, BRAF, CYCLIND1, MYC, and PTEN immunohistochemical protein expression. Nevi and ALM from 2006 to 2010 were reviewed and collected. All specimens were in the vertical growth phase, and histopathological parameters indicated that tumors were at an advanced stage at diagnosis. Average tumor thickness was 6.95 mm, 63% were ulcerated, average mitotic index was 5 mitotic cells per mm, and 43% were at Clark's level V. Compared with nevi, the χ test showed that ALM significantly correlated with SCF protein expression (P = 0.001) and expression heterogeneity (P < 0.000). Similar findings were observed for KIT (P = 0.005, P = 0.003, respectively), MYC (P < 0.000, P < 0.000), and PTEN (P = 0.005, P < 0.000). Malignancy did not correlate with BRAF and CYCLIN D1 expression (P = 0.053 and P = 0.259, respectively), but it did significantly correlate with their heterogeneous expression (P < 0.000, P = 0.024, respectively). Combined protein expression had an odds ratio of greater malignancy when BRAF and MYC were positive and/or heterogeneously expressed (OR of 78 and 95, respectively). We show that marker protein expression, when combined with heterogeneous expression as shown by immunohistochemistry, is a powerful indicator of malignancy in ALMs, especially, when protein pairs are combined.

  2. Iteration of ultrasound aberration correction methods

    NASA Astrophysics Data System (ADS)

    Maasoey, Svein-Erik; Angelsen, Bjoern; Varslot, Trond

    2004-05-01

    Aberration in ultrasound medical imaging is usually modeled by time-delay and amplitude variations concentrated on the transmitting/receiving array. This filter process is here denoted a TDA filter. The TDA filter is an approximation to the physical aberration process, which occurs over an extended part of the human body wall. Estimation of the TDA filter, and performing correction on transmit and receive, has proven difficult. It has yet to be shown that this method works adequately for severe aberration. Estimation of the TDA filter can be iterated by retransmitting a corrected signal and re-estimate until a convergence criterion is fulfilled (adaptive imaging). Two methods for estimating time-delay and amplitude variations in receive signals from random scatterers have been developed. One method correlates each element signal with a reference signal. The other method use eigenvalue decomposition of the receive cross-spectrum matrix, based upon a receive energy-maximizing criterion. Simulations of iterating aberration correction with a TDA filter have been investigated to study its convergence properties. A weak and strong human-body wall model generated aberration. Both emulated the human abdominal wall. Results after iteration improve aberration correction substantially, and both estimation methods converge, even for the case of strong aberration.

  3. A recurrent 11q aberration pattern characterizes a subset of MYC-negative high-grade B-cell lymphomas resembling Burkitt lymphoma.

    PubMed

    Salaverria, Itziar; Martin-Guerrero, Idoia; Wagener, Rabea; Kreuz, Markus; Kohler, Christian W; Richter, Julia; Pienkowska-Grela, Barbara; Adam, Patrick; Burkhardt, Birgit; Claviez, Alexander; Damm-Welk, Christine; Drexler, Hans G; Hummel, Michael; Jaffe, Elaine S; Küppers, Ralf; Lefebvre, Christine; Lisfeld, Jasmin; Löffler, Markus; Macleod, Roderick A F; Nagel, Inga; Oschlies, Ilske; Rosolowski, Maciej; Russell, Robert B; Rymkiewicz, Grzegorz; Schindler, Detlev; Schlesner, Matthias; Scholtysik, René; Schwaenen, Carsten; Spang, Rainer; Szczepanowski, Monika; Trümper, Lorenz; Vater, Inga; Wessendorf, Swen; Klapper, Wolfram; Siebert, Reiner

    2014-02-20

    The genetic hallmark of Burkitt lymphoma (BL) is the t(8;14)(q24;q32) and its variants leading to activation of the MYC oncogene. It is a matter of debate whether true BL without MYC translocation exists. Here, we identified 59 lymphomas concordantly called BL by 2 gene expression classifiers among 753 B-cell lymphomas. Only 2 (3%) of these 59 molecular BL lacked a MYC translocation, which both shared a peculiar pattern of chromosome 11q aberration characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter. We extended our analysis to 17 MYC-negative high-grade B-cell lymphomas with a similar 11q aberration and showed this aberration to be recurrently associated with morphologic and clinical features of BL. The minimal region of gain was defined by high-level amplifications in 11q23.3 and associated with overexpression of genes including PAFAH1B2 on a transcriptional and protein level. The recurrent region of loss contained a focal homozygous deletion in 11q24.2-q24.3 including the ETS1 gene, which was shown to be mutated in 4 of 16 investigated cases. These findings indicate the existence of a molecularly distinct subset of B-cell lymphomas reminiscent of BL, which is characterized by deregulation of genes in 11q.

  4. Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

    PubMed

    Tagliavia, Marcello; Cuttitta, Angela

    2016-01-01

    High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

  5. Prognostic relevance of Centromere protein H expression in esophageal carcinoma.

    PubMed

    Guo, Xian-Zhi; Zhang, Ge; Wang, Jun-Ye; Liu, Wan-Li; Wang, Fang; Dong, Ju-Qin; Xu, Li-Hua; Cao, Jing-Yan; Song, Li-Bing; Zeng, Mu-Sheng

    2008-08-13

    Many kinetochore proteins have been shown to be associated with human cancers. The aim of the present study was to clarify the expression of Centromere protein H (CENP-H), one of the fundamental components of the human active kinetochore, in esophageal carcinoma and its correlation with clinicopathological features. We examined the expression of CENP-H in immortalized esophageal epithelial cells as well as in esophageal carcinoma cells, and in 12 cases of esophageal carcinoma tissues and the paired normal esophageal tissues by RT-PCR and Western blot analysis. In addition, we analyzed CENP-H protein expression in 177 clinicopathologically characterized esophageal carcinoma cases by immunohistochemistry. Statistical analyses were applied to test for prognostic and diagnostic associations. The level of CENP-H mRNA and protein were higher in the immortalized cells, cancer cell lines and most cancer tissues than in normal control tissues. Immunohistochemistry showed that CENP-H was expressed in 127 of 171 ESCC cases (74.3%) and in 3 of 6 esophageal adenocarcinoma cases (50%). Statistical analysis of ESCC cases showed that there was a significant difference of CENP-H expression in patients categorized according to gender (P = 0.013), stage (P = 0.023) and T classification (P = 0.019). Patients with lower CENP-H expression had longer overall survival time than those with higher CENP-H expression. Multivariate analysis suggested that CENP-H expression was an independent prognostic marker for esophageal carcinoma patients. A prognostic value of CENP-H was also found in the subgroup of T3 approximately T4 and N0 tumor classification. Our results suggest that CENP-H protein is a valuable marker of esophageal carcinoma progression. CENP-H might be used as a valuable prognostic marker for esophageal carcinoma patients.

  6. Calcium affecting protein expression in longan under simulated acid rain stress.

    PubMed

    Pan, Tengfei; Li, Yongyu; Ma, Cuilan; Qiu, Dongliang

    2015-08-01

    Longan (Dimocarpus longana Lour. cv. Wulongling) of uniform one-aged seedlings grown in pots were selected to study specific proteins expressed in leaves under simulated acid rain (SiAR) stress and exogenous Ca(2+) regulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that there was a protein band specifically expressed under SiAR of pH 2.5 stress for 15 days with its molecular weight of about 23 kD. A 17 kD protein band specifically expressed after SiAR stress 5 days. Compared with pH 2.5, the pH 3.5 of SiAR made a less influence to protein expression. Two-dimensional electrophoresis (2-DE) results showed that six new specific proteins including C4 (20.2 kD pI 6.0), F (24 kD pI 6.35), B3 (22.3 kD pI 6.35), B4 (23.5 kD pI 6.5), C5 (21.8 kD pI 5.6), and C6 (20.2 kD pI 5.6) specifically expressed. C4 always expressed during SiAR stress. F expressed under the stress of pH 2.5 for 15 days and expressed in all pH SiAR stress for 20 days. The expression of proteins including B3, C5, and C6 was related to pH value and stress intensity of SiAR. The expression of B4 resulted from synergistic effects of SiAR and Ca. The expression of G1 (Mr 19.3 kD, pI 4.5), G2 (Mr 17.8 kD, pI 4.65), G3 (Mr 16.6 kD, pI 4.6), and G4 (Mr 14.7 kD, pI 4.4) enhanced under the treatment of 5 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM chlorpromazine (CPZ). These proteins showed antagonistic effects and might be relative to the Ca-calmodulin (Ca-CaM) system of longan in response to SiAR stress.

  7. Heterogeneity of proteins expressed by Brazilian Sporothrix schenckii isolates.

    PubMed

    Fernandes, Geisa Ferreira; Do Amaral, Cristiane Candida; Sasaki, Alexandre; Godoy, Patrício Martinez; De Camargo, Zoilo Pires

    2009-12-01

    The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.

  8. Chronic Nicotine Mitigates Aberrant Inhibitory Motor Learning Induced by Motor Experience under Dopamine Deficiency

    PubMed Central

    Krok, Anne C.; Xu, Jian; Contractor, Anis; McGehee, Daniel S.; Zhuang, Xiaoxi

    2016-01-01

    function and/or receptor expression of β2-containing nicotinic receptors alters presynaptic and postsynaptic striatal signaling to protect against aberrant motor learning. Moreover, these results suggest that cNIC treatment may alleviate motor symptoms and/or delay the deterioration of motor function in movement disorders by blocking aberrant motor learning. PMID:27170121

  9. Expression of HOXB2, a retinoic acid signaling target in pancreatic cancer and pancreatic intraepithelial neoplasia.

    PubMed

    Segara, Davendra; Biankin, Andrew V; Kench, James G; Langusch, Catherine C; Dawson, Amanda C; Skalicky, David A; Gotley, David C; Coleman, Maxwell J; Sutherland, Robert L; Henshall, Susan M

    2005-05-01

    Despite significant progress in understanding the molecular pathology of pancreatic cancer and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility as prognostic or therapeutic markers. Here, we used oligonucleotide microarrays to interrogate mRNA expression of pancreatic cancer tissue and normal pancreas to identify novel molecular pathways dysregulated in the development and progression of pancreatic cancer. RNA was hybridized to Affymetrix Genechip HG-U133 oligonucleotide microarrays. A relational database integrating data from publicly available resources was created to identify candidate genes potentially relevant to pancreatic cancer. The protein expression of one candidate, homeobox B2 (HOXB2), in PanIN and pancreatic cancer was assessed using immunohistochemistry. We identified aberrant expression of several components of the retinoic acid (RA) signaling pathway (RARalpha, MUC4, Id-1, MMP9, uPAR, HB-EGF, HOXB6, and HOXB2), many of which are known to be aberrantly expressed in pancreatic cancer and PanIN. HOXB2, a downstream target of RA, was up-regulated 6.7-fold in pancreatic cancer compared with normal pancreas. Immunohistochemistry revealed ectopic expression of HOXB2 in 15% of early PanIN lesions and 48 of 128 (38%) pancreatic cancer specimens. Expression of HOXB2 was associated with nonresectable tumors and was an independent predictor of poor survival in resected tumors. We identified aberrant expression of RA signaling components in pancreatic cancer, including HOXB2, which was expressed in a proportion of PanIN lesions. Ectopic expression of HOXB2 was associated with a poor prognosis for all patients with pancreatic cancer and was an independent predictor of survival in patients who underwent resection.

  10. Recombinant protein expression in Escherichia coli: advances and challenges

    PubMed Central

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555

  11. Transient expression and cellular localization of recombinant proteins in cultured insect cells

    USDA-ARS?s Scientific Manuscript database

    Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

  12. Aberrant DNA Methylation in Chronic Myeloid Leukemia: Cell Fate Control, Prognosis, and Therapeutic Response.

    PubMed

    Behzad, Masumeh Maleki; Shahrabi, Saeid; Jaseb, Kaveh; Bertacchini, Jessika; Ketabchi, Neda; Saki, Najmaldin

    2018-01-31

    Chronic myeloid leukemia (CML) is a hematopoietic stem cell malignancy characterized by the expression of the BCR-ABL1 fusion gene with different chimeric transcripts. Despite the crucial impact of constitutively active tyrosine kinase in CML pathogenesis, aberrant DNA methylation of certain genes plays an important role in disease progression and the development of drug resistance. This article reviews recent findings relevant to the effect of DNA methylation pattern of regulatory genes on various cellular activities such as cell proliferation and survival, as well as cell-signaling molecules in CML. These data might contribute to defining the role of aberrant DNA methylation in disease initiation and progression. However, further studies are needed on the validation of specific aberrant methylation markers regarding the prognosis and prediction of response among the CML patients.

  13. Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer's disease model cell line

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Genome-wide DNA methylation pattern in Alzheimer's disease model cell line. Black-Right-Pointing-Pointer Integrated analysis of CpG methylation and mRNA expression profiles. Black-Right-Pointing-Pointer Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. Black-Right-Pointing-Pointer The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterationsmore » in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2 Prime -deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory

  14. Expression of Lipid Metabolism-Related Proteins in Metastatic Breast Cancer.

    PubMed

    Jung, Yoon Yang; Kim, Hye Min; Koo, Ja Seung

    2015-01-01

    The tumor biology of metastatic breast cancers differ according to the metastatic sites, and the features of cancer metabolism may also be different. The aim of this study is to investigate the expression of lipid metabolism-related proteins in metastatic breast cancer according to metastatic site and discuss the clinical significance thereof. Immunohistochemical staining for lipid metabolism-related proteins [fatty acid synthase (FASN), hormone-sensitive lipase (HSL), carnitine palmitoyltransferase IA (CPT-1A), acyl-CoA oxidase 1 (ACOX1), fatty acid binding protein 4 (FABP4,) and perilipin 1 (PLIN1)] was performed using a tissue microarray of 149 cases of metastatic breast cancer (bone metastasis = 39, brain metastasis = 37, liver metastasis = 21, and lung metastasis = 52). The expression levels of ACOX1 (p = 0.009) and FASN (p = 0.007) varied significantly according to metastatic site, with the highest expression in brain metastasis and the lowest expression in liver metastasis. ACOX1 positivity (p = 0.005) and FASN positivity (p = 0.003) correlated with HER-2 positivity. The expression of FASN was significantly higher in HER-2 type breast cancer, and lower in luminal A and TNBC type breast cancer (p<0.001). Among lipid metabolism-related proteins, PLIN1 positivity was found to be an independent poor prognostic factor on multivariate analysis (Hazard ratio: 4.979, 95% CI: 1.054-22.59, p = 0.043). Different expression levels of lipid metabolism-related proteins were observed according to metastatic site. The expression of ACOX1 and FASN was highest in brain metastasis. These results suggest that the metastatic site should be considered when using lipid metabolism inhibitors for targeted therapy.

  15. Intrafocal heterogeneity of ERG protein expression and gene fusion pattern in prostate cancer.

    PubMed

    Suh, Ja Hee; Park, Jeong Hwan; Lee, Cheol; Moon, Kyung Chul

    2017-10-01

    Prostate cancer is considered to be highly heterogeneous, with various morphologic features and biologic behaviors. The TMPRSS2-ERG gene fusion is the most frequently observed genetic aberration in prostate cancer. The aim of this study was to elucidate the intrafocal heterogeneity of ERG gene fusion status. ERG immunohistochemistry (IHC) was performed in samples from 168 prostate cancer patients who had undergone radical prostatectomy, and 40 cases showing ERG-positive IHC staining were selected for tissue microarray (TMA) construction. Two to six representative cores were selected from each tumor focus. In the cases with heterogeneous ERG IHC staining intensity, the areas showing different intensities were separately selected. Using the TMA blocks, IHC and fluorescence in situ hybridization (FISH) were conducted to evaluate the heterogeneity of ERG protein expression and ERG fusion gene patterns, respectively, in a single tumor focus. Heterogeneity of ERG IHC staining was defined as the simultaneous presence of negative and positive cores in the same tumor focus. Heterogeneity of ERG FISH was defined by the presence of cores with positive and negative FISH signals or cores with break-apart and interstitial deletion FISH signals in the same tumor focus. A total of 202 TMA cores were isolated from 40 ERG-positive cases. Of the 202 total cores, 19 were negative for ERG IHC staining, and 46 showed 1+, 52 showed 2+, and 85 showed 3+ ERG staining intensity. Eleven cores were negative for ERG FISH signal, 119 cores showed ERG break-apart FISH signals, and the remaining 72 cores revealed interstitial deletion. Intrafocal heterogeneity of ERG IHC staining was found in 20% (8/40) of cases, and intrafocal heterogeneity of ERG gene fusion pattern was found in 32.5% (13/40) of cases. In summary, this study showed significantly frequent intrafocal heterogeneity of ERG protein expression, gene fusion status and fusion pattern. This heterogeneity can be caused by the development

  16. Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy.

    PubMed

    Dukes, Madeline J; Ramachandra, Ranjan; Baudoin, Jean-Pierre; Gray Jerome, W; de Jonge, Niels

    2011-06-01

    Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Silibinin inhibits aberrant lipid metabolism, proliferation and emergence of androgen-independence in prostate cancer cells via primarily targeting the sterol response element binding protein 1

    PubMed Central

    Nambiar, Dhanya K.; Deep, Gagan; Singh, Rana P.; Agarwal, Chapla; Agarwal, Rajesh

    2014-01-01

    Prostate cancer (PCA) kills thousands of men every year, demanding additional approaches to better understand and target this malignancy. Recently, critical role of aberrant lipogenesis is highlighted in prostate carcinogenesis, offering a unique opportunity to target it to reduce PCA. Here, we evaluated efficacy and associated mechanisms of silibinin in inhibiting lipid metabolism in PCA cells. At physiologically achievable levels in human, silibinin strongly reduced lipid and cholesterol accumulation specifically in human PCA cells but not in non-neoplastic prostate epithelial PWR-1E cells. Silibinin also decreased nuclear protein levels of sterol regulatory element binding protein 1 and 2 (SREBP1/2) and their target genes only in PCA cells. Mechanistically, silibinin activated AMPK, thereby increasing SREBP1 phosphorylation and inhibiting its nuclear translocation; AMPK inhibition reversed silibinin-mediated decrease in nuclear SREBP1 and lipid accumulation. Additionally, specific SREBP inhibitor fatostatin and stable overexpression of SREBP1 further confirmed the central role of SREBP1 in silibinin-mediated inhibition of PCA cell proliferation and lipid accumulation and cell cycle arrest. Importantly, silibinin also inhibited synthetic androgen R1881-induced lipid accumulation and completely abrogated the development of androgen-independent LNCaP cell clones via targeting SREBP1/2. Together, these mechanistic studies suggest that silibinin would be effective against PCA by targeting critical aberrant lipogenesis. PMID:25294820

  18. Silibinin inhibits aberrant lipid metabolism, proliferation and emergence of androgen-independence in prostate cancer cells via primarily targeting the sterol response element binding protein 1.

    PubMed

    Nambiar, Dhanya K; Deep, Gagan; Singh, Rana P; Agarwal, Chapla; Agarwal, Rajesh

    2014-10-30

    Prostate cancer (PCA) kills thousands of men every year, demanding additional approaches to better understand and target this malignancy. Recently, critical role of aberrant lipogenesis is highlighted in prostate carcinogenesis, offering a unique opportunity to target it to reduce PCA. Here, we evaluated efficacy and associated mechanisms of silibinin in inhibiting lipid metabolism in PCA cells. At physiologically achievable levels in human, silibinin strongly reduced lipid and cholesterol accumulation specifically in human PCA cells but not in non-neoplastic prostate epithelial PWR-1E cells. Silibinin also decreased nuclear protein levels of sterol regulatory element binding protein 1 and 2 (SREBP1/2) and their target genes only in PCA cells. Mechanistically, silibinin activated AMPK, thereby increasing SREBP1 phosphorylation and inhibiting its nuclear translocation; AMPK inhibition reversed silibinin-mediated decrease in nuclear SREBP1 and lipid accumulation. Additionally, specific SREBP inhibitor fatostatin and stable overexpression of SREBP1 further confirmed the central role of SREBP1 in silibinin-mediated inhibition of PCA cell proliferation and lipid accumulation and cell cycle arrest. Importantly, silibinin also inhibited synthetic androgen R1881-induced lipid accumulation and completely abrogated the development of androgen-independent LNCaP cell clones via targeting SREBP1/2. Together, these mechanistic studies suggest that silibinin would be effective against PCA by targeting critical aberrant lipogenesis.

  19. Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

    PubMed Central

    Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

    2013-01-01

    Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

  20. [Monochromatic aberration in accommodation. Dynamic wavefront analysis].

    PubMed

    Fritzsch, M; Dawczynski, J; Jurkutat, S; Vollandt, R; Strobel, J

    2011-06-01

    Monochromatic aberrations may influence the visual acuity of the eye. They are not stable and can be affected by different factors. The subject of the following paper is the dynamic investigation of the changes in wavefront aberration with accommodation. Dynamic measurement of higher and lower order aberrations was performed with a WASCA Wavefront Analyzer (Carl-Zeiss-Meditec) and a specially constructed target device for aligning objects in far and near distances on 25 subjects aged from 15 to 27 years old. Wavefront aberrations showed some significant changes in accommodation. In addition to the characteristic sphere reaction accompanying miosis and changes in horizontal prism (Z(1) (1)) in the sense of a convergence movement of the eyeball also occurred. Furthermore defocus rose (Z(2) (0)) and astigmatism (Z(2) (-2)) changed. In higher-order aberrations a decrease in coma-like Zernike polynomials (Z(3) (-1), Z(3) (1)) was found. The most obvious change appeared in spherical aberration (Z(4) (0)) which increased and changed from positive to negative. In addition the secondary astigmatism (Z(4) (-2)) and quadrafoil (Z(4) (4)) rise also increased. The total root mean square (RMS), as well as the higher-order aberrations (RMS-HO) significantly increased in accommodation which is associated with a theoretical reduction of visual acuity. An analysis of the influence of pupil size on aberrations showed significant increases in defocus, spherical aberration, quadrafoil, RMS and RMS HO by increasing pupil diameter. By accommodation-associated miosis, the growing aberrations are partially compensated by focusing on near objects. Temporal analysis of the accommodation process with dynamic wavefront analysis revealed significant delays in pupil response and changing of prism in relation to the sphere reaction. In accommodation to near objects a discrete time ahead of third order aberrations in relation to the sphere response was found. Using dynamic wavefront measurement

  1. High-level SLP-2 expression and HER-2/neu protein expression are associated with decreased breast cancer patient survival.

    PubMed

    Cao, Wenfeng; Zhang, Bin; Liu, Yanxue; Li, Hongtao; Zhang, Shiwu; Fu, Li; Niu, Yun; Ning, Liansheng; Cao, Xuchen; Liu, Zhihua; Sun, Baocun

    2007-09-01

    There is sufficient evidence that human stomatin-like protein 2 (SLP-2) is a novel cancer-related gene. Its protein is overexpressed in many human cancers. SLP-2 can contribute to the promotion of cell growth, cell adhesion, and tumorigenesis in esophageal squamous cell carcinoma and lymph node metastasis in laryngeal squamous cell carcinoma. Immunohistochemical detection of SLP-2, estrogen and progesterone receptors, and HER-2/neu were performed on 263 cases of primary invasive breast cancer with a tissue microarray. Of 263 cases, 138 (52.5%) showed high expression of SLP-2 protein, and 125 (47.5%) showed low or absent expression. In addition, there were significant positive associations between tumor stage and size (P = .020), lymph node metastasis (P < .001), clinical stage (P < .001), distant metastasis (P = .002), and HER-2/neu protein expression (P = .037) and high-level SLP-2 expression. High-level SLP-2 expression was associated with decreased overall survival (P = .011) and was more often found in patients with tumors larger than 20 mm, lymph node metastasis, advanced clinical stage, distant metastasis, and HER-2/neu protein-positive expression. More important, lymph node metastasis, HER-2/neu-positive expression, and high-level SLP-2 expression were associated with significantly decreased survival.

  2. Wavefront aberrations of x-ray dynamical diffraction beams.

    PubMed

    Liao, Keliang; Hong, Youli; Sheng, Weifan

    2014-10-01

    The effects of dynamical diffraction in x-ray diffractive optics with large numerical aperture render the wavefront aberrations difficult to describe using the aberration polynomials, yet knowledge of them plays an important role in a vast variety of scientific problems ranging from optical testing to adaptive optics. Although the diffraction theory of optical aberrations was established decades ago, its application in the area of x-ray dynamical diffraction theory (DDT) is still lacking. Here, we conduct a theoretical study on the aberration properties of x-ray dynamical diffraction beams. By treating the modulus of the complex envelope as the amplitude weight function in the orthogonalization procedure, we generalize the nonrecursive matrix method for the determination of orthonormal aberration polynomials, wherein Zernike DDT and Legendre DDT polynomials are proposed. As an example, we investigate the aberration evolution inside a tilted multilayer Laue lens. The corresponding Legendre DDT polynomials are obtained numerically, which represent balanced aberrations yielding minimum variance of the classical aberrations of an anamorphic optical system. The balancing of classical aberrations and their standard deviations are discussed. We also present the Strehl ratio of the primary and secondary balanced aberrations.

  3. A subgroup of pleural mesothelioma expresses ALK protein and may be targetable by combined rapamycin and crizotinib therapy.

    PubMed

    Mönch, Dina; Bode-Erdmann, Sabine; Kalla, Jörg; Sträter, Jörn; Schwänen, Carsten; Falkenstern-Ge, Roger; Klumpp, Siegfried; Friedel, Godehard; Ott, German; Kalla, Claudia

    2018-04-17

    Malignant pleural mesothelioma (MPM) is a neoplasm with inferior prognosis and notorious chemotherapeutic resistance. Targeting aberrantly overexpressed kinases to cure MPM is a promising therapeutic strategy. Here, we examined ALK, MET and mTOR as potential therapeutic targets and determined the combinatorial efficacy of ALK and mTOR targeting on tumor cell growth in vivo . First, ALK overexpression, rearrangement and mutation were studied in primary MPM by qRT-PCR, FISH, immunohistochemistry and sequence analysis; mTOR and MET expression by qRT-PCR and immunohistochemistry. Overexpression of full-length ALK transcripts was observed in 25 (19.5%) of 128 primary MPM, of which ten expressed ALK protein. ALK overexpression was not associated with gene rearrangement, amplification or kinase-domain mutation. mTOR protein was detected in 28.7% MPM, co-expressed with ALK or MET in 5% and 15% MPM, respectively. The ALK/MET inhibitor crizotinib enhanced the anti-tumor effect of the mTOR-inhibitor rapamycin in a patient-derived MPM xenograft with co-activated ALK/mTOR: combined therapy achieved tumor shrinkage in 4/5 tumors and growth stagnation in one tumor. Treatment effects on proliferation, apoptosis, autophagy and pathway signaling were assessed using Ki-67 immunohistochemistry, TUNEL assay, LC3B immunofluorescence, and immunoblotting. Co-treatment significantly suppressed cell proliferation and induced autophagy and caspase-independent, necrotic cell death. Rapamycin/crizotinib simultaneously inhibited mTORC1 (evidenced by S6 kinase and RPS6 dephosphorylation) and ALK signaling (ALK, AKT, STAT3 dephosphorylation), and crizotinib suppressed the adverse AKT activation induced by rapamycin. In conclusion, co-treatment with rapamycin and crizotinib is effective in suppressing MPM tumor growth and should be further explored as a therapeutic alternative in mesothelioma.

  4. A subgroup of pleural mesothelioma expresses ALK protein and may be targetable by combined rapamycin and crizotinib therapy

    PubMed Central

    Mönch, Dina; Bode-Erdmann, Sabine; Kalla, Jörg; Sträter, Jörn; Schwänen, Carsten; Falkenstern-Ge, Roger; Klumpp, Siegfried; Friedel, Godehard; Ott, German; Kalla, Claudia

    2018-01-01

    Malignant pleural mesothelioma (MPM) is a neoplasm with inferior prognosis and notorious chemotherapeutic resistance. Targeting aberrantly overexpressed kinases to cure MPM is a promising therapeutic strategy. Here, we examined ALK, MET and mTOR as potential therapeutic targets and determined the combinatorial efficacy of ALK and mTOR targeting on tumor cell growth in vivo. First, ALK overexpression, rearrangement and mutation were studied in primary MPM by qRT-PCR, FISH, immunohistochemistry and sequence analysis; mTOR and MET expression by qRT-PCR and immunohistochemistry. Overexpression of full-length ALK transcripts was observed in 25 (19.5%) of 128 primary MPM, of which ten expressed ALK protein. ALK overexpression was not associated with gene rearrangement, amplification or kinase-domain mutation. mTOR protein was detected in 28.7% MPM, co-expressed with ALK or MET in 5% and 15% MPM, respectively. The ALK/MET inhibitor crizotinib enhanced the anti-tumor effect of the mTOR-inhibitor rapamycin in a patient-derived MPM xenograft with co-activated ALK/mTOR: combined therapy achieved tumor shrinkage in 4/5 tumors and growth stagnation in one tumor. Treatment effects on proliferation, apoptosis, autophagy and pathway signaling were assessed using Ki-67 immunohistochemistry, TUNEL assay, LC3B immunofluorescence, and immunoblotting. Co-treatment significantly suppressed cell proliferation and induced autophagy and caspase-independent, necrotic cell death. Rapamycin/crizotinib simultaneously inhibited mTORC1 (evidenced by S6 kinase and RPS6 dephosphorylation) and ALK signaling (ALK, AKT, STAT3 dephosphorylation), and crizotinib suppressed the adverse AKT activation induced by rapamycin. In conclusion, co-treatment with rapamycin and crizotinib is effective in suppressing MPM tumor growth and should be further explored as a therapeutic alternative in mesothelioma. PMID:29755689

  5. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    PubMed

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. [Expression of goat IL-18 mature protein in insect/baculovirus and determination of bioactivity of the recombinant protein].

    PubMed

    Wang, Ting-Ting; Wang, Xi-Hui; Fan, Zhong-Ling; Chen, Jin-Long; Cao, Bing-Lei; Kong, Na; Hu, Jing-Dong; Zhao, Hong-Kun

    2011-02-01

    To express goat IL-18 in insect/baculovirus and detect the bioactivity of the recombinant protein. The mature goat interleukin-18(gIL-18) gene was cloned into the baculovirus transfer vector pFastBac Dual, and then the resulting eukaryotic expression plasmid pFastBac Dual-gIL18 was transformed into DH10Bac, followed by the identification of Bacmid-gIL18 recombinat plosmid by three antibiotics and blue-white patch. Finally, the recombinant bacmid was transfected into sf9 insect cells by Cellfectin and the transfected cells were harvested at different times. Then the expressed protein was identified by SDS-PAGE, Western blot and bioactivity assay. The recombinant protein recognized and bound to its specific antibody. Bioactivity assay showed that the recombinant protein stimulated the proliferation of lymphocytes and induced IFN-γproduction in spleen lymphocytes. The mature gIL-18 protein has been expressed successfully in insect/baculovirus expression system, and have good immunogenicity and bioactivity. The study paves a way for application of gIL-18 as an immunomodulator or immune adjuvant.

  7. Activation of D4 dopamine receptor decreases AT1 angiotensin II receptor expression in rat renal proximal tubule cells

    PubMed Central

    Chen, Ken; Deng, Kun; Wang, Xiaoyan; Wang, Zhen; Zheng, Shuo; Ren, Hongmei; He, Duofen; Han, Yu; Asico, Laureano D.; Jose, Pedro A.; Zeng, Chunyu

    2014-01-01

    The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal AT1 receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubules (RPTs) cells from Wistar-Kyoto (WKY) rats but the D4 receptor regulation of AT1 receptor is aberrant in RPT cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na+-K+ ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na+-K+ ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na+-K+ ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na+-K+ ATPase activity in SHR cells. This was confirmed in vivo; pre-treatment with PD128077 for one week augmented the anti-hypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension. PMID:25368031

  8. Green fluorescent protein as a reporter of gene expression and protein localization.

    PubMed

    Kain, S R; Adams, M; Kondepudi, A; Yang, T T; Ward, W W; Kitts, P

    1995-10-01

    The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is rapidly becoming an important reporter molecule for monitoring gene expression and protein localization in vivo, in situ and in real time. GFP emits bright green light (lambda max = 509 nm) when excited with UV or blue light (lambda max = 395 nm, minor peak at 470 nm). The fluorescence excitation and emission spectra of GFP are similar to those of fluorescein, and the conditions used to visualize this fluorophore are also suitable for GFP. Unlike other bioluminescent reporters, the chromophore in GFP is intrinsic to the primary structure of the protein, and GFP fluorescence does not require a substrate or cofactor. GFP fluorescence is stable, species-independent and can be monitored non-invasively in living cells and, in the case of transparent organisms, whole animals. Here we demonstrate GFP fluorescence in bacterial and mammalian cells and introduce our Living Colors line of GFP reporter vectors, GFP protein and anti-GFP antiserum. The reporter vectors for GFP include a promoterless GFP vector for monitoring the expression of cloned promoters/enhancers in mammalian cells and a series of six vectors for creating fusion protein to either the N or C terminus of GFP.

  9. Impact of semaphorin expression on prognostic characteristics in breast cancer.

    PubMed

    Butti, Ramesh; Kumar, Totakura Vs; Nimma, Ramakrishna; Kundu, Gopal C

    2018-01-01

    Breast cancer is one of the major causes of cancer-related deaths among women worldwide. Aberrant regulation of various growth factors, cytokines, and other proteins and their receptors in cancer cells drives the activation of various oncogenic signaling pathways that lead to cancer progression. Semaphorins are a class of proteins which are differentially expressed in various types of cancer including breast cancer. Earlier, these proteins were known to have a major function in the nerve cell adhesion, migration, and development of the central nervous system. However, their role in the regulation of several aspects of tumor progression has eventually emerged. There are over 30 genes encoding the semaphorins, which are divided into eight subclasses. It has been reported that some members of semaphorin classes are antiangiogenic and antimetastatic in nature, whereas others act as proangiogenic and prometastatic genes. Because of their differential expression and role in angiogenesis and metastasis, semaphorins emerged as one of the important prognostic factors for appraising breast cancer progression.

  10. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

    Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

  11. Fe-S Proteins that Regulate Gene Expression

    PubMed Central

    Mettert, Erin L.; Kiley, Patricia J.

    2014-01-01

    Iron-sulfur (Fe-S) cluster containing proteins that regulate gene expression are present in most organisms. The innate chemistry of their Fe-S cofactors makes these regulatory proteins ideal for sensing environmental signals, such as gases (e.g. O2 and NO), levels of Fe and Fe-S clusters, reactive oxygen species, and redox cycling compounds, to subsequently mediate an adaptive response. Here we review the recent findings that have provided invaluable insight into the mechanism and function of these highly significant Fe-S regulatory proteins. PMID:25450978

  12. In vitro protein expression: an emerging alternative to cell-based approaches.

    PubMed

    He, Mingyue

    2011-04-30

    Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Subcellular localization of transiently expressed fluorescent fusion proteins.

    PubMed

    Collings, David A

    2013-01-01

    The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.

  14. Widespread alternative and aberrant splicing revealed by lariat sequencing

    PubMed Central

    Stepankiw, Nicholas; Raghavan, Madhura; Fogarty, Elizabeth A.; Grimson, Andrew; Pleiss, Jeffrey A.

    2015-01-01

    Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures. PMID:26261211

  15. High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/ translation system.

    PubMed

    Martin, G A; Kawaguchi, R; Lam, Y; DeGiovanni, A; Fukushima, M; Mutter, W

    2001-10-01

    The Rapid Translation System (RTS 500) (Roche Molecular Biochemicals) is a high-yield protein expression system that utilizes an enhanced E. coli lysate for an in vitro transcription/translation reaction. In contrast to conventional transcription/translation, this system allows protein expression to continue for more than 24 h. We demonstrated the utility of the RTS 500 by expressing different soluble and active proteins that generally pose problems in cell-based expression systems. We first expressed GFP-lunasin, a fusion protein that, because of its toxicity, has been impossible to produce in whole cells. The second protein we expressed, human interleukin-2 (IL-2), is generally difficult to produce, either as the native molecule or as a GSTfusion protein, in a soluble form in bacteria. Finally, we demonstrated the capacity of the RTS 500 to co-express proteins, by the simultaneous production of GFP and CAT in a single reaction. This new technology appears to be particularly usefulfor the convenient production of preparative amounts (100-900 microg) of proteins that are toxic or insoluble in cell-based systems.

  16. Effect of monochromatic aberrations on photorefractive patterns

    NASA Astrophysics Data System (ADS)

    Campbell, Melanie C. W.; Bobier, W. R.; Roorda, A.

    1995-08-01

    Photorefractive methods have become popular in the measurement of refractive and accommodative states of infants and children owing to their photographic nature and rapid speed of measurement. As in the case of any method that measures the refractive state of the human eye, monochromatic aberrations will reduce the accuracy of the measurement. Monochromatic aberrations cannot be as easily predicted or controlled as chromatic aberrations during the measurement, and accordingly they will introduce measurement errors. This study defines this error or uncertainty by extending the existing paraxial optical analyses of coaxial and eccentric photorefraction. This new optical analysis predicts that, for the amounts of spherical aberration (SA) reported for the human eye, there will be a significant degree of measurement uncertainty introduced for all photorefractive methods. The dioptric amount of this uncertainty may exceed the maximum amount of SA present in the eye. The calculated effects on photorefractive measurement of a real eye with a mixture of spherical aberration and coma are shown to be significant. The ability, developed here, to predict photorefractive patterns corresponding to different amounts and types of monochromatic aberration may in the future lead to an extension of photorefractive methods to the dual measurement of refractive states and aberrations of individual eyes. aberration, retinal image quality,

  17. Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu Wenbin; Cui Zhihong; Ao Lin

    To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. Themore » prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.« less

  18. Machine learning in computational biology to accelerate high-throughput protein expression.

    PubMed

    Sastry, Anand; Monk, Jonathan; Tegel, Hanna; Uhlen, Mathias; Palsson, Bernhard O; Rockberg, Johan; Brunk, Elizabeth

    2017-08-15

    The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Combining computational biology and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template for analysis of further expression and solubility datasets. ebrunk@ucsd.edu or johanr@biotech.kth.se. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  19. Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

    PubMed Central

    Lilly, Jacob L.; Sheldon, Phillip R.; Hoversten, Liv J.; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J.

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium. PMID:25536421

  20. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    PubMed

    Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  1. The Expression and Significance of Neuronal Iconic Proteins in Podocytes

    PubMed Central

    Sun, Yu; Zhang, Hongxia; Hu, Ruimin; Sun, Jianyong; Mao, Xing; Zhao, Zhonghua; Chen, Qi; Zhang, Zhigang

    2014-01-01

    Growing evidence suggests that there are many common cell biological features shared by neurons and podocytes; however, the mechanism of podocyte foot process formation remains unclear. Comparing the mechanisms of process formation between two cell types should provide useful guidance from the progress of neuron research. Studies have shown that some mature proteins of podocytes, such as podocin, nephrin, and synaptopodin, were also expressed in neurons. In this study, using cell biological experiments and immunohistochemical techniques, we showed that some neuronal iconic molecules, such as Neuron-specific enolase, nestin and Neuron-specific nuclear protein, were also expressed in podocytes. We further inhibited the expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 by Small interfering RNA in cultured mouse podocytes and observed the significant morphological changes in treated podocytes. When podocytes were treated with Adriamycin, the protein expression of Neuron-specific enolase, nestin, synaptopodin and Ubiquitin carboxy terminal hydrolase-1 decreased over time. Meanwhile, the morphological changes in the podocytes were consistent with results of the Small interfering RNA treatment of these proteins. The data demonstrated that neuronal iconic proteins play important roles in maintaining and regulating the formation and function of podocyte processes. PMID:24699703

  2. p53 AND MDM2 PROTEIN EXPRESSION IN ACTINIC CHEILITIS

    PubMed Central

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia. PMID:19082401

  3. p53 and MDM2 protein expression in actinic cheilitis.

    PubMed

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  4. Evaluation of miRNA-196a2 and apoptosis-related target genes: ANXA1, DFFA and PDCD4 expression in gastrointestinal cancer patients: A pilot study

    PubMed Central

    Toraih, Eman A.; Ibrahiem, Afaf; Abdeldayem, Hala; Mohamed, Amany O.; Abdel-Daim, Mohamed M.

    2017-01-01

    Previous reports have suggested the significant association of miRNAs aberrant expression with tumor initiation, progression and metastasis in cancer, including gastrointestinal (GI) cancers. The current preliminary study aimed to evaluate the relative expression levels of miR-196a2 and three of its selected apoptosis-related targets; ANXA1, DFFA and PDCD4 in a sample of GI cancer patients. Quantitative real-time PCR for miR-196a2 and its selected mRNA targets, as well as immunohistochemical assay for annexin A1 protein expression were detected in 58 tissues with different GI cancer samples. In addition, correlation with the clinicopathological features and in silico network analysis of the selected molecular markers were analyzed. Stratified analyses by cancer site revealed elevated levels of miR-196a2 and low expression of the selected target genes. Annexin protein expression was positively correlated with its gene expression profile. In colorectal cancer, miR-196a over-expression was negatively correlated with annexin A1 protein expression (r = -0.738, p < 0.001), and both were indicators of unfavorable prognosis in terms of poor differentiation, larger tumor size, and advanced clinical stage. Taken together, aberrant expression of miR-196a2 and the selected apoptosis-related biomarkers might be involved in GI cancer development and progression and could have potential diagnostic and prognostic roles in these types of cancer; particularly colorectal cancer, provided the results experimentally validated and confirmed in larger multi-center studies. PMID:29091952

  5. Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice.

    PubMed

    Wei, Haiyan; Zhang, Juan; Tan, Kehong; Sun, Rongli; Yin, Lihong; Pu, Yuepu

    2015-11-12

    Benzene is a common environmental pollutant that causes hematological alterations. MicroRNAs (miRNAs) may play a role in benzene-induced hematotoxicity. In this study, C57BL/6 mice showed significant hematotoxicity after exposure to 150 mg/kg benzene for 4 weeks. Benzene exposure decreased not only the number of cells in peripheral blood but also hematopoietic progenitor cells in the bone marrow. Meanwhile, RNA from Lin(-) cells sorted from the bone marrow was applied to aberrant miRNA expression profile using Illumina sequencing. We found that 5 miRNAs were overexpressed and 45 miRNAs were downregulated in the benzene exposure group. Sequencing results were confirmed through qRT-PCR. Furthermore, we also identified five miRNAs which significantly altered in Lin(-)c-Kit⁺ cells obtained from benzene-exposed mice, including mmu-miR-34a-5p; mmu-miR-342-3p; mmu-miR-100-5p; mmu-miR-181a-5p; and mmu-miR-196b-5p. In summary, we successfully established a classical animal model to induce significant hematotoxicity by benzene injection. Benzene exposure may cause severe hematotoxicity not only to blood cells in peripheral circulation but also to hematopoietic cells in bone marrow. Benzene exposure also alters miRNA expression in hematopoietic progenitor cells. This study suggests that benzene induces alteration in hematopoiesis and hematopoiesis-associated miRNAs.

  6. Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

    PubMed Central

    Zhang, Xianghui; Fu, Jianming; Hiromasa, Yasuaki; Pan, Hongyu; Bai, Guihua

    2013-01-01

    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. PMID:24376514

  7. [Prokaryotic expression, purification and biological activity analysis of recombinant β-Lactamase protein].

    PubMed

    Zhou, Xiao-liang; Shi, Pei-ji; Wang, Hao

    2011-01-01

    To prepare RGD4CβL fusion protein using prokaryotic expression system and evaluate the biological activity of the RGD4CβL. RGD4CβL gene was cloned into pColdII to contruct β-Lactamase prokaryotic expression vector. After transformation, the recombinant vector was induced to express recombinant protein RGD4CβL by IPTG in E.coli BL(DE3). The recombinant protein was purified by Ni-NTA resin under denaturing condition and then dialyzed to renature. The tumor cell targeting ability of the recombinant protein was analyzed by flow cytometric analysis. After cleavage and purification, β-Lactamase moiety showed the expected size of 42 000 on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein specially targeted breast cancer cell line MCF-7. This research successfully estiblished a method for prokaryotic expression and purification of β-lactamase. These results suggest the potential use of the protein as an agent for ADEPT.

  8. Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

    PubMed Central

    Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

    2012-01-01

    Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

  9. Establishment of stable cell line for inducing KAP1 protein expression.

    PubMed

    Liu, Xiaoyan; Khan, Md Asaduzzaman; Cheng, Jingliang; Wei, Chunli; Zhang, Lianmei; Fu, Junjiang

    2015-06-01

    Generation of the stable cell lines is a highly efficient tool in functional studies of certain genes or proteins, where the particular genes or proteins are inducibly expressed. The KRAB-associated protein-1 (KAP1) is an important transcription regulatory protein, which is investigated in several molecular biological studies. In this study, we have aimed to generate a stable cell line for inducing KAP1 expression. The recombinant plasmid pcDNA5/FRT/TO-KAP1 was constructed at first, which was then transfected into Flp-In™T-REx™-HEK293 cells to establish an inducible pcDNA5/FRT/TO-KAP1-HEK293 cell line. The Western blot analysis showed that the protein level of KAP1 is over-expressed in the established stable cell line by doxycycline induction, both dose and time dependently. Thus we have successfully established stable pcDNA5/FRT/TO-KAP1-HEK293 cell line, which can express KAP1 inducibly. This inducible cell line might be very useful for KAP1 functional studies.

  10. Unintended changes in protein expression revealed by proteomic analysis of seeds from transgenic pea expressing a bean alpha-amylase inhibitor gene.

    PubMed

    Chen, Hancai; Bodulovic, Greg; Hall, Prudence J; Moore, Andy; Higgins, Thomas J V; Djordjevic, Michael A; Rolfe, Barry G

    2009-09-01

    Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and the corresponding alphaAI1-free segregating lines and non-GM parental line to identify unintended alterations to the proteome of GM peas due to the introduction of the gene for alphaAI1. Proteomic analysis showed that in addition to the presence of alphaAI1, 33 other proteins were differentially accumulated in the alphaAI1-expressing GM lines compared with their non-GM parental line and these were grouped into five expression classes. Among these 33 proteins, only three were found to be associated with the expression of alphaAI1 in the GM pea lines. The accumulation of the remaining 30 proteins appears to be associated with Agrobacterium-mediated transformation events. Sixteen proteins were identified after MALDI-TOF-TOF analysis. About 56% of the identified proteins with altered accumulation in the GM pea were storage proteins including legumin, vicilin or convicilin, phaseolin, cupin and valosin-containing protein. Two proteins were uniquely expressed in the alphaAI1-expressing GM lines and one new protein was present in both the alphaAI1-expressing GM lines and their alphaAI1-free segregating lines, suggesting that both transgenesis and transformation events led to demonstrable changes in the proteomes of the GM lines tested.

  11. Imaging characteristics of Zernike and annular polynomial aberrations.

    PubMed

    Mahajan, Virendra N; Díaz, José Antonio

    2013-04-01

    The general equations for the point-spread function (PSF) and optical transfer function (OTF) are given for any pupil shape, and they are applied to optical imaging systems with circular and annular pupils. The symmetry properties of the PSF, the real and imaginary parts of the OTF, and the modulation transfer function (MTF) of a system with a circular pupil aberrated by a Zernike circle polynomial aberration are derived. The interferograms and PSFs are illustrated for some typical polynomial aberrations with a sigma value of one wave, and 3D PSFs and MTFs are shown for 0.1 wave. The Strehl ratio is also calculated for polynomial aberrations with a sigma value of 0.1 wave, and shown to be well estimated from the sigma value. The numerical results are compared with the corresponding results in the literature. Because of the same angular dependence of the corresponding annular and circle polynomial aberrations, the symmetry properties of systems with annular pupils aberrated by an annular polynomial aberration are the same as those for a circular pupil aberrated by a corresponding circle polynomial aberration. They are also illustrated with numerical examples.

  12. Variation in Protein Intake Induces Variation in Spider Silk Expression

    PubMed Central

    Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

    2012-01-01

    Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

  13. ERAP1 reduces accumulation of aberrant and disulfide-linked forms of HLA-B27 on the cell surface.

    PubMed

    Tran, Tri M; Hong, Sohee; Edwan, Jehad H; Colbert, Robert A

    2016-06-01

    Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) variants contribute to the risk of ankylosing spondylitis in HLA-B27 positive individuals, implying a disease-related interaction between these gene products. The aim of this study was to determine whether reduced ERAP1 expression would alter the cell surface expression of HLA-B27 and the formation of aberrant disulfide-linked forms that have been implicated in the pathogenesis of spondyloarthritis. ERAP1 expression was knocked down in monocytic U937 cells expressing HLA-B27 and endogenous HLA class I. The effect of ERAP1 knockdown on the accumulation HLA-B alleles (B18, B51, and B27) was assessed using immunoprecipitation, isoelectric focusing, and immunoblotting, as well as flow cytometry with antibodies specific for different forms of HLA-B27. Cell surface expression of aberrant disulfide-linked HLA-B27 dimers was assessed by immunoprecipitation and electrophoresis on non-reducing polyacrylamide gels. ERAP1 knockdown increased the accumulation of HLA-B27 on the cell surface including disulfide-linked dimers, but had no effect on levels of HLA-B18 or -B51. Antibodies with unique specificity for HLA-B27 confirmed increased cell surface expression of complexes shown previously to contain long peptides. IFN-γ treatment resulted in striking increases in the expression of disulfide-linked HLA-B27 heavy chains, even in cells with normal ERAP1 expression. Our results suggest that normal levels of ERAP1 reduce the accumulation of aberrant and disulfide-linked forms of HLA-B27 in monocytes, and thus help to maintain the integrity of cell surface HLA-B27 complexes. Published by Elsevier Ltd.

  14. ERAP1 Reduces Accumulation of Aberrant and Disulfide-Linked Forms of HLA-B27 on the Cell Surface

    PubMed Central

    Tran, Tri; Hong, Sohee; Edwan, Jehad; Colbert, Robert A.

    2016-01-01

    Objective Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) variants contribute to the risk of ankylosing spondylitis in HLA-B27 positive individuals, implying a disease-related interaction between these gene products. The aim of this study was to determine whether reduced ERAP1 expression would alter the cell surface expression of HLA-B27 and the formation of aberrant disulfide-linked forms that have been implicated in the pathogenesis of spondyloarthritis. Methods ERAP1 expression was knocked down in monocytic U937 cells expressing HLA-B27 and endogenous HLA class I. The effect of ERAP1 knockdown on the accumulation HLA-B alleles (B18, B51, and B27) was assessed using immunoprecipitation, isoelectric focusing, and immunoblotting, as well as flow cytometry with antibodies specific for different forms of HLA-B27. Cell surface expression of aberrant disulfide-linked HLA-B27 dimers was assessed by immunoprecipitation and electrophoresis on non-reducing polyacrylamide gels. Results ERAP1 knockdown increased the accumulation of HLA-B27 on the cell surface including disulfide-linked dimers, but had no effect on levels of HLA-B18 or -B51. Antibodies with unique specificity for HLA-B27 confirmed increased cell surface expression of complexes shown previously to contain long peptides. IFN-γ treatment resulted in striking increases in the expression of disulfide-linked HLA-B27 heavy chains, even in cells with normal ERAP1 expression. Conclusions Our results suggest that normal levels of ERAP1 reduce the accumulation of aberrant and disulfide-linked forms of HLA-B27 in monocytes, and thus help to maintain the integrity of cell surface HLA-B27 complexes. PMID:27107845

  15. Nodal aberration theory applied to freeform surfaces

    NASA Astrophysics Data System (ADS)

    Fuerschbach, Kyle; Rolland, Jannick P.; Thompson, Kevin P.

    2014-12-01

    When new three-dimensional packages are developed for imaging optical systems, the rotational symmetry of the optical system is often broken, changing its imaging behavior and making the optical performance worse. A method to restore the performance is to use freeform optical surfaces that compensate directly the aberrations introduced from tilting and decentering the optical surfaces. In order to effectively optimize the shape of a freeform surface to restore optical functionality, it is helpful to understand the aberration effect the surface may induce. Using nodal aberration theory the aberration fields induced by a freeform surface in an optical system are explored. These theoretical predications are experimentally validated with the design and implementation of an aberration generating telescope.

  16. Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

    PubMed

    Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

    2009-02-01

    Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

  17. Corneal spherical aberration in Saudi population

    PubMed Central

    Al-Sayyari, Tarfah M.; Fawzy, Samah M.; Al-Saleh, Ahmed A.

    2014-01-01

    Purpose To find out the mean corneal spherical aberration and its changes with age in Saudi population. Setting AlHokama Eye Specialist Center, Riyadh, Saudi Arabia. Methods Three hundred (300) eyes of 185 Saudi subjects (97 men and 88 women), whose age ranged from 15 to 85 years old, with matched refractive errors, were divided into three groups according to their age, 100 for each. All the subjects were included in measuring the spherical aberration (SA) using pentacam HR (OCULUS, Germany) at the 6-mm optical zone. Results The mean corneal spherical aberration (CSA) of the fourth order (Z40) of the whole groups was 0.252 ± 0.1154 μm. Patients from 15 to 35 years old have root mean square (RMS) of CSA of 0.2068 ± 0.07151 μm, 0.2370 ± 0.08023 μm was the RMS of CSA of the patients from 35 to 50 years old, while those from 50 to 85 years old have a CSA-RMS of 0.31511 ± 0.1503 μm (P < 0.0001). A positive correlation was found between the spherical aberration (Z40) and the progress of age (r = 0.3429, P < 0.0001). The high order aberration (HOA) presented 28.1% of the total corneal aberrations. While the fourth order corneal spherical aberration constituted 57% of the HOA and 16% of the total aberration. The pupil diameter shows a negative correlation with the increase in age (P = 0.0012). Conclusion Our results showed a CSA (Z40) that is varied among the population, comparable to other studies, and significantly correlates to the progress of age. PMID:25278799

  18. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    López, Claudia S., E-mail: lopezcl@ohsu.edu; Sloan, Rachel; Cylinder, Isabel

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag proteinmore » expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.« less

  19. Hippocampal expression of a virus-derived protein impairs memory in mice.

    PubMed

    Bétourné, Alexandre; Szelechowski, Marion; Thouard, Anne; Abrial, Erika; Jean, Arnaud; Zaidi, Falek; Foret, Charlotte; Bonnaud, Emilie M; Charlier, Caroline M; Suberbielle, Elsa; Malnou, Cécile E; Granon, Sylvie; Rampon, Claire; Gonzalez-Dunia, Daniel

    2018-02-13

    The analysis of the biology of neurotropic viruses, notably of their interference with cellular signaling, provides a useful tool to get further insight into the role of specific pathways in the control of behavioral functions. Here, we exploited the natural property of a viral protein identified as a major effector of behavioral disorders during infection. We used the phosphoprotein (P) of Borna disease virus, which acts as a decoy substrate for protein kinase C (PKC) when expressed in neurons and disrupts synaptic plasticity. By a lentiviral-based strategy, we directed the singled-out expression of P in the dentate gyrus of the hippocampus and we examined its impact on mouse behavior. Mice expressing the P protein displayed increased anxiety and impaired long-term memory in contextual and spatial memory tasks. Interestingly, these effects were dependent on P protein phosphorylation by PKC, as expression of a mutant form of P devoid of its PKC phosphorylation sites had no effect on these behaviors. We also revealed features of behavioral impairment induced by P protein expression but that were independent of its phosphorylation by PKC. Altogether, our findings provide insight into the behavioral correlates of viral infection, as well as into the impact of virus-mediated alterations of the PKC pathway on behavioral functions.

  20. BAX protein expression and clinical outcome in epithelial ovarian cancer.

    PubMed

    Tai, Y T; Lee, S; Niloff, E; Weisman, C; Strobel, T; Cannistra, S A

    1998-08-01

    Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer. BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed. BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified. The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.

  1. Transcranial phase aberration correction using beam simulations and MR-ARFI

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vyas, Urvi, E-mail: urvi.vyas@gmail.com; Kaye, Elena; Pauly, Kim Butts

    2014-03-15

    Purpose: Transcranial magnetic resonance-guided focused ultrasound surgery is a noninvasive technique for causing selective tissue necrosis. Variations in density, thickness, and shape of the skull cause aberrations in the location and shape of the focal zone. In this paper, the authors propose a hybrid simulation-MR-ARFI technique to achieve aberration correction for transcranial MR-guided focused ultrasound surgery. The technique uses ultrasound beam propagation simulations with MR Acoustic Radiation Force Imaging (MR-ARFI) to correct skull-caused phase aberrations. Methods: Skull-based numerical aberrations were obtained from a MR-guided focused ultrasound patient treatment and were added to all elements of the InSightec conformal bone focusedmore » ultrasound surgery transducer during transmission. In the first experiment, the 1024 aberrations derived from a human skull were condensed into 16 aberrations by averaging over the transducer area of 64 elements. In the second experiment, all 1024 aberrations were applied to the transducer. The aberrated MR-ARFI images were used in the hybrid simulation-MR-ARFI technique to find 16 estimated aberrations. These estimated aberrations were subtracted from the original aberrations to result in the corrected images. Each aberration experiment (16-aberration and 1024-aberration) was repeated three times. Results: The corrected MR-ARFI image was compared to the aberrated image and the ideal image (image with zero aberrations) for each experiment. The hybrid simulation-MR-ARFI technique resulted in an average increase in focal MR-ARFI phase of 44% for the 16-aberration case and 52% for the 1024-aberration case, and recovered 83% and 39% of the ideal MR-ARFI phase for the 16-aberrations and 1024-aberration case, respectively. Conclusions: Using one MR-ARFI image and noa priori information about the applied phase aberrations, the hybrid simulation-MR-ARFI technique improved the maximum MR-ARFI phase of the beam's focus.« less

  2. Aberrant RNA splicing and mutations in spliceosome complex in acute myeloid leukemia.

    PubMed

    Zhou, Jianbiao; Chng, Wee-Joo

    2017-01-01

    The spliceosome, the cellular splicing machinery, regulates RNA splicing of messenger RNA precursors (pre-mRNAs) into maturation of protein coding RNAs. Recurrent mutations and copy number changes in genes encoding spliceosomal proteins and splicing regulatory factors have tumor promoting or suppressive functions in hematological malignancies, as well as some other cancers. Leukemia stem cell (LSC) populations, although rare, are essential contributors of treatment failure and relapse. Recent researches have provided the compelling evidence that link the erratic spicing activity to the LSC phenotype in acute myeloid leukemia (AML). In this article, we describe the diverse roles of aberrant splicing in hematological malignancies, particularly in AML and their contributions to the characteristics of LSC. We review these promising strategies to exploit the addiction of aberrant spliceosomal machinery for anti-leukemic therapy with aim to eradicate LSC. However, given the complexity and plasticity of spliceosome and not fully known functions of splicing in cancer, the challenges facing the development of the therapeutic strategies targeting RAN splicing are highlighted and future directions are discussed too.

  3. Aberrant excitatory rewiring of layer V pyramidal neurons early after neocortical trauma

    PubMed Central

    Takahashi, D. Koji; Isabel, Feng Gu; Parada, Shri Vyas; Prince, David A.

    2016-01-01

    Lesioned neuronal circuits form new functional connections after a traumatic brain injury (TBI). In humans and animal models, aberrant excitatory connections that form after TBI may contribute to the pathogenesis of post-traumatic epilepsy. Partial neocortical isolation (“undercut” or “UC”) leads to altered neuronal circuitry and network hyperexcitability recorded in vivo and in brain slices from chronically lesioned neocortex. Recent data suggest a critical period for maladaptive excitatory circuit formation within the first 3 days post UC injury (Graber and Prince, 1999, 2004; Li et al., 2011, 2012b). The present study focuses on alterations in excitatory connectivity within this critical period. Immunoreactivity (IR) for growth-associated protein (GAP)-43 was increased in the UC cortex 3 days after injury. Some GAP-43-expressing excitatory terminals targeted the somata of layer V pyramidal (Pyr) neurons, a domain usually innervated predominantly by inhibitory terminals. Immunocytochemical analysis of pre- and postsynaptic markers showed that putative excitatory synapses were present on somata of these neurons in UC neocortex. Excitatory postsynaptic currents from UC layer V Pyr cells displayed properties consistent with perisomatic inputs and also reflected an increase in the number of synaptic contacts. Laser scanning photostimulation (LSPS) experiments demonstrated reorganized excitatory connectivity after injury within the UC. Concurrent with these changes, spontaneous epileptiform bursts developed in UC slices. Results suggest that aberrant reorganization of excitatory connectivity contributes to early neocortical hyperexcitability in this model. The findings are relevant for understanding the pathophysiology of neocortical post-traumatic epileptogenesis and are important in terms of the timing of potential prophylactic treatments. PMID:26956396

  4. Protein Expression Profile using Two-Dimensional Gel Analysis in Squamous Cervical Cancer Patients

    PubMed Central

    Bae, Su-Mi; Min, Hyun-Jin; Ding, Guo Hua; Kwak, Sun-Young; Cho, Young-Lae; Nam, Kye-Hyun; Park, Choong Hak; Kim, Yong-Wan; Kim, Chong-Kook; Han, Byoung-Don; Lee, Young-Joo; Kim, Do Kang

    2006-01-01

    Purpose Screening in cervical cancer is now progressing to discover candidate genes and proteins that may serve as biological markers and that play a role in tumor progression. We examined the protein expression patterns of the squamous cell carcinoma (SCC) tissues from Korean women with using two- dimensional polyacrylamide gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight (MALDI- TOF) mass spectrometer. Materials and Methods Normal cervix and SCC tissues were solubilized and 2-DE was performed using pH 3~10 linear IPG strips of 17 cm length. The protein expression was evaluated using PDQuest 2-D software™. The differentially expressed protein spots were identified with a MALDI-TOF mass spectrometer, and the peptide mass spectra identifications were performed using the Mascot program and by searching the Swiss-prot or NCBInr databases. Results A total of 35 proteins were detected in SCC. 17 proteins were up-regulated and 18 proteins weredown-regulated. Among the proteins that were identified, 12 proteins (pigment epithelium derived factor, annexin A2 and A5, keratin 19 and 20, heat shock protein 27, smooth muscle protein 22 alpha, α-enolase, squamous cell carcinoma antigen 1 and 2, glutathione S-transferase and apolipoprotein a1) were protein previously known to be involved in tumor, and 21 proteins were newly identified in this study. Conclusion 2-DE offers the total protein expression profiles of SCC tissues; further characterization of these differentially expressed proteins will give a chance to identify the badly needed tumor-specific diagnostic markers for SCC. PMID:19771267

  5. Anti-forensics of chromatic aberration

    NASA Astrophysics Data System (ADS)

    Mayer, Owen; Stamm, Matthew C.

    2015-03-01

    Over the past decade, a number of information forensic techniques have been developed to identify digital image manipulation and falsification. Recent research has shown, however, that an intelligent forger can use anti-forensic countermeasures to disguise their forgeries. In this paper, an anti-forensic technique is proposed to falsify the lateral chromatic aberration present in a digital image. Lateral chromatic aberration corresponds to the relative contraction or expansion between an image's color channels that occurs due to a lens's inability to focus all wavelengths of light on the same point. Previous work has used localized inconsistencies in an image's chromatic aberration to expose cut-and-paste image forgeries. The anti-forensic technique presented in this paper operates by estimating the expected lateral chromatic aberration at an image location, then removing deviations from this estimate caused by tampering or falsification. Experimental results are presented that demonstrate that our anti-forensic technique can be used to effectively disguise evidence of an image forgery.

  6. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Raymond, Amy; Lovell, Scott; Lorimer, Don

    2009-12-01

    With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. colimore » and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.« less

  7. In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector.

    PubMed

    Zhang, G; Leung, C; Murdin, L; Rovinski, B; White, K A

    2000-02-01

    Plant viruses show significant potential as expression vectors for the production of foreign proteins (e.g., antigens) in plants. The HIV-1 p24 nucleocapsid protein is an important early marker of HIV infection and has been used as an antigen in the development of HIV vaccines. Toward developing a plant-based expression system for the production of p24, we have investigated the use of a (positive)-strand RNA plant virus, tomato bushy stunt virus (TBSV), as an expression vector. The HIV p24 open reading frame (ORF) was introduced into a cloned cDNA copy of the TBSV genome as an in-frame fusion with a 5'-terminal portion of the TBSV coat protein ORF. In vitro-generated RNA transcripts corresponding to the engineered virus vector were infectious when inoculated into plant protoplasts; Northern and Western blot analyses verified the accumulation of a predicted p24-encoding viral subgenomic mRNA and the production of p24 fusion product. Whole-plant infections with the viral vector led to the accumulation of p24 fusion protein in inoculated leaves, which cross-reacted with p24-specific antibodies, thus confirming the maintenance of key antigenic determinants. This study is the first to demonstrate that TBSV can be engineered to express a complete foreign protein of clinical importance. Strategies for optimizing protein yield from this viral vector are discussed.

  8. PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins

    PubMed Central

    Janer, Alexandre; Martin, Elodie; Muriel, Marie-Paule; Latouche, Morwena; Fujigasaki, Hiroto; Ruberg, Merle; Brice, Alexis; Trottier, Yvon; Sittler, Annie

    2006-01-01

    The pathogenesis of spinocerebellar ataxia type 7 and other neurodegenerative polyglutamine (polyQ) disorders correlates with the aberrant accumulation of toxic polyQ-expanded proteins in the nucleus. Promyelocytic leukemia protein (PML) nuclear bodies are often present in polyQ aggregates, but their relation to pathogenesis is unclear. We show that expression of PML isoform IV leads to the formation of distinct nuclear bodies enriched in components of the ubiquitin-proteasome system. These bodies recruit soluble mutant ataxin-7 and promote its degradation by proteasome-dependent proteolysis, thus preventing the aggregate formation. Inversely, disruption of the endogenous nuclear bodies with cadmium increases the nuclear accumulation and aggregation of mutant ataxin-7, demonstrating their role in ataxin-7 turnover. Interestingly, β-interferon treatment, which induces the expression of endogenous PML IV, prevents the accumulation of transiently expressed mutant ataxin-7 without affecting the level of the endogenous wild-type protein. Therefore, clastosomes represent a potential therapeutic target for preventing polyQ disorders. PMID:16818720

  9. The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

    PubMed

    Mlynek, Georg; Lehner, Anita; Neuhold, Jana; Leeb, Sarah; Kostan, Julius; Charnagalov, Alexej; Stolt-Bergner, Peggy; Djinović-Carugo, Kristina; Pinotsis, Nikos

    2014-06-01

    Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

  10. Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

    PubMed

    Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

    2010-10-01

    HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

  11. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  12. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

    PubMed

    Schroeter, Christina B; Koehler, Sybille; Kann, Martin; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T; Rinschen, Markus M

    2018-04-25

    Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions at a depth of >7000 proteins. Similar to mouse podocytes, human podocyte differentiation involved a shift in proteostasis: undifferentiated podocytes have high expression of proteasomal proteins, whereas differentiated podocytes have high expression of lysosomal proteins. Additional analyses with pulsed stable-isotope labeling by amino acids in cell culture and protein degradation assays determined protein dynamics and half-lives. These studies unraveled a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal, and membrane proteins were stabilized, particularly in differentiated podocytes. Importantly, protein half-lives strongly contributed to protein abundance in each state. These data suggest that regulation of protein turnover of particular cellular functions determines podocyte differentiation, a paradigm involving mitophagy and, potentially, of importance in conditions of increased podocyte stress and damage.-Schroeter, C. B., Koehler, S., Kann, M., Schermer, B., Benzing, T., Brinkkoetter, P. T., Rinschen, M. M. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

  13. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Protein corona of airborne nanoscale PM2.5 induces aberrant proliferation of human lung fibroblasts based on a 3D organotypic culture.

    PubMed

    Li, Yan; Wang, Pengcheng; Hu, Chuanlin; Wang, Kun; Chang, Qing; Liu, Lieju; Han, Zhenggang; Shao, Yang; Zhai, Ying; Zuo, Zhengyu; Mak, Michael; Gong, Zhiyong; Wu, Yang

    2018-01-31

    Exposure to PM2.5 has become one of the most important factors affecting public health in the world. Both clinical and research studies have suggested that PM2.5 inhalation is associated with impaired lung function. In this study, material characterization identified the existence of nanoscale particulate matter (NPM) in airborne PM2.5 samples. When coming into contact with protein-rich fluids, the NPM becomes covered by a protein layer that forms a "protein corona". Based on a 3D organotypic cell culture, the protein corona was shown to mitigate NPM cytotoxicity and further stimulate the proliferation of human lung fibroblasts (HLFs). ROS-activated alpha-smooth muscle actin (α-SMA) is considered to be one of the proliferation pathways. In this research, 3D cell cultures exhibited more tissue-like properties compared with the growth in 2D models. Animal models have been widely used in toxicological research. However, species differences make it impossible to directly translate discoveries from animals to humans. In this research, the 3D HLF model could partly simulate the biological responses of NPM-protein corona-induced aberrant HLF proliferation in the human lung. Our 3D cellular results provide auxiliary support for an animal model in research on PM2.5-induced impaired lung function, particularly in lung fibrosis.

  15. Caenorhabditis elegans TRPV Channels Function in a Modality-Specific Pathway to Regulate Response to Aberrant Sensory Signaling

    PubMed Central

    Ezak , Meredith J.; Hong , Elizabeth; Chaparro-Garcia , Angela; Ferkey , Denise M.

    2010-01-01

    Olfaction and some forms of taste (including bitter) are mediated by G protein-coupled signal transduction pathways. Olfactory and gustatory ligands bind to chemosensory G protein-coupled receptors (GPCRs) in specialized sensory cells to activate intracellular signal transduction cascades. G protein-coupled receptor kinases (GRKs) are negative regulators of signaling that specifically phosphorylate activated GPCRs to terminate signaling. Although loss of GRK function usually results in enhanced cellular signaling, Caenorhabditis elegans lacking GRK-2 function are not hypersensitive to chemosensory stimuli. Instead, grk-2 mutant animals do not chemotax toward attractive olfactory stimuli or avoid aversive tastes and smells. We show here that loss-of-function mutations in the transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 selectively restore grk-2 behavioral avoidance of bitter tastants, revealing modality-specific mechanisms for TRPV channel function in the regulation of C. elegans chemosensation. Additionally, a single amino acid point mutation in OCR-2 that disrupts TRPV channel-mediated gene expression, but does not decrease channel function in chemosensory primary signal transduction, also restores grk-2 bitter taste avoidance. Thus, loss of GRK-2 function may lead to changes in gene expression, via OSM-9/OCR-2, to selectively alter the levels of signaling components that transduce or regulate bitter taste responses. Our results suggest a novel mechanism and multiple modality-specific pathways that sensory cells employ in response to aberrant signal transduction. PMID:20176974

  16. Expression of c-Fes protein isoforms correlates with differentiation in myeloid leukemias.

    PubMed

    Carlson, Anne; Berkowitz, Jeanne McAdara; Browning, Damaris; Slamon, Dennis J; Gasson, Judith C; Yates, Karen E

    2005-05-01

    The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells. Therefore, we compared c-Fes expression in cells with an undifferentiated, blast phenotype (acute myelogenous leukemia--AML) to cells with a differentiated phenotype (chronic myelogenous leukemia--CML). Instead of differences in p93 expression levels, we found complex patterns of c-Fes immunoreactive proteins that corresponded with differentiation in normal and leukemic myeloid cells. The "blast" pattern consisted of c-Fes immunoreactive proteins p93, p74, and p70; the "differentiated" pattern showed two additional c-Fes immunoreactive proteins, p67 and p62. Using mRNA from mouse and human cell lines, we found deletion of one or more exons in the c-fes mRNA. Those deletions predicted truncation of conserved domains (CDC15/FCH and SH2) involved in protein-protein interactions. No deletions were found, however, within the kinase domain. We infer that alternative splicing generates a family of c-Fes proteins. This may be a mechanism to direct the c-Fes kinase domain to different subcellular locations and/or substrates at specific stages of myeloid cell differentiation.

  17. Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis.

    PubMed

    Sun, Xiaoyan; Wray, Curtis; Tian, Xintian; Hasselgren, Per-Olof; Lu, James

    2003-09-01

    Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.

  18. Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce.

    PubMed

    Sun, Hyeon-Jin; Cui, Min-Long; Ma, Biao; Ezura, Hiroshi

    2006-01-23

    Taste-modifying proteins are a natural alternative to artificial sweeteners and flavor enhancers and have been used in some cultures for centuries. The taste-modifying protein, miraculin, has the unusual property of being able to modify a sour taste into a sweet taste. Here, we report the use of a plant expression system for the production of miraculin. A synthetic gene encoding miraculin was placed under the control of constitutive promoters and transferred to lettuce. Expression of this gene in transgenic lettuce resulted in the accumulation of significant amounts of miraculin protein in the leaves. The miraculin expressed in transgenic lettuce possessed sweetness-inducing activity. These results demonstrate that the production of miraculin in edible plants can be a good alternative strategy to enhance the availability of this protein.

  19. Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

    PubMed

    Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

    2015-01-01

    Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

  20. Heat shock proteins and toll-like receptors.

    PubMed

    Asea, Alexzander

    2008-01-01

    Researchers have only just begun to elucidate the relationship between heat shock proteins (HSP) and Toll-like receptors (TLR). HSP were originally described as an intracellular molecular chaperone of naïve, aberrantly folded, or mutated proteins and primarily implicated as a cytoprotective protein when cells are exposed to stressful stimuli. However, recent studies have ascribed novel functions to the Hsp70 protein depending on its localization: Surface-bound Hsp70 specifically activate natural killer (NK) cells, while Hsp70 released into the extracellular milieu specifically bind to Toll-like receptors (TLR) 2 and 4 on antigen-presenting cells (APC) and exerts immunoregulatory effects, including upregulation of adhesion molecules, co-stimulatory molecule expression, and cytokine and chemokine release-a process known as the chaperokine activity of Hsp70. This chapter discusses the most recent advances in the understanding of heat shock protein (HSP) and TLR interactions in general and highlights recent findings that demonstrate Hsp70 is a ligand for TLR and its biological significance.

  1. Performance analysis of grazing incidence imaging systems. [X ray telescope aberrations

    NASA Technical Reports Server (NTRS)

    Winkler, C. E.; Korsch, D.

    1977-01-01

    An exact expression relating the coordinates of a point on the incident ray, a point of reflection from an arbitrary surface, and a point on the reflected ray is derived. The exact relation is then specialized for the case of grazing incidence, and first order and third order systematic analyses are carried out for a single reflective surface and then for a combination of two surfaces. The third order treatment yields a complete set of primary aberrations for single element and two element systems. The importance of a judicious choice for a coordinate system in showing field curvature to clearly be the predominant aberration for a two element system is discussed. The validity of the theory is verified through comparisons with the exact ray trace results for the case of the telescope.

  2. Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation.

    PubMed

    Keates, Tracy; Cooper, Christopher D O; Savitsky, Pavel; Allerston, Charles K; Phillips, Claire; Hammarström, Martin; Daga, Neha; Berridge, Georgina; Mahajan, Pravin; Burgess-Brown, Nicola A; Müller, Susanne; Gräslund, Susanne; Gileadi, Opher

    2012-06-15

    The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation

    PubMed Central

    Keates, Tracy; Cooper, Christopher D.O.; Savitsky, Pavel; Allerston, Charles K.; Phillips, Claire; Hammarström, Martin; Daga, Neha; Berridge, Georgina; Mahajan, Pravin; Burgess-Brown, Nicola A.; Müller, Susanne; Gräslund, Susanne; Gileadi, Opher

    2012-01-01

    The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome. PMID:22027370

  4. Wrecked regulation of intrinsically disordered proteins in diseases: pathogenicity of deregulated regulators

    PubMed Central

    Uversky, Vladimir N.

    2014-01-01

    Biologically active proteins without stable tertiary structure are common in all known proteomes. Functions of these intrinsically disordered proteins (IDPs) are typically related to regulation, signaling, and control. Cellular levels of these important regulators are tightly regulated by a variety mechanisms ranging from firmly controlled expression to precisely targeted degradation. Functions of IDPs are controlled by binding to specific partners, alternative splicing, and posttranslational modifications among other means. In the norm, right amounts of precisely activated IDPs have to be present in right time at right places. Wrecked regulation brings havoc to the ordered world of disordered proteins, leading to protein misfolding, misidentification, and missignaling that give rise to numerous human diseases, such as cancer, cardiovascular disease, neurodegenerative diseases, and diabetes. Among factors inducing pathogenic transformations of IDPs are various cellular mechanisms, such as chromosomal translocations, damaged splicing, altered expression, frustrated posttranslational modifications, aberrant proteolytic degradation, and defective trafficking. This review presents some of the aspects of deregulated regulation of IDPs leading to human diseases. PMID:25988147

  5. Exercise alters myostatin protein expression in sedentary and exercised streptozotocin-diabetic rats.

    PubMed

    Bassi, Daniela; Bueno, Patricia de Godoy; Nonaka, Keico Okino; Selistre-Araujo, Heloisa Sobreiro; Leal, Angela Merice de Oliveira

    2015-04-01

    The aim of this study was to analyze the effect of exercise on the pattern of muscle myostatin (MSTN) protein expression in two important metabolic disorders, i.e., obesity and diabetes mellitus. MSTN, is a negative regulator of skeletal muscle mass. We evaluated the effect of exercise on MSTN protein expression in diabetes mellitus and high fat diet-induced obesity. MSTN protein expression in gastrocnemius muscle was analyzed by Western Blot. P < 0.05 was assumed. Exercise induced a significant decrease in glycemia in both diabetic and obese animals. The expression of precursor and processed protein forms of MSTN and the weight of gastrocnemius muscle did not vary in sedentary or exercised obese animals. Diabetes reduced gastrocnemius muscle weight in sedentary animals. However, gastrocnemius muscle weight increased in diabetic exercised animals. Both the precursor and processed forms of muscle MSTN protein were significantly higher in sedentary diabetic rats than in control rats. The precursor form was significantly lower in diabetic exercised animals than in diabetic sedentary animals. However, the processed form did not change. These results demonstrate that exercise can modulate the muscle expression of MSTN protein in diabetic rats and suggest that MSTN may be involved in energy homeostasis.

  6. The correction of aberrations computed in the aperture plane of multifrequency microwave radiometer antennas

    NASA Technical Reports Server (NTRS)

    Schmidt, R. F.

    1984-01-01

    An analytical/numerical approach to identifying and correcting the aberrations introduced by a general displacement of the feed from the focal point of a single offset paraboloid antenna used in deployable radiometer systems is developed. A 15 meter reflector with 18 meter focal length is assumed for the analysis, which considers far field radiation pattern quality, focal region fields, and aberrations appearing in the aperture plane. The latter are obtained by ray tracing in the transmit mode and are expressed in terms of optical notation. Attention is given to the physical restraints imposed on corrective elements by real microwave systems and to the intermediate near field aspects of the problem in three dimensions. The subject of wave fronts and caustics in the receive mode is introduced for comparative purposes. Several specific examples are given for aberration reduction at eight beamwidths of scan at a frequency of 1.414 GHz.

  7. Formula for the rms blur circle radius of Wolter telescope based on aberration theory

    NASA Technical Reports Server (NTRS)

    Shealy, David L.; Saha, Timo T.

    1990-01-01

    A formula for the rms blur circle for Wolter telescopes has been derived using the transverse ray aberration expressions of Saha (1985), Saha (1984), and Saha (1986). The resulting formula for the rms blur circle radius over an image plane and a formula for the surface of best focus based on third-, fifth-, and seventh-order aberration theory predict results in good agreement with exact ray tracing. It has also been shown that one of the two terms in the empirical formula of VanSpeybroeck and Chase (1972), for the rms blur circle radius of a Wolter I telescope can be justified by the aberration theory results. Numerical results are given comparing the rms blur radius and the surface of best focus vs the half-field angle computed by skew ray tracing and from analytical formulas for grazing incidence Wolter I-II telescopes and a normal incidence Cassegrain telescope.

  8. Aberrant Wnt/β-catenin signaling and elevated expression of stem cell proteins are associated with osteosarcoma side population cells of high tumorigenicity.

    PubMed

    Yi, Xi-Jun; Zhao, Yu-Hua; Qiao, Li-Xiang; Jin, Chun-Lei; Tian, Jing; Li, Qiu-Shi

    2015-10-01

    According to the cancer stem cell theory, the presence of a small sub‑population of cancer cells, termed cancer stem cells (CSCs), have a significant implication on cancer treatment and are responsible for tumor recurrence. Previous studies have reported that alterations in the Wnt/β‑catenin signaling are crucial in the maintenance of CSCs. In the present study, the characteristic features and activation of Wnt/β‑catenin signaling in CSCs from osteosarcoma, an aggressive human bone tumor, were investigated. In total, ~2.1% of the cancer stem‑like side population (SP) cells were identified in the osteosarcoma samples. The results of subsequent western blot and reverse transcription‑quantitative polymerase chain reaction analyses revealed that the protein levels of β‑catenin and cyclin D1 were markedly upregulated in the fluorescence‑activated cell sorted osteosarcoma SP cells. In addition, the elevated expression levels of stem cell proteins, including CD133, nestin Oct‑4, Sox‑2 and Nanog were significantly higher in the SP cells, which contributed to self‑renewal and enhanced the proliferation rate of the SP cells. Furthermore, the SP cells were found to be highly invasive and able to form tumors in vivo. Taken together, these data suggested that the identification of novel anticancer drugs, which suppress the Wnt/β‑catenin signaling and its downstream pathway may assist in eradicating osteosarcoma stem cells.

  9. Plasmids for variable expression of proteins targeted to the mitochondrial matrix or intermembrane space.

    PubMed

    Newman, Laura E; Schiavon, Cara; Kahn, Richard A

    2016-01-01

    We describe the construction and uses of a series of plasmids for directing expression to varied levels of exogenous proteins targeted to the mitochondrial matrix or intermembrane space. We found that the level of protein expression achieved, the kinetics of expression and mitochondrial import, and half-life after import can each vary with the protein examined. These factors should be considered when directing localization of an exogenous protein to mitochondria for rescue, proteomics, or other approaches. We describe the construction of a collection of plasmids for varied expression of proteins targeted to the mitochondrial matrix or intermembrane space, using previously defined targeting sequences and strength CMV promoters. The limited size of these compartments makes them particularly vulnerable to artifacts from over-expression. We found that different proteins display different kinetics of expression and import that should be considered when analyzing results from this approach. Finally, this collection of plasmids has been deposited in the Addgene plasmid repository to facilitate the ready access and use of these tools.

  10. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McKenna, Declan J., E-mail: dj.mckenna@ulster.ac.uk; Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen's University Belfast, Belfast BT9 7BL; Patel, Daksha, E-mail: d.patel@qub.ac.uk

    2014-01-05

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in themore » cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.« less

  11. The Triangle of Death in Alzheimer's Disease Brain: The Aberrant Cross-Talk Among Energy Metabolism, Mammalian Target of Rapamycin Signaling, and Protein Homeostasis Revealed by Redox Proteomics.

    PubMed

    Di Domenico, Fabio; Barone, Eugenio; Perluigi, Marzia; Butterfield, D Allan

    2017-03-10

    Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder and represents one of the most disabling conditions. AD shares many features in common with systemic insulin resistance diseases, suggesting that it can be considered as a metabolic disease, characterized by reduced insulin-stimulated growth and survival signaling, increased oxidative stress (OS), proinflammatory cytokine activation, mitochondrial dysfunction, impaired energy metabolism, and altered protein homeostasis. Recent Advances: Reduced glucose utilization and energy metabolism in AD have been associated with the buildup of amyloid-β peptide and hyperphosphorylated tau, increased OS, and the accumulation of unfolded/misfolded proteins. Mammalian target of rapamycin (mTOR), which is aberrantly activated in AD since early stages, plays a key role during AD neurodegeneration by, on one side, inhibiting insulin signaling as a negative feedback mechanism and, on the other side, regulating protein homeostasis (synthesis/clearance). It is likely that the concomitant and mutual alterations of energy metabolism-mTOR signaling-protein homeostasis might represent a self-sustaining triangle of harmful events that trigger the degeneration and death of neurons and the development and progression of AD. Intriguingly, the altered cross-talk between the components of such a triangle of death, beyond altering the redox homeostasis of the neuron, is further exacerbated by increased levels of OS that target and impair key components of the pathways involved. Redox proteomic studies in human samples and animal models of AD-like dementia led to identification of oxidatively modified components of the pathways composing the triangle of death, therefore revealing the crucial role of OS in fueling this aberrant vicious cycle. The identification of compounds able to restore the function of the pathways targeted by oxidative damage might represent a valuable therapeutic approach to slow or delay AD. Antioxid

  12. Aberrant dopamine D2-like receptor function in a rodent model of schizophrenia.

    PubMed

    Perez, Stephanie M; Lodge, Daniel J

    2012-11-01

    Based on the observation that antipsychotic medications display antagonist properties at dopamine D2-like receptors, aberrant dopamine signaling has been proposed to underlie psychosis in patients with schizophrenia. Thus, it is not surprising that considerable research has been devoted to understanding the mechanisms involved in the antipsychotic action of these compounds. It is important to note that the majority of these studies have been performed in "normal" experimental animals. Given that these animals do not possess the aberrant neuronal information processing typically associated with schizophrenia, the aim of the current study was to examine the dopamine D2 receptor system in a rodent model of schizophrenia. Here, we demonstrate that methylazoxymethanol acetate (MAM)-treated rats display an enhanced effect of quinpirole on dopamine neuron activity and an aberrant locomotor response to D2-like receptor activation, suggesting changes in postsynaptic D2-like receptor function. To better understand the mechanisms underlying the enhanced response to D2-like ligands in MAM-treated rats, we examined the expression of D2, D3, and dopamine transporter mRNA in the nucleus accumbens and ventral tegmental area by quantitative reverse transcription-polymerase chain reaction. MAM-treated rats displayed a significant increase in dopamine D3 receptor mRNA expression in the nucleus accumbens with no significant changes in the expression of the D2 receptor. Taken together, these data demonstrate robust alterations in dopamine D2-like receptor function in a rodent model of schizophrenia and provide evidence that preclinical studies examining the mechanisms of antipsychotic drug action should be performed in animal models that mirror aspects of the abnormal neuronal transmission thought to underlie symptoms of schizophrenia.

  13. Aberrated laser beams in terms of Zernike polynomials

    NASA Astrophysics Data System (ADS)

    Alda, Javier; Alonso, Jose; Bernabeu, Eusebio

    1996-11-01

    The characterization of light beams has devoted a lot of attention in the past decade. Several formalisms have been presented to treat the problem of parameter invariance and characterization in the propagation of light beam along ideal, ABCD, optical systems. The hard and soft apertured optical systems have been treated too. Also some aberrations have been analyzed, but it has not appeared a formalism able to treat the problem as a whole. In this contribution we use a classical approach to describe the problem of aberrated, and therefore apertured, light beams. The wavefront aberration is included in a pure phase term expanded in terms of the Zernike polynomials. Then, we can use the relation between the lower order Zernike polynomia and the Seidel or third order aberrations. We analyze the astigmatism, the spherical aberration and the coma, and we show how higher order aberrations can be taken into account. We have calculated the divergence, and the radius of curvature of such aberrated beams and the influence of these aberrations in the quality of the light beam. Some numerical simulations have been done to illustrate the method.

  14. Whole eye wavefront aberrations in Mexican male subjects.

    PubMed

    Cantú, Roberto; Rosales, Marco A; Tepichín, Eduardo; Curioca, Andrée; Montes, Victor; Bonilla, Julio

    2004-01-01

    To analyze the characteristics, incidence, and appearance of wavefront aberrations in undilated, normal, unoperated eyes. Eighty-eight eyes of 44 healthy male Mexican subjects (mean age 25.32 years, range 18 to 36 yr) were divided into three groups based on uncorrected visual acuity of greater than or equal to 20/20, 20/30, or 20/40. UCVA measurements were obtained using an Acuity Max computer screen chart. Wavefront aberrations were measured with the Nidek OPD-Scan ARK 10000, Ver. 1.11b. All measurements were carried out at the same center by the same technician during a single session, following manufacturer instructions. Background illumination was 3 Lux. Wavefront aberration measurements for each group were statistically analyzed using StatView; an average eye was characterized and the resulting aberrations were simulated using MATLAB. We obtained wavefront aberration maps for the 20/20 undilated normal unoperated eyes for total, low, and high order aberration coefficients. Wavefront maps for right eyes were practically the same as those for left eyes. Higher aberrations did not contribute substantially to total wavefront analysis. Average aberrations of this "normal eye" will be used as criteria to decide the necessity of wavefront-guided ablation in our facilities. We will focus on the nearly zero average of high order aberrations in this normal whole eye as a reference to be matched.

  15. The recombinant expression and activity detection of MAF-1 fusion protein.

    PubMed

    Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

    2015-10-01

    This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

  16. Accommodation to wavefront vergence and chromatic aberration.

    PubMed

    Wang, Yinan; Kruger, Philip B; Li, James S; Lin, Peter L; Stark, Lawrence R

    2011-05-01

    Longitudinal chromatic aberration (LCA) provides a cue to accommodation with small pupils. However, large pupils increase monochromatic aberrations, which may obscure chromatic blur. In this study, we examined the effect of pupil size and LCA on accommodation. Accommodation was recorded by infrared optometer while observers (nine normal trichromats) viewed a sinusoidally moving Maltese cross target in a Badal stimulus system. There were two illumination conditions: white (3000 K; 20 cd/m) and monochromatic (550 nm with 10 nm bandwidth; 20 cd/m) and two artificial pupil conditions (3 and 5.7 mm). Separately, static measurements of wavefront aberration were made with the eye accommodating to targets between 0 and 4 D (COAS, Wavefront Sciences). Large individual differences in accommodation to wavefront vergence and to LCA are a hallmark of accommodation. LCA continues to provide a signal at large pupil sizes despite higher levels of monochromatic aberrations. Monochromatic aberrations may defend against chromatic blur at high spatial frequencies, but accommodation responds best to optical vergence and to LCA at 3 c/deg where blur from higher order aberrations is less.

  17. Protein-protein interaction network of gene expression in the hydrocortisone-treated keloid.

    PubMed

    Chen, Rui; Zhang, Zhiliang; Xue, Zhujia; Wang, Lin; Fu, Mingang; Lu, Yi; Bai, Ling; Zhang, Ping; Fan, Zhihong

    2015-01-01

    In order to explore the molecular mechanism of hydrocortisone in keloid tissue, the gene expression profiles of keloid samples treated with hydrocortisone were subjected to bioinformatics analysis. Firstly, the gene expression profiles (GSE7890) of five samples of keloid treated with hydrocortisone and five untreated keloid samples were downloaded from the Gene Expression Omnibus (GEO) database. Secondly, data were preprocessed using packages in R language and differentially expressed genes (DEGs) were screened using a significance analysis of microarrays (SAM) protocol. Thirdly, the DEGs were subjected to gene ontology (GO) function and KEGG pathway enrichment analysis. Finally, the interactions of DEGs in samples of keloid treated with hydrocortisone were explored in a human protein-protein interaction (PPI) network, and sub-modules of the DEGs interaction network were analyzed using Cytoscape software. Based on the analysis, 572 DEGs in the hydrocortisone-treated samples were screened; most of these were involved in the signal transduction and cell cycle. Furthermore, three critical genes in the module, including COL1A1, NID1, and PRELP, were screened in the PPI network analysis. These findings enhance understanding of the pathogenesis of the keloid and provide references for keloid therapy. © 2015 The International Society of Dermatology.

  18. Mutation of a chitinase-like gene causes ectopic deposition of lignin, aberrant cell shapes, and overproduction of ethylene.

    PubMed

    Zhong, Ruiqin; Kays, Stanley J; Schroeder, Betty P; Ye, Zheng-Hua

    2002-01-01

    Chitinase-like proteins have long been proposed to play roles in normal plant growth and development, but no mutations in chitinase-like genes have been obtained previously to support this hypothesis. In this study, we have shown that the gene responsible for the elp1 mutation in Arabidopsis encodes a chitinase-like protein (AtCTL1). Mutation of this chitinase-like gene caused ectopic deposition of lignin and aberrant shapes of cells with incomplete cell walls in the pith of inflorescence stems. The AtCTL1 gene was expressed in all organs during normal plant growth and development, but it was not induced by wounding, salicylic acid, pectin fragments, or ethylene. Consistent with its ubiquitous expression pattern, mutation of the AtCTL1 gene affected many aspects of plant growth and development, including exaggerated hook curvature, reduced length and increased diameter of hypocotyls in dark-grown seedlings, and reduced root length and increased number of root hairs in light-grown seedlings. The mutant phenotypes could be rescued partially by ethylene inhibitors, and ethylene production in the mutant was significantly greater than in the wild type. Together, these results suggest that AtCTL1, a chitinase-like gene, is essential for normal plant growth and development in Arabidopsis.

  19. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression

    PubMed Central

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-01-01

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm’-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. PMID:27231931

  20. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression.

    PubMed

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-05-24

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm'-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment.

  1. PIWI Proteins and PIWI-Interacting RNA: Emerging Roles in Cancer.

    PubMed

    Han, Yi-Neng; Li, Yuan; Xia, Sheng-Qiang; Zhang, Yuan-Yuan; Zheng, Jun-Hua; Li, Wei

    2017-01-01

    P-Element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are a type of noncoding RNAs (ncRNAs) and interact with PIWI proteins. piRNAs were primarily described in the germline, but emerging evidence revealed that piRNAs are expressed in a tissue-specific manner among multiple human somatic tissue types as well and play important roles in transposon silencing, epigenetic regulation, gene and protein regulation, genome rearrangement, spermatogenesis and germ stem-cell maintenance. PIWI proteins were first discovered in Drosophila and they play roles in spermatogenesis, germline stem-cell maintenance, self-renewal, retrotransposons silencing and the male germline mobility control. A growing number of studies have demonstrated that several piRNA and PIWI proteins are aberrantly expressed in various kinds of cancers and may probably serve as a novel biomarker and therapeutic target for cancer treatment. Nevertheless, their specific mechanisms and functions need further investigation. In this review, we discuss about the biogenesis, functions and the emerging role of piRNAs and PIWI proteins in cancer, providing novel insights into the possible applications of piRNAs and PIWI proteins in cancer diagnosis and clinical treatment. © 2017 The Author(s). Published by S. Karger AG, Basel.

  2. Metabolic Genetic Screens Reveal Multidimensional Regulation of Virulence Gene Expression in Listeria monocytogenes and an Aminopeptidase That Is Critical for PrfA Protein Activation.

    PubMed

    Friedman, Sivan; Linsky, Marika; Lobel, Lior; Rabinovich, Lev; Sigal, Nadejda; Herskovits, Anat A

    2017-06-01

    Listeria monocytogenes is an environmental saprophyte and intracellular bacterial pathogen. Upon invading mammalian cells, the bacterium senses abrupt changes in its metabolic environment, which are rapidly transduced to regulation of virulence gene expression. To explore the relationship between L. monocytogenes metabolism and virulence, we monitored virulence gene expression dynamics across a library of genetic mutants grown under two metabolic conditions known to activate the virulent state: charcoal-treated rich medium containing glucose-1-phosphate and minimal defined medium containing limiting concentrations of branched-chain amino acids (BCAAs). We identified over 100 distinct mutants that exhibit aberrant virulence gene expression profiles, the majority of which mapped to nonessential metabolic genes. Mutants displayed enhanced, decreased, and early and late virulence gene expression profiles, as well as persistent levels, demonstrating a high plasticity in virulence gene regulation. Among the mutants, one was noteworthy for its particularly low virulence gene expression level and mapped to an X-prolyl aminopeptidase (PepP). We show that this peptidase plays a role in posttranslational activation of the major virulence regulator, PrfA. Specifically, PepP mediates recruitment of PrfA to the cytoplasmic membrane, a step identified as critical for PrfA protein activation. This study establishes a novel step in the complex mechanism of PrfA activation and further highlights the cross regulation of metabolism and virulence. Copyright © 2017 American Society for Microbiology.

  3. Differentially expressed proteins among normal cervix, cervical intraepithelial neoplasia and cervical squamous cell carcinoma.

    PubMed

    Zhao, Q; He, Y; Wang, X-L; Zhang, Y-X; Wu, Y-M

    2015-08-01

    To explore the differentially expressed proteins in normal cervix, cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) tissues by differential proteomics technique. Cervical tissues (including normal cervix, CIN and CSCC) were collected in Department of Gynecologic Oncology of Beijing Obstetrics and Gynecology Hospital. Two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) and DeCyder software were used to detect the differentially expressed proteins. Matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) was used to identify the differentially expressed proteins. Western blot (WB) and immunohistochemistry (IHC) were performed to validate the expressions of selected proteins among normal cervix, CIN and CSCC. 2-D DIGE images with high resolution and good repeatability were obtained. Forty-six differentially expressed proteins (27 up-regulated and 19 down-regulated) were differentially expressed among the normal cervix, CIN and CSCC. 26 proteins were successfully identified by MALDI-TOF/TOF MS. S100A9 (S100 calcium-binding protein A9) was the most significantly up-regulated protein. Eukaryotic elongation factor 1-alpha-1 (eEF1A1) was the most significantly down-regulated protein. Pyruvate kinase isozymes M2 (PKM2) was both up-regulated and down-regulated. The results of WB showed that with the increase in the severity of cervical lesions, the expression of S100A9 protein was significantly increased among the three groups (P = 0.010). The expression of eEF1A1 was reduced but without significant difference (P = 0.861). The expression of PKM2 was significantly reduced (P = 0.000). IHC showed that protein S100A9 was mainly expressed in the cytoplasm, and its positive expression rate was 20.0 % in normal cervix, 70.0 % in CIN and 100.0 % in CSCC, with a significant difference among them (P = 0.006). eEF1A1 was mainly expressed in the cell plasma, and its

  4. Benzene-Induced Aberrant miRNA Expression Profile in Hematopoietic Progenitor Cells in C57BL/6 Mice

    PubMed Central

    Wei, Haiyan; Zhang, Juan; Tan, Kehong; Sun, Rongli; Yin, Lihong; Pu, Yuepu

    2015-01-01

    Benzene is a common environmental pollutant that causes hematological alterations. MicroRNAs (miRNAs) may play a role in benzene-induced hematotoxicity. In this study, C57BL/6 mice showed significant hematotoxicity after exposure to 150 mg/kg benzene for 4 weeks. Benzene exposure decreased not only the number of cells in peripheral blood but also hematopoietic progenitor cells in the bone marrow. Meanwhile, RNA from Lin− cells sorted from the bone marrow was applied to aberrant miRNA expression profile using Illumina sequencing. We found that 5 miRNAs were overexpressed and 45 miRNAs were downregulated in the benzene exposure group. Sequencing results were confirmed through qRT-PCR. Furthermore, we also identified five miRNAs which significantly altered in Lin−c-Kit+ cells obtained from benzene-exposed mice, including mmu-miR-34a-5p; mmu-miR-342-3p; mmu-miR-100-5p; mmu-miR-181a-5p; and mmu-miR-196b-5p. In summary, we successfully established a classical animal model to induce significant hematotoxicity by benzene injection. Benzene exposure may cause severe hematotoxicity not only to blood cells in peripheral circulation but also to hematopoietic cells in bone marrow. Benzene exposure also alters miRNA expression in hematopoietic progenitor cells. This study suggests that benzene induces alteration in hematopoiesis and hematopoiesis-associated miRNAs. PMID:26569237

  5. Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

    PubMed

    Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

    2016-06-01

    Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Junction region of EWS-FLI1 fusion protein has a dominant negative effect in Ewing's sarcoma in vitro.

    PubMed

    Jully, Babu; Vijayalakshmi, Ramshankar; Gopal, Gopisetty; Sabitha, Kesavan; Rajkumar, Thangarajan

    2012-11-12

    Ewing's sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein. In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251-343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251-280 ectopically in Ewing's sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed. Our modelling analysis indicated that Junction region (aa 251-343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing's sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect. Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing's Sarcoma.

  7. [Survival of patients with primary central nervous system diffuse large B-cell lymphoma: impact of gene aberrations and protein overexpression of bcl-2 and C-MYC, and selection of chemotherapy regimens].

    PubMed

    Yin, W J; Zhu, X; Yang, H Y; Sun, W Y; Wu, M J

    2018-01-08

    bcl-2 gene translocation and ICN were found in 30 patients. Four ICNs of C-MYC gene were found in 28 patients. Elevated protein in cerebrospinal fluid (CSF) was found in 13 patients. LDH increased in 10 cases. Follow-up period was 2-90 months with the average survival time of (23.0±3.7) months and two-year survival rate of 39.0%. Univariate survival analysis showed that overexpression of bcl-2 protein (≥70%) and MYC protein (≥40%), bcl-2 gene abnormality (including copy number increase and translocation), C-MYC gene copy number increased were adverse factors for survival. C-MYC/ bcl-2 gene double hit was seen in 2 cases. Bivariate survival analysis found that of bcl-2/MYC protein double expression and bcl-2 and C-MYC genes double aberration were significantly associated with adverse outcomes. Cox multivariate risk regression analysis found that gender, cerebrospinal fluid protein increasing, and ICN of C-MYC gene were independent poor prognostic factors. DH-MTX based comprehensive chemotherapy was associated with better prognosis. Conclusions: Double hit at genomic level (copy number variations and gene rearrangements) and double protein expression of bcl-2 and C-MYC in PCNS-DLBCL are significantly associated with an adverse outcome. DH-MTX based comprehensive treatment may prolong the patient survival.

  8. SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy

    PubMed Central

    Wang, Xiaoxin X.; Levi, Jonathan; Luo, Yuhuan; Myakala, Komuraiah; Herman-Edelstein, Michal; Qiu, Liru; Wang, Dong; Peng, Yingqiong; Grenz, Almut; Lucia, Scott; Dobrinskikh, Evgenia; D'Agati, Vivette D.; Koepsell, Hermann; Kopp, Jeffrey B.; Rosenberg, Avi Z.; Levi, Moshe

    2017-01-01

    There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice. PMID:28196866

  9. Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lovrinovic, Marina; Niemeyer, Christof M.

    2005-09-30

    We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter weremore » ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.« less

  10. Automated aberration compensation in high numerical aperture systems for arbitrary laser modes (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hering, Julian; Waller, Erik H.; von Freymann, Georg

    2017-02-01

    Since a large number of optical systems and devices are based on differently shaped focal intensity distributions (point-spread-functions, PSF), the PSF's quality is crucial for the application's performance. E.g., optical tweezers, optical potentials for trapping of ultracold atoms as well as stimulated-emission-depletion (STED) based microscopy and lithography rely on precisely controlled intensity distributions. However, especially in high numerical aperture (NA) systems, such complex laser modes are easily distorted by aberrations leading to performance losses. Although different approaches addressing phase retrieval algorithms have been recently presented[1-3], fast and automated aberration compensation for a broad variety of complex shaped PSFs in high NA systems is still missing. Here, we report on a Gerchberg-Saxton[4] based algorithm (GSA) for automated aberration correction of arbitrary PSFs, especially for high NA systems. Deviations between the desired target intensity distribution and the three-dimensionally (3D) scanned experimental focal intensity distribution are used to calculate a correction phase pattern. The target phase distribution plus the correction pattern are displayed on a phase-only spatial-light-modulator (SLM). Focused by a high NA objective, experimental 3D scans of several intensity distributions allow for characterization of the algorithms performance: aberrations are reliably identified and compensated within less than 10 iterations. References 1. B. M. Hanser, M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, "Phase-retrieved pupil functions in wide-field fluorescence microscopy," J. of Microscopy 216(1), 32-48 (2004). 2. A. Jesacher, A. Schwaighofer, S. Frhapter, C. Maurer, S. Bernet, and M. Ritsch-Marte, "Wavefront correction of spatial light modulators using an optical vortex image," Opt. Express 15(9), 5801-5808 (2007). 3. A. Jesacher and M. J. Booth, "Parallel direct laser writing in three dimensions with spatially dependent

  11. Patterning of inflorescences and flowers by the F-Box protein DOUBLE TOP and the LEAFY homolog ABERRANT LEAF AND FLOWER of petunia.

    PubMed

    Souer, Erik; Rebocho, Alexandra B; Bliek, Mattijs; Kusters, Elske; de Bruin, Robert A M; Koes, Ronald

    2008-08-01

    Angiosperms display a wide variety of inflorescence architectures differing in the positions where flowers or branches arise. The expression of floral meristem identity (FMI) genes determines when and where flowers are formed. In Arabidopsis thaliana, this is regulated via transcription of LEAFY (LFY), which encodes a transcription factor that promotes FMI. We found that this is regulated in petunia (Petunia hybrida) via transcription of a distinct gene, DOUBLE TOP (DOT), a homolog of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Mutation of DOT or its tomato (Solanum lycopersicum) homolog ANANTHA abolishes FMI. Ubiquitous expression of DOT or UFO in petunia causes very early flowering and transforms the inflorescence into a solitary flower and leaves into petals. Ectopic expression of DOT or UFO together with LFY or its homolog ABERRANT LEAF AND FLOWER (ALF) in petunia seedlings activates genes required for identity or outgrowth of organ primordia. DOT interacts physically with ALF, suggesting that it activates ALF by a posttranslational mechanism. Our findings suggest a wider role than previously thought for DOT and UFO in the patterning of flowers and indicate that the different roles of LFY and UFO homologs in the spatiotemporal control of floral identity in distinct species result from their divergent expression patterns.

  12. Cloning, Expression, and Purification of Brucella suis Outer Membrane Proteins

    DTIC Science & Technology

    2005-01-01

    13-09-20061 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Cloning, expression and purification of Brucella suis outer membrane proteins 5b. GRANT NUMBER...attractive for this purpose. In this study, we cloned, expressed and purified seven predicted OMPs of Brucella suis . The recombinant proteins were...fused with 6-his and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based

  13. The E4 protein; structure, function and patterns of expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genomemore » amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1

  14. Dopamine Signaling Leads to Loss of Polycomb Repression and Aberrant Gene Activation in Experimental Parkinsonism

    PubMed Central

    Lerdrup, Mads; Gomes, Ana-Luisa; Kryh, Hanna; Spigolon, Giada; Caboche, Jocelyne; Fisone, Gilberto; Hansen, Klaus

    2014-01-01

    Polycomb group (PcG) proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation. The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP) experiments showed that increased H3K27me3S28p was accompanied by reduced PcG binding to regulatory regions of genes. An analysis of the genome wide distribution of L-DOPA-induced H3K27me3S28 phosphorylation by ChIP sequencing (ChIP-seq) in combination with expression analysis by RNA-sequencing (RNA-seq) showed that the induction of H3K27me3S28p correlated with increased expression of a subset of PcG repressed genes. We found that induction of H3K27me3S28p persisted during chronic L-DOPA administration to parkisonian mice and correlated with aberrant gene expression. We propose that dopaminergic transmission can activate PcG repressed genes in the adult brain and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson's disease. PMID:25254549

  15. Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

    PubMed Central

    2013-01-01

    Background In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co

  16. Different architectures in the assembly of infectious bursal disease virus capsid proteins expressed in insect cells.

    PubMed

    Martinez-Torrecuadrada, J L; Castón, J R; Castro, M; Carrascosa, J L; Rodriguez, J F; Casal, J I

    2000-12-20

    Infectious bursal disease virus (IBDV) capsid is formed by the processing of a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructs were made using two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, the expression of the capsid proteins gave rise to different types of particles in each system, as observed by electron microscopy and immunofluorescence. FastBac expression led to the production of only rigid tubular structures, similar to those described as type I in viral infection. Western blot analysis revealed that these rigid tubules are formed exclusively by VPX. These tubules revealed a hexagonal arrangement of units that are trimer clustered, similar to those observed in IBDV virions. In contrast, pAcYM1 expression led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of IBDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only on the inner surfaces of VLPs and caps of the tubular structures. In summary, assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2. Copyright 2000 Academic Press.

  17. Simple method based on intensity measurements for characterization of aberrations from micro-optical components.

    PubMed

    Perrin, Stephane; Baranski, Maciej; Froehly, Luc; Albero, Jorge; Passilly, Nicolas; Gorecki, Christophe

    2015-11-01

    We report a simple method, based on intensity measurements, for the characterization of the wavefront and aberrations produced by micro-optical focusing elements. This method employs the setup presented earlier in [Opt. Express 22, 13202 (2014)] for measurements of the 3D point spread function, on which a basic phase-retrieval algorithm is applied. This combination allows for retrieval of the wavefront generated by the micro-optical element and, in addition, quantification of the optical aberrations through the wavefront decomposition with Zernike polynomials. The optical setup requires only an in-motion imaging system. The technique, adapted for the optimization of micro-optical component fabrication, is demonstrated by characterizing a planoconvex microlens.

  18. Heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence.

    PubMed

    Faratian, Dana; Christiansen, Jason; Gustavson, Mark; Jones, Christine; Scott, Christopher; Um, InHwa; Harrison, David J

    2011-10-25

    Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor(1,2), and some of these sub-clones give rise to metastatic (and therefore lethal) disease. Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension(3), or on macrodissection(4). A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue(5), providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi

  19. Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

    PubMed Central

    McMains, Emily; Krishnan, Vijai; Prasad, Sujitha; Gleason, Evanna

    2011-01-01

    Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue. PMID:21408174

  20. Functional evaluation of candidate ice structuring proteins using cell-free expression systems.

    PubMed

    Brödel, A K; Raymond, J A; Duman, J G; Bier, F F; Kubick, S

    2013-02-10

    Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Spherical aberrations of human astigmatic corneas.

    PubMed

    Zhao, Huawei; Dai, Guang-Ming; Chen, Li; Weeber, Henk A; Piers, Patricia A

    2011-11-01

    To evaluate whether the average spherical aberration of human astigmatic corneas is statistically equivalent to human nonastigmatic corneas. Spherical aberrations of 445 astigmatic corneas prior to laser vision correction were retrospectively investigated to determine Zernike coefficients for central corneal areas 6 mm in diameter using CTView (Sarver and Associates). Data were divided into groups according to cylinder power (0.01 to 0.25 diopters [D], 0.26 to 0.75 D, 0.76 to 1.06 D, 1.07 to 1.53 D, 1.54 to 2.00 D, and >2.00 D) and according to age by decade. Spherical aberrations were correlated with age and astigmatic power among groups and the entire population. Statistical analyses were conducted, and P<.05 was considered statistically significant. Mean patient age was 42.6±11 years. Astigmatic corneas had an average astigmatic power of 0.78±0.58 D and mean spherical aberration was 0.25±0.13 μm for the entire population and approximately the same (0.27 μm) for individual groups, ranging from 0.23 to 0.29 μm (P>.05 for all tested groups). Mean spherical aberration of astigmatic corneas was not correlated significantly with cylinder power or age (P>.05). Spherical aberrations are similar to those of nonastigmatic corneas, permitting the use of these additional data in the design of aspheric toric intra-ocular lenses. Copyright 2011, SLACK Incorporated.

  2. Transition from two to one integument in Prunus species: expression pattern of INNER NO OUTER (INO), ABERRANT TESTA SHAPE (ATS) and ETTIN (ETT).

    PubMed

    Lora, Jorge; Hormaza, José I; Herrero, Maria

    2015-10-01

    While gymnosperm ovules have one integument, in most angiosperms two integuments surround the ovules. Unitegmic ovules have arisen independently several times during the evolution of angiosperms, but the ultimate genetic cause of the presence of a single integument remains elusive. We compared species of the genus Prunus that have different numbers of integuments: bitegmic species, such as Prunus armeniaca (apricot) and Prunus persica (peach), and unitegmic species, such as Prunus incisa, analyzing the expression pattern of genes that are involved in integument development in Arabidopsis thaliana: INNER NO OUTER (INO), ABERRANT TESTA SHAPE (ATS) and ETTIN (ETT). Bitegmic and unitegmic species showed similar INO expression patterns, indicative of the conservation of an outer integument. However, expression of ETT, which occurs in the boundary of the outer and inner integuments, was altered in unitegmic ovules, which showed lack of ETT expression. These results strongly suggest that the presence of a single integument could be attributable to the amalgamation of two integuments and support the role of ETT in the fusion of the outer and inner integuments in unitegmic ovules, a situation that could be widespread in other unitegmic species of angiosperms. © 2015 Consejo Superior de Investigaciones Cientificas. New Phytologist © 2015 New Phytologist Trust.

  3. Aberrant Gene Expression in Dogs with Portosystemic Shunts

    PubMed Central

    Grinwis, Guy C. M.; Kummeling, Anne; van Gils, Ingrid H. M.; Koerkamp, Marian J. A. Groot.; van Leenen, Dik; Holstege, Frank C. P.; Penning, Louis C.; Rothuizen, Jan; Leegwater, Peter A. J.; Spee, Bart

    2013-01-01

    Congenital portosystemic shunts are developmental anomalies of the splanchnic vascular system that cause portal blood to bypass the liver. Large-breed dogs are predisposed for intrahepatic portosystemic shunts (IHPSS) and small-breed dogs for extrahepatic portosystemic shunts (EHPSS). While the phenotype resulting from portal bypass of the liver of the two types of shunt is identical, the genotype and molecular pathways involved are probably different. The aim of this study was to gain insight into the pathways involved in the different types of portosystemic shunting. Microarray analysis of mRNA expression in liver tissue from dogs with EHPSS and IHPSS revealed that the expression of 26 genes was altered in either IHPSS or EHPSS samples compared with that in liver samples from control dogs. Quantitative real-time PCR of these genes in 14 IHPSS, 17 EHPSS, and 8 control liver samples revealed a significant differential expression of ACBP, CCBL1, GPC3, HAMP, PALLD, VCAM1, and WEE1. Immunohistochemistry and Western blotting confirmed an increased expression of VCAM1 in IHPSS but its absence in EHPSS, an increased WEE1 expression in IHPSS but not in EHPSS, and a decreased expression of CCBL1 in both shunt types. Regarding their physiologic functions, these findings may indicate a causative role for VCAM1 in IHPSS and WEE1 for IHPSS. CCBL1 could be an interesting candidate to study not yet elucidated aspects in the pathophysiology of hepatic encephalopathy. PMID:23451256

  4. Expression of small heat shock proteins from pea seedlings under gravity altered conditions

    NASA Astrophysics Data System (ADS)

    Talalaev, Alexandr S.

    2005-08-01

    A goal of our study was to evaluate the stress gene expression in Pisum sativum seedlings exposed to altered gravity and temperature elevation. We investigate message for the two inducible forms of the cytosolic small heat shock proteins (sHsp), sHsp 17.7 and sHsp 18.1. Both proteins are able to enhance the refolding of chemically denatured proteins in an ATP- independent manner, in other words they can function as molecular chaperones. We studied sHsps expression in pea seedlings cells by Western blotting. Temperature elevation, as the positive control, significantly increased PsHsp 17.7 and PsHsp 18.1 expression. Expression of the housekeeping protein, actin was constant and comparable to unstressed controls for all treatments. We concluded that gravitational perturbations incurred by clinorotation did not change sHsp genes expression.

  5. The role of Myc-induced protein synthesis in cancer

    PubMed Central

    Ruggero, Davide

    2009-01-01

    Deregulation in different steps of translational control is an emerging mechanism for cancer formation. One example of an oncogene with a direct role in control of translation is the Myc transcription factor. Myc directly increases protein synthesis rates by controlling the expression of multiple components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, Pol III and rDNA. However, the contribution of Myc-dependent increases in protein synthesis towards the multi-step process leading to cancer has remained unknown. Recent evidence strongly suggests that Myc oncogenic signaling may monopolize the translational machinery to elicit cooperative effects on cell growth, cell cycle progression, and genome instability as a mechanism for cancer initiation. Moreover, new genetic tools to restore aberrant increases in protein synthesis control are now available, which should enable the dissection of important mechanisms in cancer that rely on the translational machinery. PMID:19934336

  6. High order aberration and straylight evaluation after cataract surgery with implantation of an aspheric, aberration correcting monofocal intraocular lens

    PubMed Central

    Kretz, Florian T A; Tandogan, Tamer; Khoramnia, Ramin; Auffarth, Gerd U

    2015-01-01

    AIM To evaluate the quality of vision in respect to high order aberrations and straylight perception after implantation of an aspheric, aberration correcting, monofocal intraocular lens (IOL). METHODS Twenty-one patients (34 eyes) aged 50 to 83y underwent cataract surgery with implantation of an aspheric, aberration correcting IOL (Tecnis ZCB00, Abbott Medical Optics). Three months after surgery they were examined for uncorrected (UDVA) and corrected distance visual acuity (CDVA), contrast sensitivity (CS) under photopic and mesopic conditions with and without glare source, ocular high order aberrations (HOA, Zywave II) and retinal straylight (C-Quant). RESULTS Postoperatively, patients achieved a postoperative CDVA of 0.0 logMAR or better in 97.1% of eyes. Mean values of high order abberations were +0.02±0.27 (primary coma components) and -0.04±0.16 (spherical aberration term). Straylight values of the C-Quant were 1.35±0.44 log which is within normal range of age matched phakic patients. The CS measurements under mesopic and photopic conditions in combination with and without glare did not show any statistical significance in the patient group observed (P≥0.28). CONCLUSION The implantation of an aspherical aberration correcting monofocal IOL after cataract surgery resulted in very low residual higher order aberration (HOA) and normal straylight. PMID:26309872

  7. Differentiation of EL4 lymphoma cells by tumoral environment is associated with inappropriate expression of the large chondroitin sulfate proteoglycan PG-M and the tumor-associated antigen HTgp-175.

    PubMed

    Rottiers, P; Verfaillie, T; Contreras, R; Revets, H; Desmedt, M; Dooms, H; Fiers, W; Grooten, J

    1998-11-09

    Progression to malignancy of transformed cells involves complex genetic alterations and aberrant gene expression patterns. While aberrant gene expression is often caused by alterations in individual genes, the contribution of the tumoral environment to the triggering of this gene expression is less well established. The stable but heterogeneous expression in cultured EL4/13 cells of a novel tumor-associated antigen, designated as HTgp-175, was chosen for the investigation of gene expression during tumor formation. Homogeneously HTgp-175-negative EL4/13 cells, isolated by cell sorting or obtained by subcloning, acquired HTgp-175 expression as a result of tumor formation. The tumorigenicity of HTgp-175-negative vs. HTgp-175-positive EL4 variants was identical, indicating that induction but not selection accounted for the phenotypic switch from HTgp-175-negative to HTgp-175-positive. Although mutagenesis experiments showed that the protein was not essential for tumor establishment, tumor-derived cells showed increased malignancy, linking HTgp-175 expression with genetic changes accompanying tumor progression. This novel gene expression was not an isolated event, since it was accompanied by ectopic expression of the large chondroitin sulfate proteoglycan PG-M and of normal differentiation antigens. We conclude that signals derived from the tumoral microenvironment contribute significantly to the aberrant gene expression pattern of malignant cells, apparently by fortuitous activation of differentiation processes and cause expression of novel differentiation antigens as well as of inappropriate tumor-associated and ectopic antigens.

  8. Accommodation to Wavefront Vergence and Chromatic Aberration

    PubMed Central

    Wang, Yinan; Kruger, Philip B.; Li, James S.; Lin, Peter L.; Stark, Lawrence R.

    2011-01-01

    Purpose Longitudinal chromatic aberration (LCA) provides a cue to accommodation with small pupils. However, large pupils increase monochromatic aberrations, which may obscure chromatic blur. In the present study, we examined the effect of pupil size and LCA on accommodation. Methods Accommodation was recorded by infrared optometer while observers (nine normal trichromats) viewed a sinusoidally moving Maltese cross target in a Badal stimulus system. There were two illumination conditions: white (3000 K; 20 cd/m2) and monochromatic (550 nm with 10 nm bandwidth; 20 cd/m2) and two artificial pupil conditions (3 mm and 5.7 mm). Separately, static measurements of wavefront aberration were made with the eye accommodating to targets between 0 and 4 D (COAS, Wavefront Sciences). Results Large individual differences in accommodation to wavefront vergence and to LCA are a hallmark of accommodation. LCA continues to provide a signal at large pupil sizes despite higher levels of monochromatic aberrations. Conclusions Monochromatic aberrations may defend against chromatic blur at high spatial frequencies, but accommodation responds best to optical vergence and to LCA at 3 c/deg where blur from higher order aberrations is less. PMID:21317666

  9. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes

    PubMed Central

    Soltani, Mohammad; Vargas-Garcia, Cesar A.; Antunes, Duarte; Singh, Abhyudai

    2016-01-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  10. Immunogenicity of Newcastle disease virus vectors expressing Norwalk virus capsid protein in the presence or absence of VP2 protein.

    PubMed

    Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y; Samal, Siba K

    2015-10-01

    Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirus-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Immunogenicity of Newcastle Disease Virus Vectors Expressing Norwalk Virus Capsid Protein in the Presence or Absence of VP2 Protein

    PubMed Central

    Kim, Shin-Hee; Chen, Shun; Jiang, Xi; Green, Kim Y.; Samal, Siba K.

    2015-01-01

    Noroviruses are the most common cause of acute gastroenteritis in humans. Development of an effective vaccine is required for reducing their outbreaks. In order to develop a GI norovirus vaccine, Newcastle disease virus vectors, rLaSota and modified rBC, were used to express VP1 protein of Norwalk virus. Co-expression of VP1 and VP2 proteins by Newcastle disease virus vectors resulted in enhanced expression of Norwalk virus VP1 protein and self-assembly of VP1 protein into virus-like particles. Furthermore, the Norwalk virus-specific IgG response induced in mice by Newcastle disease virus vectors was similar to that induced by baculovirs-expressed virus-like particles in mice. However, the modified rBC vector in the presence of VP2 protein induced significantly higher levels of cellular and mucosal immune responses than those induced by baculovirus-expressed VLPs. These results indicate that Newcastle disease virus has great potential for developing a live Norwalk virus vaccine by inducing humoral, cellular and mucosal immune responses in humans. PMID:26099695

  12. Functional Heterologous Protein Expression by Genetically Engineered Probiotic Yeast Saccharomyces boulardii

    PubMed Central

    Hudson, Lauren E.; Fasken, Milo B.; McDermott, Courtney D.; McBride, Shonna M.; Kuiper, Emily G.; Guiliano, David B.; Corbett, Anita H.; Lamb, Tracey J.

    2014-01-01

    Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders. PMID:25391025

  13. Functional heterologous protein expression by genetically engineered probiotic yeast Saccharomyces boulardii.

    PubMed

    Hudson, Lauren E; Fasken, Milo B; McDermott, Courtney D; McBride, Shonna M; Kuiper, Emily G; Guiliano, David B; Corbett, Anita H; Lamb, Tracey J

    2014-01-01

    Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.

  14. Comparison of Aberrations After Standard and Customized Refractive Surgery

    NASA Astrophysics Data System (ADS)

    Fang, L.; He, X.; Wang, Y.

    2013-09-01

    To detect possible differences in residual wavefront aberrations between standard and customized laser refractive surgery based onmathematical modeling, the residual optical aberrations after conventional and customized laser refractive surgery were compared accordingto the ablation profile with transition zone. The results indicated that ablation profile has a significant impact on the residual aberrations.The amount of residual aberrations for conventional correction is higher than that for customized correction. Additionally, the residualaberrations for high myopia eyes are markedly larger than those for moderate myopia eyes. For a 5 mm pupil, the main residual aberrationterm is coma and yet it is spherical aberration for a 7 mm pupil. When the pupil diameter is the same as optical zone or greater, themagnitudes of residual aberrations is obviously larger than that for a smaller pupil. In addition, the magnitudes of the residual fifth orsixth order aberrations are relatively large, especially secondary coma in a 6 mm pupil and secondary spherical aberration in a 7 mm pupil.Therefore, the customized ablation profile may be superior to the conventional correction even though the transition zone and treatmentdecentration are taken into account. However, the customized ablation profile will still induce significant amount of residual aberrations.

  15. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  16. Genome-wide analysis of aberrantly expressed lncRNAs and miRNAs with associated co-expression and ceRNA networks in β-thalassemia and hereditary persistence of fetal hemoglobin.

    PubMed

    Lai, Ketong; Jia, Siyuan; Yu, Shanjuan; Luo, Jianming; He, Yunyan

    2017-07-25

    The implications of lncRNAs regarding fetal hemoglobin (HbF) induction in hemoglobin disorders remain poorly understood. In this study, microarray analysis was performed to profile lncRNAs, miRNAs and mRNAs in individuals with hereditary persistence of fetal hemoglobin (HPFH), β-thalassemia carriers with high HbF levels and healthy controls. The results show aberrant expression of 862 lncRNAs, 568 mRNAs and 63 miRNAs in the high-HbF group compared with the control group. Altered NR_001589, NR_120526, T315543, miR-486-3p, miR-19b-1-5p and miR-20a-3p expression was confirmed by quantitative reverse transcription-polymerase chain reaction, and Spearman correlation coefficients revealed significant positive correlations with HbF. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed the hematopoietic cell lineage and apoptosis to be most significantly dysregulated in HbF induction. We analyzed coding genes near the lncRNAs and constructed a coding-noncoding co-expression network. Based on the results, lncRNAs likely contribute to increased HbF levels by activating expression of HBE1 and hematopoietic cell lineage-inducible molecules and by inhibiting that of apoptosis-inducible molecules. Finally, through construction of a competing endogenous RNA network, we found that 6 lncRNAs could bind competitively with miR-486-3p, resulting in increased HbF levels. Taken together, our findings provide new insights into the mechanisms of HbF induction and potentially provide new targets for the treatment of β-thalassemia major.

  17. Genome-wide analysis of aberrantly expressed lncRNAs and miRNAs with associated co-expression and ceRNA networks in β-thalassemia and hereditary persistence of fetal hemoglobin

    PubMed Central

    Yu, Shanjuan; Luo, Jianming; He, Yunyan

    2017-01-01

    The implications of lncRNAs regarding fetal hemoglobin (HbF) induction in hemoglobin disorders remain poorly understood. In this study, microarray analysis was performed to profile lncRNAs, miRNAs and mRNAs in individuals with hereditary persistence of fetal hemoglobin (HPFH), β-thalassemia carriers with high HbF levels and healthy controls. The results show aberrant expression of 862 lncRNAs, 568 mRNAs and 63 miRNAs in the high-HbF group compared with the control group. Altered NR_001589, NR_120526, T315543, miR-486-3p, miR-19b-1-5p and miR-20a-3p expression was confirmed by quantitative reverse transcription-polymerase chain reaction, and Spearman correlation coefficients revealed significant positive correlations with HbF. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed the hematopoietic cell lineage and apoptosis to be most significantly dysregulated in HbF induction. We analyzed coding genes near the lncRNAs and constructed a coding-noncoding co-expression network. Based on the results, lncRNAs likely contribute to increased HbF levels by activating expression of HBE1 and hematopoietic cell lineage-inducible molecules and by inhibiting that of apoptosis-inducible molecules. Finally, through construction of a competing endogenous RNA network, we found that 6 lncRNAs could bind competitively with miR-486-3p, resulting in increased HbF levels. Taken together, our findings provide new insights into the mechanisms of HbF induction and potentially provide new targets for the treatment of β-thalassemia major. PMID:28624809

  18. The protein expression landscape of mitosis and meiosis in diploid budding yeast.

    PubMed

    Becker, Emmanuelle; Com, Emmanuelle; Lavigne, Régis; Guilleux, Marie-Hélène; Evrard, Bertrand; Pineau, Charles; Primig, Michael

    2017-03-06

    Saccharomyces cerevisiae is an established model organism for the molecular analysis of fundamental biological processes. The genomes of numerous strains have been sequenced, and the transcriptome and proteome ofmajor phases during the haploid and diploid yeast life cycle have been determined. However, much less is known about dynamic changes of the proteome when cells switch from mitotic growth to meiotic development. We report a quantitative protein profiling analysis of yeast cell division and differentiation based on mass spectrometry. Information about protein levels was integrated with strand-specific tiling array expression data. We identified a total of 2366 proteins in at least one condition, including 175 proteins showing a statistically significant>5-fold change across the sample set, and 136 proteins detectable in sporulating but not respiring cells. We correlate protein expression patterns with biological processes and molecular function by Gene Ontology term enrichment, chemoprofiling, transcription interference and the formation of double stranded RNAs by overlapping sense/antisense transcripts. Our work provides initial quantitative insight into protein expression in diploid respiring and differentiating yeast cells. Critically, it associates developmentally regulated induction of antisense long noncoding RNAs and double stranded RNAs with fluctuating protein concentrations during growth and development. This integrated genomics analysis helps better understand how the transcriptome and the proteome correlate in diploid yeast cells undergoing mitotic growth in the presence of acetate (respiration) versus meiotic differentiation (Meiosis I and II). The study (i) provides quantitative expression data for 2366 proteins and their cognate mRNAs in at least one sample, (ii) shows strongly fluctuating protein levels during growth and differentiation for 175 cases, and (iii) identifies 136 proteins absent in mitotic but present in meiotic yeast cells. We

  19. Theoretical investigation of aberrations upon ametropic human eyes

    NASA Astrophysics Data System (ADS)

    Tan, Bo; Chen, Ying-Ling; Lewis, J. W. L.; Baker, Kevin

    2003-11-01

    The human eye aberrations are important for visual acuity and ophthalmic diagnostics and surgical procedures. Reported monochromatic aberration data of the normal 20/20 human eyes are scarce. There exist even fewer reports of the relation between ametropic conditions and aberrations. We theoretically investigate the monochromatic and chromatic aberrations of human eyes for refractive errors of -10 to +10 diopters. Schematic human eye models are employed using optical design software for axial, index, and refractive types of ametropia.

  20. ChiPPI: a novel method for mapping chimeric protein-protein interactions uncovers selection principles of protein fusion events in cancer.

    PubMed

    Frenkel-Morgenstern, Milana; Gorohovski, Alessandro; Tagore, Somnath; Sekar, Vaishnovi; Vazquez, Miguel; Valencia, Alfonso

    2017-07-07

    Fusion proteins, comprising peptides deriving from the translation of two parental genes, are produced in cancer by chromosomal aberrations. The expressed fusion protein incorporates domains of both parental proteins. Using a methodology that treats discrete protein domains as binding sites for specific domains of interacting proteins, we have cataloged the protein interaction networks for 11 528 cancer fusions (ChiTaRS-3.1). Here, we present our novel method, chimeric protein-protein interactions (ChiPPI) that uses the domain-domain co-occurrence scores in order to identify preserved interactors of chimeric proteins. Mapping the influence of fusion proteins on cell metabolism and pathways reveals that ChiPPI networks often lose tumor suppressor proteins and gain oncoproteins. Furthermore, fusions often induce novel connections between non-interactors skewing interaction networks and signaling pathways. We compared fusion protein PPI networks in leukemia/lymphoma, sarcoma and solid tumors finding distinct enrichment patterns for each disease type. While certain pathways are enriched in all three diseases (Wnt, Notch and TGF β), there are distinct patterns for leukemia (EGFR signaling, DNA replication and CCKR signaling), for sarcoma (p53 pathway and CCKR signaling) and solid tumors (FGFR and EGFR signaling). Thus, the ChiPPI method represents a comprehensive tool for studying the anomaly of skewed cellular networks produced by fusion proteins in cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. SRY protein is expressed in ovotestis and streak gonads from human sex-reversal.

    PubMed

    Salas-Cortés, L; Jaubert, F; Nihoul-Feketé, C; Brauner, R; Rosemblatt, M; Fellous, M

    2000-01-01

    In mammals, a master gene located on the Y chromosome, the testis-determining gene SRY, controls sex determination. SRY protein is expressed in the genital ridge before testis determination, and in the testis it is expressed in Sertoli and germ cells. Completely sex-reversed patients are classified as either 46,XX males or 46,XY females. SRY mutations have been described in only 15% of patients with 46,XY complete or partial gonadal dysgenesis. However, although incomplete or partial sex-reversal affects 46,XX true hermaphrodites, 46,XY gonadal dysgenesis, and 46,XX/46,XY mosaicism, only 15% of the 46,XX true hermaphrodites analyzed have the SRY gene. Here, we demonstrate that the SRY protein is expressed in the tubules of streak gonads and rete testis, indicating that the SRY protein is normally expressed early during testis determination. Based on these results, we propose that some factors downstream from SRY may be mutated in these 46,XY sex-reversal patients. We have also analyzed SRY protein expression in the ovotestis from 46,XX true hermaphrodites and 46,XX/46,XY mosaicism, demonstrating SRY protein expression in both testicular and ovarian portions in these patients. This suggests that the SRY protein does not inhibit ovary development. These results confirm that other factors are needed for complete testis development, in particular, those downstream of the SRY protein. Copyright 2001 S. Karger AG, Basel

  2. Anterior Corneal, Posterior Corneal, and Lenticular Contributions to Ocular Aberrations.

    PubMed

    Atchison, David A; Suheimat, Marwan; Mathur, Ankit; Lister, Lucas J; Rozema, Jos

    2016-10-01

    To determine the corneal surfaces and lens contributions to ocular aberrations. There were 61 healthy participants with ages ranging from 20 to 55 years and refractions -8.25 diopters (D) to +3.25 D. Anterior and posterior corneal topographies were obtained with an Oculus Pentacam, and ocular aberrations were obtained with an iTrace aberrometer. Raytracing through models of corneas provided total corneal and surface component aberrations for 5-mm-diameter pupils. Lenticular contributions were given as differences between ocular and corneal aberrations. Theoretical raytracing investigated influence of object distance on aberrations. Apart from defocus, the highest aberration coefficients were horizontal astigmatism, horizontal coma, and spherical aberration. Most correlations between lenticular and ocular parameters were positive and significant, with compensation of total corneal aberrations by lenticular aberrations for 5/12 coefficients. Anterior corneal aberrations were approximately three times higher than posterior corneal aberrations and usually had opposite signs. Corneal topographic centers were displaced from aberrometer pupil centers by 0.32 ± 0.19 mm nasally and 0.02 ± 0.16 mm inferiorly; disregarding corneal decentration relative to pupil center was significant for oblique astigmatism, horizontal coma, and horizontal trefoil. An object at infinity, rather than at the image in the anterior cornea, gave incorrect aberration estimates of the posterior cornea. Corneal and lenticular aberration magnitudes are similar, and aberrations of the anterior corneal surface are approximately three times those of the posterior surface. Corneal decentration relative to pupil center has significant effects on oblique astigmatism, horizontal coma, and horizontal trefoil. When estimating component aberrations, it is important to use correct object/image conjugates and heights at surfaces.

  3. Human XPA and XRCC1 DNA repair proteins expressed in yeast, Saccharomyces cerevisiae.

    PubMed

    Pushnova, E A; Ostanin, K; Thelen, M P

    2001-11-01

    Human XPA and XRCC1 DNA repair proteins have been expressed in a series of novel yeast episomal vectors. Expression of XPA cDNA resulted in synthesis of anti-XPA crossreacting polypeptides of 40 and 42 kDa, the status of the native protein found in human cells. Likewise, the majority of the recombinant XRCC1 found in the yeast intracellular fraction corresponded to the molecular mass of the full-length human protein. Recombinant XPA protein expressed as an NH(2)-terminal polyhistidine fusion could be affinity purified using Ni(2+) agarose. Copyright 2001 Academic Press.

  4. Significance of aquaporins’ expression in the prognosis of gastric cancer

    PubMed Central

    Thapa, Saroj; Chetry, Mandika; Huang, Kaiyu; Peng, Yangpei; Wang, Jinsheng; Wang, Jiaoni; Zhou, Yingying; Shen, Yigen; Xue, Yangjing; Ji, Kangting

    2018-01-01

    Gastric carcinoma is one of the most lethal malignancy at present with leading cause of cancer-related deaths worldwide. Aquaporins (AQPs) are a family of small, integral membrane proteins, which have been evidenced to play a crucial role in cell migration and proliferation of different cancer cells including gastric cancers. However, the aberrant expression of specific AQPs and its correlation to detect predictive and prognostic significance in gastric cancer remains elusive. In the present study, we comprehensively explored immunohistochemistry based map of protein expression profiles in normal tissues, cancer and cell lines from publicly available Human Protein Atlas (HPA) database. Moreover, to improve our understanding of general gastric biology and guide to find novel predictive prognostic gastric cancer biomarker, we also retrieved ‘The Kaplan–Meier plotter’ (KM plotter) online database with specific AQPs mRNA to overall survival (OS) in different clinicopathological features. We revealed that ubiquitous expression of AQPs protein can be effective tools to generate gastric cancer biomarker. Furthermore, high level AQP3, AQP9, and AQP11 mRNA expression were correlated with better OS in all gastric patients, whereas AQP0, AQP1, AQP4, AQP5, AQP6, AQP8, and AQP10 mRNA expression were associated with poor OS. With regard to the clinicopathological features including Laurens classification, clinical stage, human epidermal growth factor receptor 2 (HER2) status, and different treatment strategy, we could illustrate significant role of individual AQP mRNA expression in the prognosis of gastric cancer patients. Thus, our results indicated that AQP’s protein and mRNA expression in gastric cancer patients provide effective role to predict prognosis and act as an essential agent to therapeutic strategy. PMID:29678898

  5. Expression of SET Protein in the Ovaries of Patients with Polycystic Ovary Syndrome

    PubMed Central

    Boqun, Xu; Xiaonan, Dai; YuGui, Cui; Lingling, Gao; Xue, Dai; Gao, Chao; Feiyang, Diao; Jiayin, Liu; Gao, Li; Li, Mei; Zhang, Yuan; Ma, Xiang

    2013-01-01

    Background. We previously found that expression of SET gene was up-regulated in polycystic ovaries by using microarray. It suggested that SET may be an attractive candidate regulator involved in the pathophysiology of polycystic ovary syndrome (PCOS). In this study, expression and cellular localization of SET protein were investigated in human polycystic and normal ovaries. Method. Ovarian tissues, six normal ovaries and six polycystic ovaries, were collected during transsexual operation and surgical treatment with the signed consent form. The cellular localization of SET protein was observed by immunohistochemistry. The expression levels of SET protein were analyzed by Western Blot. Result. SET protein was expressed predominantly in the theca cells and oocytes of human ovarian follicles in both PCOS ovarian tissues and normal ovarian tissues. The level of SET protein expression in polycystic ovaries was triple higher than that in normal ovaries (P < 0.05). Conclusion. SET was overexpressed in polycystic ovaries more than that in normal ovaries. Combined with its localization in theca cells, SET may participate in regulating ovarian androgen biosynthesis and the pathophysiology of hyperandrogenism in PCOS. PMID:23861679

  6. Expression of SET Protein in the Ovaries of Patients with Polycystic Ovary Syndrome.

    PubMed

    Boqun, Xu; Xiaonan, Dai; Yugui, Cui; Lingling, Gao; Xue, Dai; Gao, Chao; Feiyang, Diao; Jiayin, Liu; Gao, Li; Li, Mei; Zhang, Yuan; Ma, Xiang

    2013-01-01

    Background. We previously found that expression of SET gene was up-regulated in polycystic ovaries by using microarray. It suggested that SET may be an attractive candidate regulator involved in the pathophysiology of polycystic ovary syndrome (PCOS). In this study, expression and cellular localization of SET protein were investigated in human polycystic and normal ovaries. Method. Ovarian tissues, six normal ovaries and six polycystic ovaries, were collected during transsexual operation and surgical treatment with the signed consent form. The cellular localization of SET protein was observed by immunohistochemistry. The expression levels of SET protein were analyzed by Western Blot. Result. SET protein was expressed predominantly in the theca cells and oocytes of human ovarian follicles in both PCOS ovarian tissues and normal ovarian tissues. The level of SET protein expression in polycystic ovaries was triple higher than that in normal ovaries (P < 0.05). Conclusion. SET was overexpressed in polycystic ovaries more than that in normal ovaries. Combined with its localization in theca cells, SET may participate in regulating ovarian androgen biosynthesis and the pathophysiology of hyperandrogenism in PCOS.

  7. Tuberous sclerosis: aberrant metabolism of ornithine, proline and glutamate in cultured fibroblasts.

    PubMed

    Tanaka, H; Nakazawa, K; Arima, M; Hayashi, A

    1987-01-01

    To investigate aberrant metabolism of proline (Pro) and its precursors in tuberous sclerosis (TS), 6, 7 and 5 strains of control, TS (normal skin) and TS (tumor) fibroblasts, respectively, were cultured in Eagle's MEM containing dialyzed fetal bovine serum with or without 0.1 mM ornithine (Orn). Ornithine aminotransferase (OAT) activity was decreased in TS, especially in TS (tumor) after mild sonication treatment. The yield of the OAT protein was inhibited in TS (tumor) when cultured without Orn. Free glutamate (Glu) in the medium was significantly increased in TS (tumor). Free proline (Pro) in cells was significantly decreased in TS (tumor) when cultured with Orn, but protein-bound Pro was not. The relative concentration of free Glu to glutamine (Gln) in the medium and that of free Glu to Pro in cells cultured with Orn were increased in TS (tumor). These results suggest that the requirement for Orn, increased turnover of Pro to Glu and increased elimination of Glu into the medium occur in TS (tumor). Aberrant regulation or turnover of Pro and Glu metabolism may occur in TS, especially in tumor cells.

  8. Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

    PubMed

    Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

    2012-01-01

    Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization. Copyright © 2011 S. Karger AG, Basel.

  9. Abiotic stresses modulate expression of major intrinsic proteins in barley (Hordeum vulgare).

    PubMed

    Ligaba, Ayalew; Katsuhara, Maki; Shibasaka, Mineo; Djira, Gemechis

    2011-02-01

    In one of the most important crops, barley (Hordeum vulgare L.), gene expression and physiological roles of most major intrinsic proteins (MIPs) remained to be elucidated. Here we studied expression of five tonoplast intrinsic protein isoforms (HvTIP1;2, HvTIP2;1, HvTIP2;2, HvTIP2;3 and HvTIP4;1), a NOD26-like intrinsic protein (HvNIP2;1) and a plasma membrane intrinsic protein (HvPIP2;1) by using the quantitative real-time RT-PCR. Five-day-old seedlings were exposed to abiotic stresses (salt, heavy metals and nutrient deficiency), abscisic acid (ABA) and gibberellic acid (GA) for 24 h. Treatment with 100 mM NaCl, 0.1 mM ABA and 1 mM GA differentially regulated gene expression in roots and shoots. Nitrogen and prolonged P-deficiency downregulated expression of most MIP genes in roots. Intriguingly, gene expression was restored to the values in the control three days after nutrient supply was resumed. Heavy metals (0.2 mM each of Cd, Cu, Zn and Cr) downregulated the transcript levels by 60-80% in roots, whereas 0.2 mM Hg upregulated expressions of most genes in roots. This was accompanied by a 45% decrease in the rate of transpiration. In order to study the physiological role of the MIPs, cDNA of three genes (HvTIP2;1, HvTIP2;3 and HvNIP2;1) have been cloned and heterologous expression was performed in Xenopus laevis oocytes. Osmotic water permeability was determined by a swelling assay. However, no water uptake activity was observed for the three proteins. Hence, the possible physiological role of the proteins is discussed. Copyright © 2010 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  10. Tunable Control of an Escherichia coli Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant lac Repressor.

    PubMed

    Kim, Seong Keun; Lee, Dae-Hee; Kim, Oh Cheol; Kim, Jihyun F; Yoon, Sung Ho

    2017-09-15

    Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.

  11. Clinicopathologic significance of minichromosome maintenance protein 2 and Tat-interacting protein 30 expression in benign and malignant lesions of the gallbladder.

    PubMed

    Liu, Dong-cai; Yang, Zhu-lin

    2011-11-01

    Gallbladder cancers are aggressive tumors with a poor prognosis and high mortality rate. To find specific biological markers for early diagnosis and prognosis and to develop possible alternative treatment strategies, we examined minichromosome maintenance protein 2 (MCM2) and Tat-interacting protein 30 (TIP30) expression in 108 gallbladder adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues using immunohistochemistry. Expression of MCM2 was significantly higher in adenocarcinomas than in peritumoral tissues (χ² = 8.41; P < .01), adenomatous polyps (χ² = 6.81; P < .01), and chronic cholecystitis (χ² = 21.00; P < .01). In contrast, Tat-interacting protein 30 expression was significantly less in adenocarcinomas than in peritumoral tissues (χ² = 13.26; P < .01), adenomatous polyps (χ² = 4.76; P < .05), and chronic cholecystitis (χ² = 18.93; P < .01). The benign lesions in gallbladder epithelium with positive MCM2 or negative Tat-interacting protein 30 expression showed moderate to severe atypical hyperplasia. Expression of MCM2 and absence of Tat-interacting protein 30 were significantly associated with poor differentiation, large tumor mass, lymph node metastasis, and invasion of adenocarcinoma. Univariate Kaplan-Meier analysis showed that either elevated MCM2 (P = .006) or lowered Tat-interacting protein 30 (P = .006) expression was closely associated with shorter overall survival. Multivariate Cox regression analysis revealed that expression of MCM2 (P = .007) or nonexpression of Tat-interacting protein 30 (P = .009) was an independent predictor of a poor prognosis in adenocarcinoma. Our results suggest that overexpression of MCM2 or loss of expression of Tat-interacting protein 30 is closely related to carcinogenesis, progression, biological behavior, and prognosis of gallbladder adenocarcinoma. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Aberrant excitatory rewiring of layer V pyramidal neurons early after neocortical trauma.

    PubMed

    Takahashi, D Koji; Gu, Feng; Parada, Isabel; Vyas, Shri; Prince, David A

    2016-07-01

    Lesioned neuronal circuits form new functional connections after a traumatic brain injury (TBI). In humans and animal models, aberrant excitatory connections that form after TBI may contribute to the pathogenesis of post-traumatic epilepsy. Partial neocortical isolation ("undercut" or "UC") leads to altered neuronal circuitry and network hyperexcitability recorded in vivo and in brain slices from chronically lesioned neocortex. Recent data suggest a critical period for maladaptive excitatory circuit formation within the first 3days post UC injury (Graber and Prince 1999, 2004; Li et al. 2011, 2012b). The present study focuses on alterations in excitatory connectivity within this critical period. Immunoreactivity (IR) for growth-associated protein (GAP)-43 was increased in the UC cortex 3days after injury. Some GAP-43-expressing excitatory terminals targeted the somata of layer V pyramidal (Pyr) neurons, a domain usually innervated predominantly by inhibitory terminals. Immunocytochemical analysis of pre- and postsynaptic markers showed that putative excitatory synapses were present on somata of these neurons in UC neocortex. Excitatory postsynaptic currents from UC layer V Pyr cells displayed properties consistent with perisomatic inputs and also reflected an increase in the number of synaptic contacts. Laser scanning photostimulation (LSPS) experiments demonstrated reorganized excitatory connectivity after injury within the UC. Concurrent with these changes, spontaneous epileptiform bursts developed in UC slices. Results suggest that aberrant reorganization of excitatory connectivity contributes to early neocortical hyperexcitability in this model. The findings are relevant for understanding the pathophysiology of neocortical post-traumatic epileptogenesis and are important in terms of the timing of potential prophylactic treatments. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Changes in protein expression during honey bee larval development.

    PubMed

    Chan, Queenie W T; Foster, Leonard J

    2008-10-29

    The honey bee (Apis mellifera), besides its role in pollination and honey production, serves as a model for studying the biochemistry of development, metabolism, and immunity in a social organism. Here we use mass spectrometry-based quantitative proteomics to quantify nearly 800 proteins during the 5- to 6-day larval developmental stage, tracking their expression profiles. We report that honey bee larval growth is marked by an age-correlated increase of protein transporters and receptors, as well as protein nutrient stores, while opposite trends in protein translation activity and turnover were observed. Levels of the immunity factors prophenoloxidase and apismin are positively correlated with development, while others surprisingly were not significantly age-regulated, suggesting a molecular explanation for why bees are susceptible to major age-associated bee bacterial infections such as American Foulbrood or fungal diseases such as chalkbrood. Previously unreported findings include the reduction of antioxidant and G proteins in aging larvae. These data have allowed us to integrate disparate findings in previous studies to build a model of metabolism and maturity of the immune system during larval development. This publicly accessible resource for protein expression trends will help generate new hypotheses in the increasingly important field of honey bee research.

  14. Aberration corrected STEM by means of diffraction gratings

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Linck, Martin; Ercius, Peter A.; Pierce, Jordan S.

    In the past 15 years, the advent of aberration correction technology in electron microscopy has enabled materials analysis on the atomic scale. This is made possible by precise arrangements of multipole electrodes and magnetic solenoids to compensate the aberrations inherent to any focusing element of an electron microscope. In this paper, we describe an alternative method to correct for the spherical aberration of the objective lens in scanning transmission electron microscopy (STEM) using a passive, nanofabricated diffractive optical element. This holographic device is installed in the probe forming aperture of a conventional electron microscope and can be designed to removemore » arbitrarily complex aberrations from the electron's wave front. In this work, we show a proof-of-principle experiment that demonstrates successful correction of the spherical aberration in STEM by means of such a grating corrector (GCOR). Our GCOR enables us to record aberration-corrected high-resolution high-angle annular dark field (HAADF-) STEM images, although yet without advancement in probe current and resolution. Finally, improvements in this technology could provide an economical solution for aberration-corrected high-resolution STEM in certain use scenarios.« less

  15. Aberration corrected STEM by means of diffraction gratings

    DOE PAGES

    Linck, Martin; Ercius, Peter A.; Pierce, Jordan S.; ...

    2017-06-12

    In the past 15 years, the advent of aberration correction technology in electron microscopy has enabled materials analysis on the atomic scale. This is made possible by precise arrangements of multipole electrodes and magnetic solenoids to compensate the aberrations inherent to any focusing element of an electron microscope. In this paper, we describe an alternative method to correct for the spherical aberration of the objective lens in scanning transmission electron microscopy (STEM) using a passive, nanofabricated diffractive optical element. This holographic device is installed in the probe forming aperture of a conventional electron microscope and can be designed to removemore » arbitrarily complex aberrations from the electron's wave front. In this work, we show a proof-of-principle experiment that demonstrates successful correction of the spherical aberration in STEM by means of such a grating corrector (GCOR). Our GCOR enables us to record aberration-corrected high-resolution high-angle annular dark field (HAADF-) STEM images, although yet without advancement in probe current and resolution. Finally, improvements in this technology could provide an economical solution for aberration-corrected high-resolution STEM in certain use scenarios.« less

  16. A phosphatase-independent gain-of-function mutation in PTEN triggers aberrant cell growth in astrocytes through an autocrine IGF-1 loop.

    PubMed

    Fernández, S; Genis, L; Torres-Alemán, I

    2014-08-07

    Loss-of-function mutations in the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome10) contribute to aberrant cell growth in part through upregulation of the mitogenic IGF-1/PI3K/Akt pathway. In turn, this pathway exerts a homeostatic feedback over PTEN. Using mutagenesis analysis to explore a possible impact of this mutual control on astrocyte growth, we found that truncation of the C-terminal region of PTEN (Δ51) associates with a marked increase in NFκB activity, a transcription factor overactivated in astrocyte tumors. Whereas mutations of PTEN are considered to lead to a loss-of-function, PTENΔ51, a truncation that comprises a region frequently mutated in human gliomas, displayed a neomorphic (gain-of-function) activity that was independent of its phosphatase activity. This gain-of-function of PTENΔ51 includes stimulation of IGF-1 synthesis through protein kinase A activation of the IGF-1 promoter. Increased IGF-1 originates an autocrine loop that activates Akt and NFκB. Constitutive activation of NFκB in PTENΔ51-expressing astrocytes leads to aberrant cell growth; astrocytes expressing this mutant PTEN generate colonies in vitro and tumors in vivo. Mutations converting a tumor suppressor such as PTEN into a tumor promoter through a gain-of-function involving IGF-1 production may further our understanding of the role played by this growth factor in glioma growth and help us define druggable targets for personalized therapy.

  17. Of mothers and myelin: Aberrant myelination phenotypes in mouse model of Angelman syndrome are dependent on maternal and dietary influences.

    PubMed

    Grier, Mark D; Carson, Robert P; Lagrange, Andre H

    2015-09-15

    Angelman syndrome (AS) is a neurodevelopmental disorder characterized by a number of neurological problems, including developmental delay, movement disorders, and epilepsy. AS results from the loss of UBE3A (an imprinted gene) expressed from the maternal chromosome in neurons. Given the ubiquitous expression of Ube3a and the devastating nature of AS, the role of environmental and maternal effects has been largely ignored. Severe ataxia, anxiety-like behaviors and learning deficits are well-documented in patients and AS mice. More recently, clinical imaging studies of AS patients suggest myelination may be delayed or reduced. Utilizing a mouse model of AS, we found disrupted expression of cortical myelin proteins, the magnitude of which is influenced by maternal status, in that the aberrant myelination in the AS pups of AS affected mothers were more pronounced than those seen in AS pups raised by unaffected (Ube3a (m+/p-)) Carrier mothers. Furthermore, feeding the breeding mothers a higher fat (11% vs 5%) diet normalizes these myelin defects. These effects are not limited to myelin proteins. Since AS mice have abnormal stress responses, including altered glucocorticoid receptor (GR) expression, we measured GR expression in pups from Carrier and affected AS mothers. AS pups had higher GR expression than their WT littermates. However, we also found an effect of maternal status, with reduced GR levels in pups from affected mothers compared to genotypically identical pups raised by unaffected Carrier mothers. Taken together, our findings suggest that the phenotypes observed in AS mice may be modulated by factors independent of Ube3a genotype. Published by Elsevier B.V.

  18. Aberration correction for charged particle lithography

    NASA Astrophysics Data System (ADS)

    Munro, Eric; Zhu, Xieqing; Rouse, John A.; Liu, Haoning

    2001-12-01

    At present, the throughput of projection-type charge particle lithography systems, such as PREVAIL and SCALPEL, is limited primarily by the combined effects of field curvature in the projection lenses and Coulomb interaction in the particle beam. These are fundamental physical limitations, inherent in charged particle optics, so there seems little scope for significantly improving the design of such systems, using conventional rotationally symmetric electron lenses. This paper explores the possibility of overcoming the field aberrations of round electron lense, by using a novel aberration corrector, proposed by Professor H. Rose of University of Darmstadt, called a hexapole planator. In this scheme, a set of round lenses is first used to simultaneously correct distortion and coma. The hexapole planator is then used to correct the field curvature and astigmatism, and to create a negative spherical aberration. The size of the transfer lenses around the planator can then be adjusted to zero the residual spherical aberration. In a way, an electron optical projection system is obtained that is free of all primary geometrical aberrations. In this paper, the feasibility of this concept has been studied with a computer simulation. The simulations verify that this scheme can indeed work, for both electrostatic and magnetic projection systems. Two design studies have been carried out. The first is for an electrostatic system that could be used for ion beam lithography, and the second is for a magnetic projection system for electron beam lithography. In both cases, designs have been achieved in which all primary third-order geometrical aberrations are totally eliminated.

  19. Aberration Compensation in Aplanatic Solid Immersion Lens Microscopy

    DTIC Science & Technology

    2013-11-08

    model and ray tracing software ( Zemax ) to understand how much aberrations are in the system and how much can be compensated by the DM. Subsequently...aberration. Table 2 shows the Zemax simulation on this particular case. With aberration compensation, the finest resolvable group is at 252 nm

  20. Glucose transporter 3 (GLUT3) protein expression in human placenta across gestation

    PubMed Central

    Brown, Kelecia; Heller, Debra S.; Zamudio, Stacy; Illsley, Nicholas P.

    2012-01-01

    Conflicting information regarding expression of GLUT3 protein in the human placenta has been reported and the localization and pattern of expression of GLUT3 protein across gestation has not been clearly defined. The objective of this study was characterization of syncytial GLUT3 protein expression across gestation. We hypothesized that GLUT3 protein is present in the syncytial microvillous membrane and that its expression decreases over gestation. GLUT3 protein was measured in samples from a range of gestational ages (first to third trimester), with human brain and human bowel used as a positive and negative control respectively. As an additional measure of specificity, we transfected BeWo choriocarcinoma cells, a trophoblast cell line expressing GLUT3, with siRNA directed against GLUT3 and analyzed expression by Western blotting. GLUT3 was detected in the syncytiotrophoblast at all gestational ages by immunohistochemistry. Using Western blotting GLUT3 was detected as an integral membrane protein at a molecular weight of ~50kDa in microvillous membranes from all trimesters but not in syncytial basal membranes. The identity of the primary antibody target was confirmed by demonstrating that expression of the immunoblotting signal in GLUT3 siRNA-treated BeWo was decreased to 18 ± 6% (mean ± SEM) of that seen in cells transfected with a non-targeting siRNA. GLUT3 expression in microvillous membranes detected by Western blot decreased through the trimesters such that expression in the second trimester (wks 14–26) was 48 ± 7% of that in the first trimester and by the third trimester (wks 31–40) only 34 ± 10% of first trimester expression. In addition, glucose uptake into BeWo cells treated with GLUT3 siRNA was reduced to 60% of that measured in cells treated with the non-targeting siRNA. This suggests that GLUT3-mediated uptake comprises approximately 50% of glucose uptake into BeWo cells. These results confirm the hypothesis that GLUT3 is present in the

  1. Expression Atlas: gene and protein expression across multiple studies and organisms

    PubMed Central

    Tang, Y Amy; Bazant, Wojciech; Burke, Melissa; Fuentes, Alfonso Muñoz-Pomer; George, Nancy; Koskinen, Satu; Mohammed, Suhaib; Geniza, Matthew; Preece, Justin; Jarnuczak, Andrew F; Huber, Wolfgang; Stegle, Oliver; Brazma, Alvis; Petryszak, Robert

    2018-01-01

    Abstract Expression Atlas (http://www.ebi.ac.uk/gxa) is an added value database that provides information about gene and protein expression in different species and contexts, such as tissue, developmental stage, disease or cell type. The available public and controlled access data sets from different sources are curated and re-analysed using standardized, open source pipelines and made available for queries, download and visualization. As of August 2017, Expression Atlas holds data from 3,126 studies across 33 different species, including 731 from plants. Data from large-scale RNA sequencing studies including Blueprint, PCAWG, ENCODE, GTEx and HipSci can be visualized next to each other. In Expression Atlas, users can query genes or gene-sets of interest and explore their expression across or within species, tissues, developmental stages in a constitutive or differential context, representing the effects of diseases, conditions or experimental interventions. All processed data matrices are available for direct download in tab-delimited format or as R-data. In addition to the web interface, data sets can now be searched and downloaded through the Expression Atlas R package. Novel features and visualizations include the on-the-fly analysis of gene set overlaps and the option to view gene co-expression in experiments investigating constitutive gene expression across tissues or other conditions. PMID:29165655

  2. Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

    PubMed Central

    Culver, J N; Lehto, K; Close, S M; Hilf, M E; Dawson, W O

    1993-01-01

    Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes. Images Fig. 2 Fig. 3 Fig. 4 PMID:8446627

  3. The expression and function of epithelial membrane protein 1 in laryngeal carcinoma.

    PubMed

    Li, Hong; Zhang, Xiaowen; Jiang, Xuejun; Ji, Xu

    2017-01-01

    In this study, we compared the expression of epithelial membrane protein 1 (EMP1) on the steady-state mRNA level (by quantitative real-time PCR) and on the protein level (by western immunoblot and immunohistochemistry) in 51 pairs of laryngeal carcinoma tissues and matched cancer-free peritumor tissues, and we analyzed the correlation between EMP1 expression and different clinicopathological factors. Furthermore, we ectopically expressed EMP1 in human laryngeal carcinoma Hep-2 cells and examined the effects on cell viability, apoptosis, colonogenicity, and motility, by MTT assay, flow cytometry, colony formation assay and Transwell migration assay, respectively. EMP1 expression (on both the mRNA and protein levels) was significantly lower in the cancer tissues than in matched peritumor tissues (P<0.05). In laryngeal cancers, the level of EMP1 protein was correlated with histological grade (P<0.05), but not with age, gender, clinical stage, cancer subtype or lymph node metastasis (P>0.05). Functionally, ectopic expression of EMP1 in Hep-2 cells significantly reduced cell viability, colony formation, and migration, but enhanced apoptosis. Therefore, EMP1 is a tumor suppressor in laryngeal carcinoma. Boosting EMP1 expression in laryngeal carcinoma initiates multiple anticancer phenotypes and thus presents a promising therapeutic strategy for laryngeal cancer.

  4. A protein and mRNA expression-based classification of gastric cancer.

    PubMed

    Setia, Namrata; Agoston, Agoston T; Han, Hye S; Mullen, John T; Duda, Dan G; Clark, Jeffrey W; Deshpande, Vikram; Mino-Kenudson, Mari; Srivastava, Amitabh; Lennerz, Jochen K; Hong, Theodore S; Kwak, Eunice L; Lauwers, Gregory Y

    2016-07-01

    The overall survival of gastric carcinoma patients remains poor despite improved control over known risk factors and surveillance. This highlights the need for new classifications, driven towards identification of potential therapeutic targets. Using sophisticated molecular technologies and analysis, three groups recently provided genetic and epigenetic molecular classifications of gastric cancer (The Cancer Genome Atlas, 'Singapore-Duke' study, and Asian Cancer Research Group). Suggested by these classifications, here, we examined the expression of 14 biomarkers in a cohort of 146 gastric adenocarcinomas and performed unsupervised hierarchical clustering analysis using less expensive and widely available immunohistochemistry and in situ hybridization. Ultimately, we identified five groups of gastric cancers based on Epstein-Barr virus (EBV) positivity, microsatellite instability, aberrant E-cadherin, and p53 expression; the remaining cases constituted a group characterized by normal p53 expression. In addition, the five categories correspond to the reported molecular subgroups by virtue of clinicopathologic features. Furthermore, evaluation between these clusters and survival using the Cox proportional hazards model showed a trend for superior survival in the EBV and microsatellite-instable related adenocarcinomas. In conclusion, we offer as a proposal a simplified algorithm that is able to reproduce the recently proposed molecular subgroups of gastric adenocarcinoma, using immunohistochemical and in situ hybridization techniques.

  5. Structural centrosome aberrations favor proliferation by abrogating microtubule-dependent tissue integrity of breast epithelial mammospheres

    PubMed Central

    Schnerch, D; Nigg, E A

    2016-01-01

    Structural centrosome aberrations are frequently observed in early stage carcinomas, but their role in malignant transformation is poorly understood. Here, we examined the impact of overexpression of Ninein-like protein (Nlp) on the architecture of polarized epithelia in three-dimensional mammospheres. When Nlp was overexpressed to levels resembling those seen in human tumors, it formed striking centrosome-related bodies (CRBs), which sequestered Ninein and affected the kinetics of microtubule (MT) nucleation and release. In turn, the profound reorganization of the MT cytoskeleton resulted in mislocalization of several adhesion and junction proteins as well as the tumor suppressor Scribble, resulting in the disruption of epithelial polarity, cell-cell interactions and mammosphere architecture. Remarkably, cells harboring Nlp-CRBs displayed an enhanced proliferative response to epidermal growth factor. These results demonstrate that structural centrosome aberrations cause not only the disruption of epithelial polarity but also favor overproliferation, two phenotypes typically associated with human carcinomas. PMID:26364601

  6. Recombinant G protein-coupled receptor expression in Saccharomyces cerevisiae for protein characterization.

    PubMed

    Blocker, Kory M; Britton, Zachary T; Naranjo, Andrea N; McNeely, Patrick M; Young, Carissa L; Robinson, Anne S

    2015-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that mediate signaling across the cellular membrane and facilitate cellular responses to external stimuli. Due to the critical role that GPCRs play in signal transduction, therapeutics have been developed to influence GPCR function without an extensive understanding of the receptors themselves. Closing this knowledge gap is of paramount importance to improving therapeutic efficacy and specificity, where efforts to achieve this end have focused chiefly on improving our knowledge of the structure-function relationship. The purpose of this chapter is to review methods for the heterologous expression of GPCRs in Saccharomyces cerevisiae, including whole-cell assays that enable quantitation of expression, localization, and function in vivo. In addition, we describe methods for the micellular solubilization of the human adenosine A2a receptor and for reconstitution of the receptor in liposomes that have enabled its biophysical characterization. © 2015 Elsevier Inc. All rights reserved.

  7. An XPA gene splicing mutation resulting in trace protein expression in an elderly patient with xeroderma pigmentosum group A without neurological abnormalities.

    PubMed

    Takahashi, Y; Endo, Y; Kusaka-Kikushima, A; Nakamaura, S; Nakazawa, Y; Ogi, T; Uryu, M; Tsuji, G; Furue, M; Moriwaki, S

    2017-07-01

    A certain relationship between XPA gene mutations and the severity of symptoms has been observed in patients with xeroderma pigmentosum group A (XP-A). Patients with mutations within the DNA-binding domain usually exhibit severe symptoms, whereas splicing mutations in the same domain sometimes cause very mild symptoms. This inconsistency can be explained by a small amount of functional XPA protein produced from normally spliced transcripts. We herein report the case of an adult Japanese patient with XP-A with unusually mild symptoms. We identified a homozygous c.529G>A mutation in exon 4 of the XPA gene, which resulted in aberrant splicing with a 29-bp deletion in exon 4 causing a frameshift. Intact mRNA was observable, but a Western blot analysis failed to detect any normal XPA protein. We therefore evaluated the DNA repair capacity in normal cells in which the XPA expression was artificially diminished. The repair capacity was still present in cells with trace levels of the XPA protein. The repair capacity of the cells derived from our patient with mild symptoms was poor by comparison, but still significant compared with that of the cells derived from a patient with XP-A with severe symptoms. These results provide strong evidence that a trace level of XPA protein can still exert a relatively strong repair capacity, resulting in only a mild phenotype. © 2016 British Association of Dermatologists.

  8. Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

    PubMed

    Merchant, Mark; Kinney, Clint; Sanders, Paige

    2009-12-01

    Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

  9. Dexamethasone Regulates Cochlear Expression of Deafness-associated Proteins Myelin Protein Zero and Heat Shock Protein 70, as Revealed by iTRAQ Proteomics.

    PubMed

    Maeda, Yukihide; Fukushima, Kunihiro; Kariya, Shin; Orita, Yorihisa; Nishizaki, Kazunori

    2015-08-01

    Using proteomics, we aimed to identify the proteins differentially regulated by dexamethasone in the mouse cochlea based on mass-spectrometry data. Glucocorticoid therapy is widely used for many forms of sensorineural hearing loss; however, the molecular mechanism of its action in the cochlea remains poorly understood. Dexamethasone or control saline was intratympanically applied to the cochleae of mice. Twelve hours after application, proteins differentially regulated by dexamethasone in the cochlea were analyzed by isobaric Tag for Relative and Absolute Quantitation (iTRAQ)-mass spectrometry. Next, dexamethasone-dependent regulation of these proteins was verified in the cochleae of mice with noise-induced hearing loss (NIHL) and systemic administration of dexamethasone by western blotting. Immunolocalizations of these proteins were examined in cochleae with NIHL. A total of 247 proteins with a greater than 95% confidence interval of protein identification were found, and 11 differentially expressed proteins by dexamethasone were identified by the iTRAQ-mass spectrometry. One protein, myelin protein zero (Mpz), was upregulated (1.870 ± 0.201-fold change, p < 0.01) at 6 hours post-systemic dexamethasone and noise exposure in a mouse model of NIHL. Heat shock protein 70 (Hsp70) was downregulated (0.511 ± 0.274-fold change, p < 0.05) at 12 hours post-systemic dexamethasone. Immunohistochemistry confirmed Mpz localization to the efferent and afferent processes of the spiral neurons, whereas Hsp70 showed a more ubiquitous expression pattern in the cochlea. Both Mpz and Hsp70 have been reported to be closely associated with sensorineural hearing loss in humans. Dexamethasone significantly modulated the expression levels of these proteins in the cochleae of mice.

  10. Genomics Analysis of Genes Expressed in Maize Endosperm Identifies Novel Seed Proteins and Clarifies Patterns of Zein Gene Expression

    PubMed Central

    Woo, Young-Min; Hu, David Wang-Nan; Larkins, Brian A.; Jung, Rudolf

    2001-01-01

    We analyzed cDNA libraries from developing endosperm of the B73 maize inbred line to evaluate the expression of storage protein genes. This study showed that zeins are by far the most highly expressed genes in the endosperm, but we found an inverse relationship between the number of zein genes and the relative amount of specific mRNAs. Although α-zeins are encoded by large multigene families, only a few of these genes are transcribed at high or detectable levels. In contrast, relatively small gene families encode the γ- and δ-zeins, and members of these gene families, especially the γ-zeins, are highly expressed. Knowledge of expressed storage protein genes allowed the development of DNA and antibody probes that distinguish between closely related gene family members. Using in situ hybridization, we found differences in the temporal and spatial expression of the α-, γ-, and δ-zein gene families, which provides evidence that γ-zeins are synthesized throughout the endosperm before α- and δ-zeins. This observation is consistent with earlier studies that suggested that γ-zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD γ-zein, an 18-kD α-globulin, and a legumin-related protein. Immunolocalization of the 50-kD γ-zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other γ-zeins. The 18-kD α-globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm. PMID:11595803

  11. Differential expression of genes and proteins associated with wool follicle cycling.

    PubMed

    Liu, Nan; Li, Hegang; Liu, Kaidong; Yu, Juanjuan; Cheng, Ming; De, Wei; Liu, Jifeng; Shi, Shuyan; He, Yanghua; Zhao, Jinshan

    2014-08-01

    Sheep are valuable resources for the wool industry. Wool growth of Aohan fine wool sheep has cycled during different seasons in 1 year. Therefore, identifying genes that control wool growth cycling might lead to ways for improving the quality and yield of fine wool. In this study, we employed Agilent sheep gene expression microarray and proteomic technology to compare the gene expression patterns of the body side skins at August and December time points in Aohan fine wool sheep (a Chinese indigenous breed). Microarray study revealed that 2,223 transcripts were differentially expressed, including 1,162 up-regulated and 1,061 down-regulated transcripts, comparing body side skin at the August time point to the December one (A/D) in Aohan fine wool sheep. Then seven differentially expressed genes were selected to validated the reliability of the gene chip data. The majority of the genes possibly related to follicle development and wool growth could be assigned into the categories including regulation of receptor binding, extracellular region, protein binding and extracellular space. Proteomic study revealed that 84 protein spots showed significant differences in expression levels. Of the 84, 63 protein spots were upregulated and 21 were downregulated in A/D. Finally, 55 protein points were determined through MALDI-TOF/MS analyses. Furthermore, the regulation mechanism of hair follicle might resemble that of fetation.

  12. Secreted Proteins Defy the Expression Level–Evolutionary Rate Anticorrelation

    PubMed Central

    Feyertag, Felix; Berninsone, Patricia M.; Alvarez-Ponce, David

    2017-01-01

    The rates of evolution of the proteins of any organism vary across orders of magnitude. A primary factor influencing rates of protein evolution is expression. A strong negative correlation between expression levels and evolutionary rates (the so-called E–R anticorrelation) has been observed in virtually all studied organisms. This effect is currently attributed to the abundance-dependent fitness costs of misfolding and unspecific protein–protein interactions, among other factors. Secreted proteins are folded in the endoplasmic reticulum, a compartment where chaperones, folding catalysts, and stringent quality control mechanisms promote their correct folding and may reduce the fitness costs of misfolding. In addition, confinement of secreted proteins to the extracellular space may reduce misinteractions and their deleterious effects. We hypothesize that each of these factors (the secretory pathway quality control and extracellular location) may reduce the strength of the E–R anticorrelation. Indeed, here we show that among human proteins that are secreted to the extracellular space, rates of evolution do not correlate with protein abundances. This trend is robust to controlling for several potentially confounding factors and is also observed when analyzing protein abundance data for 6 human tissues. In addition, analysis of mRNA abundance data for 32 human tissues shows that the E–R correlation is always less negative, and sometimes nonsignificant, in secreted proteins. Similar observations were made in Caenorhabditis elegans and in Escherichia coli, and to a lesser extent in Drosophila melanogaster, Saccharomyces cerevisiae and Arabidopsis thaliana. Our observations contribute to understand the causes of the E–R anticorrelation. PMID:28007979

  13. Recombinant Protein Expression in Escherichia coli (E.coli): What We Need to Know.

    PubMed

    Hayat, Seyed Mohammad Gheibi; Farahani, Najmeh; Golichenari, Behrouz; Sahebkar, Amir Hosein

    2018-01-31

    Host, vector, and culture conditions (including cultivation media) are considered among the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the most common hosts to produce recombinant therapeutic proteins is Escherichia coli. A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli. Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along with current pros and cons of different types of these factors leading to success of recombinant protein expression in Escherichia coli were discussed. Successful protein expression in Escherichia coli necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as well as factors related to media including time, temperature, and inducer. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. High SRPX2 protein expression predicts unfavorable clinical outcome in patients with prostate cancer

    PubMed Central

    Zhang, Meng; Li, Xiaoli; Fan, Zhirui; Zhao, Jing; Liu, Shuzheng; Zhang, Mingzhi; Li, Huixiang; Goscinski, Mariusz Adam; Fan, Huijie; Suo, Zhenhe

    2018-01-01

    Background Sushi repeat-containing protein X-linked 2 (SRPX2) is overexpressed in a variety of different tumor tissues and correlated with poor prognosis in patients. Little research focuses on the role of SRPX2 expression in prostate cancer (PCa), and the clinicopathological significance of the protein expression in this tumor is relatively unknown. However, our previous transcriptome data from those cancer stem-like cells indicated the role of SRPX2 in PCa. Materials and methods In this study, RT-PCR and Western blotting were firstly used to examine the SRPX2 expression in three PCa cell lines including LNCaP, DU145, and PC3, and then SRPX2 protein expression was immunohistochemically investigated and statistically analyzed in a series of 106 paraffin-embedded PCa tissue specimens. Results Significantly lower levels of SRPX2 expression were verified in the LNCaP cells, compared with the expression in the aggressive DU145 and PC3 cells, in both mRNA and protein levels. Immunohistochemically, there were variable SRPX2 protein expressions in the clinical samples. Moreover, high levels of SRPX2 expression in the PCa tissues were significantly associated with Gleason score (P=0.008), lymph node metastasis (P=0.009), and distant metastasis (P=0.021). Furthermore, higher levels of SRPX2 expression in the PCa tissues were significantly associated with shorter overall survival (OS) (P<0.001). Conclusion Our results demonstrate that SRPX2 is highly expressed in aggressive PCa cells in vitro, and its protein expression in PCa is significantly associated with malignant clinical features and shorter OS, strongly indicating its prognostic value in prostate cancers. PMID:29881288

  15. Double promoter expression systems for recombinant protein production by industrial microorganisms.

    PubMed

    Öztürk, Sibel; Ergün, Burcu Gündüz; Çalık, Pınar

    2017-10-01

    Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain. While bacterial double promoter systems have been constructed as chimeric tandem promoters, yeast dual promoter systems have been developed as separate expression cassettes. To increase production and productivity, the optimal transcriptional activity should be justified either by simultaneously satisfying the requirements of both promoters, or by consecutively stimulating the changeover from one to another in a biphasic process or via successive-iterations. Thus, considering the dynamics of a fermentation process, double promoters can be classified according to their operational mechanisms, as: i) consecutively operating double promoter systems, and ii) simultaneously operating double promoter systems. Among these metabolic design strategies, extending the expression period with two promoters activated under different conditions, or enhancing the transcriptional activity with two promoters activated under similar conditions within the production domain, can be applied independently from the host. Novel studies with new insights, which aim a rational systematic design and construction of dual promoter expression vectors with tailored transcriptional activity, will empower r-protein production with enhanced production and productivity. Finally, the current state-of-the-art review emphasizes the advantages of double promoter systems along with the necessity for discovering new promoters for the development of more

  16. Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

    PubMed

    Gupta, Sanjeev K; Shukla, Pratyoosh

    2016-12-01

    Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.

  17. A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

    NASA Astrophysics Data System (ADS)

    Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

    2000-05-01

    RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

  18. Apical Plasma Membrane Mispolarization of NaK-ATPase in Polycystic Kidney Disease Epithelia Is Associated with Aberrant Expression of the β2 Isoform

    PubMed Central

    Wilson, Patricia D.; Devuyst, Olivier; Li, Xiaohong; Gatti, Laura; Falkenstein, Doris; Robinson, Shawn; Fambrough, Douglas; Burrow, Christopher R.

    2000-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease of the kidney, characterized by cystic enlargement of renal tubules, aberrant epithelial proliferation, and ion and fluid secretion into the lumen. Previous studies have shown abnormalities in polarization of membrane proteins, including mislocalization of the NaK-ATPase to the apical plasma membranes of cystic epithelia. Apically located NaK-ATPase has previously been shown to be fully functional in vivo and in membrane-grown ADPKD epithelial cells in vitro, where basal-to-apical 22Na transport was inhibited by application of ouabain to the apical membrane compartment. Studies were conducted with polymerase chain reaction-generated specific riboprobes and polyclonal peptide antibodies against human sequences of α1, α3, β1, and β2 subunits of NaK-ATPase. High levels of expression of α1 and β1 messenger RNA were detected in ADPKD and age-matched normal adult kidneys in vivo, whereas β2 messenger RNA was detected only in ADPKD kidneys. Western blot analysis and immunocytochemical studies showed that, in normal adult kidneys, peptide subunit-specific antibodies against α1 and β1 localized to the basolateral membranes of normal renal tubules, predominantly thick ascending limbs of Henle’s loop. In ADPKD kidneys, α1 and β2 subunits were localized to the apical epithelial cell membranes, whereas β1 was distributed throughout the cytoplasm and predominantly in the endoplasmic reticulum, but was not seen associated with cystic epithelial cell membranes or in cell membrane fractions. Polarizing, renal-derived epithelial Madin Darby canine kidney cells, stably expressing normal or N-terminally truncated chicken β1 subunits, showed selective accumulation in the basolateral Madin Darby canine kidney cell surface, whereas c-myc epitope-tagged chicken β2 or human β2 subunits accumulated selectively in the apical cell surface. Similarly, human ADPKD epithelial cell lines, which

  19. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  20. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  1. Rooting Out Aberrant Behavior in Training.

    ERIC Educational Resources Information Center

    Kokalis, Jerry, Jr.; Paquin, Dave

    1989-01-01

    Discusses aberrant, or disruptive, behavior in an industrial/business, classroom-based, instructor-led training setting. Three examples of aberrant behavior are described, typical case studies are provided for each, and preventive (long-term) and corrective (on-the-spot) strategies for dealing with the problems are discussed. (LRW)

  2. Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity

    PubMed Central

    Pérez-Pérez, Rafael; López, Juan A.; García-Santos, Eva; Camafeita, Emilio; Gómez-Serrano, María; Ortega-Delgado, Francisco J.; Ricart, Wifredo; Fernández-Real, José M.; Peral, Belén

    2012-01-01

    Background Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals. Methodology and Findings To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples. Conclusions ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity

  3. Automation of large scale transient protein expression in mammalian cells

    PubMed Central

    Zhao, Yuguang; Bishop, Benjamin; Clay, Jordan E.; Lu, Weixian; Jones, Margaret; Daenke, Susan; Siebold, Christian; Stuart, David I.; Yvonne Jones, E.; Radu Aricescu, A.

    2011-01-01

    Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI− cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative. PMID:21571074

  4. Human eyes do not need monochromatic aberrations for dynamic accommodation.

    PubMed

    Bernal-Molina, Paula; Marín-Franch, Iván; Del Águila-Carrasco, Antonio J; Esteve-Taboada, Jose J; López-Gil, Norberto; Kruger, Philip B; Montés-Micó, Robert

    2017-09-01

    To determine if human accommodation uses the eye's own monochromatic aberrations to track dynamic accommodative stimuli. Wavefront aberrations were measured while subjects monocularly viewed a monochromatic Maltese cross moving sinusoidally around 2D of accommodative demand with 1D amplitude at 0.2 Hz. The amplitude and phase (delay) of the accommodation response were compared to the actual vergence of the stimulus to obtain gain and temporal phase, calculated from wavefront aberrations recorded over time during experimental trials. The tested conditions were as follows: Correction of all the subject's aberrations except defocus (C); Correction of all the subject's aberrations except defocus and habitual second-order astigmatism (AS); Correction of all the subject's aberrations except defocus and odd higher-order aberrations (HOAs); Correction of all the subject's aberrations except defocus and even HOAs (E); Natural aberrations of the subject's eye, i.e., the adaptive-optics system only corrected the optical system's aberrations (N); Correction of all the subject's aberrations except defocus and fourth-order spherical aberration (SA). The correction was performed at 20 Hz and each condition was repeated six times in randomised order. Average gain (±2 standard errors of the mean) varied little across conditions; between 0.55 ± 0.06 (SA), and 0.62 ± 0.06 (AS). Average phase (±2 standard errors of the mean) also varied little; between 0.41 ± 0.02 s (E), and 0.47 ± 0.02 s (O). After Bonferroni correction, no statistically significant differences in gain or phase were found in the presence of specific monochromatic aberrations or in their absence. These results show that the eye's monochromatic aberrations are not necessary for accommodation to track dynamic accommodative stimuli. © 2017 The Authors. Ophthalmic and Physiological Optics published by John Wiley & Sons Ltd on behalf of College of Optometrists.

  5. Differential expression patterns of metastasis suppressor proteins in basal cell carcinoma.

    PubMed

    Bozdogan, Onder; Yulug, Isik G; Vargel, Ibrahim; Cavusoglu, Tarik; Karabulut, Ayse A; Karahan, Gurbet; Sayar, Nilufer

    2015-08-01

    Basal cell carcinomas (BCCs) are common malignant skin tumors. Despite having a significant invasion capacity, they metastasize only rarely. Our aim in this study was to detect the expression patterns of the NM23-H1, NDRG1, E-cadherin, RHOGDI2, CD82/KAI1, MKK4, and AKAP12 metastasis suppressor proteins in BCCs. A total of 96 BCC and 10 normal skin samples were included for the immunohistochemical study. Eleven frozen BCC samples were also studied by quantitative real time polymerase chain reaction (qRT-PCR) to detect the gene expression profile. NM23-H1 was strongly and diffusely expressed in all types of BCC. Significant cytoplasmic expression of NDRG1 and E-cadherin was also detected. However, AKAP12 and CD82/KAI1 expression was significantly decreased. The expressions of the other proteins were somewhere between the two extremes. Similarly, qRT-PCR analysis showed down-regulation of AKAP12 and up-regulation of NM23-H1 and NDRG1 in BCC. Morphologically aggressive BCCs showed significantly higher cytoplasmic NDRG1 expression scores and lower CD82/KAI1 scores than non-aggressive BCCs. The relatively preserved levels of NM23-H1, NDRG1, and E-cadherin proteins may have a positive effect on the non-metastasizing features of these tumors. © 2014 The International Society of Dermatology.

  6. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    PubMed

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  7. Tandem SUMO fusion vectors for improving soluble protein expression and purification.

    PubMed

    Guerrero, Fernando; Ciragan, Annika; Iwaï, Hideo

    2015-12-01

    Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Third-rank chromatic aberrations of electron lenses.

    PubMed

    Liu, Zhixiong

    2018-02-01

    In this paper the third-rank chromatic aberration coefficients of round electron lenses are analytically derived and numerically calculated by Mathematica. Furthermore, the numerical results are cross-checked by the differential algebraic (DA) method, which verifies that all the formulas for the third-rank chromatic aberration coefficients are completely correct. It is hoped that this work would be helpful for further chromatic aberration correction in electron microscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Ascl1-induced neuronal differentiation of P19 cells requires expression of a specific inhibitor protein of cAMP-dependent protein kinase

    PubMed Central

    Huang, Holly S.; Turner, David L.; Thompson, Robert C.; Uhler, Michael D.

    2011-01-01

    cAMP-dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix-loop-helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal β-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)β. PKIβ-targeted shRNA constructs both reduced the levels of PKIβ expression and blocked the neuronal differentiation of P19 cells. This inhibition of differentiation was rescued by transfection of a shRNA-resistant expression vector for the PKIβ protein, and this rescue required the PKA-specific inhibitory sequence of the PKIβprotein. PKIβ played a very specific role in the Ascl1-mediated differentiation process since other PKI isoforms were unable to rescue the deficit conferred by shRNA-mediated knockdown of PKIβ. Our results define a novel requirement for PKIβ and its inhibition of PKA during neuronal differentiation of P19 cells. PMID:21623794

  10. Prognostic impact of MYC protein expression in central nervous system diffuse large B-cell lymphoma: comparison with MYC rearrangement and MYC mRNA expression.

    PubMed

    Son, Seung-Myoung; Ha, Sang-Yun; Yoo, Hae-Yong; Oh, Dongryul; Kim, Seok-Jin; Kim, Won-Seog; Ko, Young-Hyeh

    2017-01-01

    The prognostic role of MYC has been well documented in non-central nervous system diffuse large B-cell lymphoma; however, it remains controversial in central nervous system diffuse large B-cell lymphoma. To investigate the prognostic value of MYC, we analyzed the MYC protein expression by immunohistochemistry, mRNA expression by RNA in situ hybridization, and gene status by fluorescence in situ hybridization in 74 cases of central nervous system diffuse large B-cell lymphoma. Moreover, we examined the correlation between MYC translocation, mRNA expression, and protein expression. The mean percentage of MYC immunopositive cells was 49%. Using a 44% cutoff value, 49 (66%) cases showed MYC protein overexpression. The result of mRNA in situ hybridization using the RNA scope technology was obtained using the H-scoring system; the median value was 34.2. Using the cutoff value of 63.5, 16 (22%) cases showed MYC mRNA overexpression. MYC gene rearrangement was detected in five out of 68 (7%) cases. MYC translocation showed no statistically significant correlation with mRNA expression; however, all MYC translocation-positive cases showed MYC protein overexpression, with a higher mean percentage of MYC protein expression than that of translocation-negative cases (78 vs 48%, P=0.001). The level of MYC mRNA expression was moderately correlated with the level of MYC protein expression (P<0.001). The mean percentage of MYC protein expression in the high MYC mRNA group was higher than that in the low MYC mRNA group (70 vs 47%, P<0.001). A univariate analysis showed that age over 60 years, Eastern Cooperative Oncology Group (ECOG) performance status ≥2 and MYC protein overexpression were significantly associated with an increased risk of death. MYC translocation and MYC mRNA expression had no prognostic significance. On multivariate analysis, MYC protein overexpression and ECOG score retained prognostic significance.

  11. An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding.

    PubMed

    Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad

    2017-09-01

    The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Ubiquitin over-expression phenotypes and ubiquitin gene molecular misreading during aging in Drosophila melanogaster

    PubMed Central

    Hoe, Nicholas; Huang, Chung M.; Landis, Gary; Verhage, Marian; Ford, Daniel; Yang, Junsheng; van Leeuwen, Fred W.; Tower, John

    2011-01-01

    Molecular Misreading (MM) is the inaccurate conversion of genomic information into aberrant proteins. For example, when RNA polymerase II transcribes a GAGAG motif it synthesizes at low frequency RNA with a two-base deletion. If the deletion occurs in a coding region, translation will result in production of misframed proteins. During mammalian aging, misframed versions of human amyloid precursor protein (hApp) and ubiquitin (hUbb) accumulate in the aggregates characteristic of neurodegenerative diseases, suggesting dysfunctional degradation or clearance. Here cDNA clones encoding wild-type hUbb and the frame-shifted version hUbb+1 were expressed in transgenic Drosophila using the doxycycline-regulated system. Misframed proteins were abundantly produced, both from the transgenes and from endogenous Drosophila ubiquitin-encoding genes, and their abundance increased during aging in whole-fly extracts. Over-expression of wild-type hUbb, but not hUbb+1, was toxic during fly development. In contrast, when over-expressed specifically in adult flies, hUbb+1 caused small decreases in life span, whereas hUbb was associated with small increases, preferentially in males. The data suggest that MM occurs in Drosophila and that the resultant misframed proteins accumulate with age. MM of the ubiquitin gene can produce alternative ubiquitin gene products with different and sometimes opposing phenotypic effects. PMID:21415465

  13. The Distribution of Chromosomal Aberrations in Human Cells Predicted by a Generalized Time-Dependent Model of Radiation-Induced Formation of Aberrations

    NASA Technical Reports Server (NTRS)

    Ponomarev, Artem L.; George, K.; Cucinotta, F. A.

    2011-01-01

    New experimental data show how chromosomal aberrations for low- and high-LET radiation are dependent on DSB repair deficiencies in wild-type, AT and NBS cells. We simulated the development of chromosomal aberrations in these cells lines in a stochastic track-structure-dependent model, in which different cells have different kinetics of DSB repair. We updated a previously formulated model of chromosomal aberrations, which was based on a stochastic Monte Carlo approach, to consider the time-dependence of DSB rejoining. The previous version of the model had an assumption that all DSBs would rejoin, and therefore we called it a time-independent model. The chromosomal-aberrations model takes into account the DNA and track structure for low- and high-LET radiations, and provides an explanation and prediction of the statistics of rare and more complex aberrations. We compared the program-simulated kinetics of DSB rejoining to the experimentally-derived bimodal exponential curves of the DSB kinetics. We scored the formation of translocations, dicentrics, acentric and centric rings, deletions, and inversions. The fraction of DSBs participating in aberrations was studied in relation to the rejoining time. Comparisons of simulated dose dependence for simple aberrations to the experimental dose-dependence for HF19, AT and NBS cells will be made.

  14. Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

    PubMed

    Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

    2006-01-01

    Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell

  15. Harmonic oscillator states in aberration optics

    NASA Technical Reports Server (NTRS)

    Wolf, Kurt Bernardo

    1993-01-01

    The states of the three-dimensional quantum harmonic oscillator classify optical aberrations of axis-symmetric systems due to the isomorphism between the two mathematical structures. Cartesian quanta and angular momentum classifications have their corresponding aberration classifications. The operation of concatenation of optical elements introduces a new operation between harmonic oscillator states.

  16. Ethylene Regulates Monomeric GTP-Binding Protein Gene Expression and Activity in Arabidopsis1

    PubMed Central

    Moshkov, Igor E.; Mur, Luis A.J.; Novikova, Galina V.; Smith, Aileen R.; Hall, Michael A.

    2003-01-01

    Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction. PMID:12692329

  17. [Arf6, RalA and BIRC5 protein expression in non small cell lung cancer].

    PubMed

    Knizhnik, A V; Kovaleva, O B; Laktionov, K K; Mochal'nikova, V V; Komel'kov, A V; Chevkina, E M; Zborovskaia, I B

    2011-01-01

    Evaluation of tumor markers expression pattern which determines individual progression parameters is one of the major topics in molecular oncopathology research. This work presents research on expression analysis of several Ras-Ral associated signal transduction pathway proteins (Arf6, RalA and BIRC5) in accordance with clinical criteria in non small cell lung cancer patients. Using Western-blot analysis and RT-PCR Arf6, RalA and BIRC5 expression has been analyzed in parallel in 53 non small cell lung cancer samples of different origin. Arf6 protein expression was elevated in 55% non small cell lung cancer tumor samples in comparison with normal tissue. In the group of squamous cell lung cancer Arf6 expression elevation was observed more often. RalA protein expression was decreased in comparison to normal tissue samples in 64% of non small cell lung cancer regardless to morphological structure. Correlation between RalA protein expression decrease and absence of regional metastases was revealed for squamous cell lung cancer. BIRC5 protein expression in tumor samples versus corresponding normal tissue was 1.3 times more often elevated in the squamous cell lung cancer group (in 76% tumor samples). At the same time elevation of BIRC5 expression was fixed only in 63% of adenocarcinoma tumor samples. A statistically significant decrease (p = 0.0158) of RalA protein expression and increase (p = 0.0498) of Arf6 protein expression in comparison with normal tissue was found for T1-2N0M0 and T1-2N1-2M0 groups of squamous cell lung cancer correspondingly.

  18. Mutations in protein-binding hot-spots on the hub protein Smad3 differentially affect its protein interactions and Smad3-regulated gene expression.

    PubMed

    Schiro, Michelle M; Stauber, Sara E; Peterson, Tami L; Krueger, Chateen; Darnell, Steven J; Satyshur, Kenneth A; Drinkwater, Norman R; Newton, Michael A; Hoffmann, F Michael

    2011-01-01

    Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for

  19. Analysis of disease-associated protein expression using quantitative proteomics—fibulin-5 is expressed in association with hepatic fibrosis.

    PubMed

    Bracht, Thilo; Schweinsberg, Vincent; Trippler, Martin; Kohl, Michael; Ahrens, Maike; Padden, Juliet; Naboulsi, Wael; Barkovits, Katalin; Megger, Dominik A; Eisenacher, Martin; Borchers, Christoph H; Schlaak, Jörg F; Hoffmann, Andreas-Claudius; Weber, Frank; Baba, Hideo A; Meyer, Helmut E; Sitek, Barbara

    2015-05-01

    Hepatic fibrosis and cirrhosis are major health problems worldwide. Until now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. To develop noninvasive diagnostic assays for the assessment of liver fibrosis, it is urgently necessary to identify molecules that are robustly expressed in association with the disease. We analyzed biopsied tissue samples from 95 patients with HBV/HCV-associated hepatic fibrosis using three different quantification methods. We performed a label-free proteomics discovery study to identify novel disease-associated proteins using a subset of the cohort (n = 27). Subsequently, gene expression data from all available clinical samples were analyzed (n = 77). Finally, we performed a targeted proteomics approach, multiple reaction monitoring (MRM), to verify the disease-associated expression in samples independent from the discovery approach (n = 68). We identified fibulin-5 (FBLN5) as a novel protein expressed in relation to hepatic fibrosis. Furthermore, we confirmed the altered expression of microfibril-associated glycoprotein 4 (MFAP4), lumican (LUM), and collagen alpha-1(XIV) chain (COL14A1) in association to hepatic fibrosis. To our knowledge, no tissue-based quantitative proteomics study for hepatic fibrosis has been performed using a cohort of comparable size. By this means, we add substantial evidence for the disease-related expression of the proteins examined in this study.

  20. Disruption of an Evolutionarily Novel Synaptic Expression Pattern in Autism

    PubMed Central

    Jiang, Xi; Hu, Haiyang; Guijarro, Patricia; Mitchell, Amanda; Ely, John J.; Sherwood, Chet C.; Hof, Patrick R.; Qiu, Zilong; Pääbo, Svante; Akbarian, Schahram; Khaitovich, Philipp

    2016-01-01

    Cognitive defects in autism spectrum disorder (ASD) include socialization and communication: key behavioral capacities that separate humans from other species. Here, we analyze gene expression in the prefrontal cortex of 63 autism patients and control individuals, as well as 62 chimpanzees and macaques, from natal to adult age. We show that among all aberrant expression changes seen in ASD brains, a single aberrant expression pattern overrepresented in genes involved synaptic-related pathways is enriched in nucleotide variants linked to autism. Furthermore, only this pattern contains an excess of developmental expression features unique to humans, thus resulting in the disruption of human-specific developmental programs in autism. Several members of the early growth response (EGR) transcription factor family can be implicated in regulation of this aberrant developmental change. Our study draws a connection between the genetic risk architecture of autism and molecular features of cortical development unique to humans. PMID:27685936