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Sample records for high baalc expression

  1. mRNA overexpression of BAALC: A novel prognostic factor for pediatric acute lymphoblastic leukemia

    OpenAIRE

    Azizi, Zahra; Rahgozar, Soheila; Moafi, Alireza; DABAGHI, MOHAMMAD; NADIMI, MOTAHAREH

    2015-01-01

    BAALC is a novel molecular marker in leukemia that is highly expressed in patients with acute leukemia. Increased expression levels of BAALC are known as poor prognostic factors in adult acute myeloid and lymphoid leukemia. The purpose of the present study was to evaluate the prognostic significance of the BAALC gene expression levels in pediatric acute lymphoblastic leukemia (ALL) and its association with MDR1. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the...

  2. mRNA overexpression of BAALC: A novel prognostic factor for pediatric acute lymphoblastic leukemia

    Science.gov (United States)

    AZIZI, ZAHRA; RAHGOZAR, SOHEILA; MOAFI, ALIREZA; DABAGHI, MOHAMMAD; NADIMI, MOTAHAREH

    2015-01-01

    BAALC is a novel molecular marker in leukemia that is highly expressed in patients with acute leukemia. Increased expression levels of BAALC are known as poor prognostic factors in adult acute myeloid and lymphoid leukemia. The purpose of the present study was to evaluate the prognostic significance of the BAALC gene expression levels in pediatric acute lymphoblastic leukemia (ALL) and its association with MDR1. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the mRNA expression levels of BAALC and MRD1 were measured in bone marrow samples of 28 new diagnosed childhood ALL patients and 13 children without cancer. Minimal residual disease (MRD) was measured one year after the initiation of the chemotherapy using the RT-qPCR method. The high level expression of BAALC had a significant association with the pre-B-ALL subtype, leukocytosis and positive MRD after one year of treatment in leukemic patients. In addition, a positive correlation between BAALC and MDR1 mRNA expression was shown in this group. In conclusion, to the best of our knowledge, the increase of BAALC expression as a poor prognostic factor for childhood ALL is shown for the first time. Additionally, the correlation between BAALC and MDR1 in mRNA expression levels can aid for an improved understanding of the mechanism through which BAALC may function in ALL and multidrug resistance. PMID:26137238

  3. mRNA overexpression of BAALC: A novel prognostic factor for pediatric acute lymphoblastic leukemia.

    Science.gov (United States)

    Azizi, Zahra; Rahgozar, Soheila; Moafi, Alireza; Dabaghi, Mohammad; Nadimi, Motahareh

    2015-05-01

    BAALC is a novel molecular marker in leukemia that is highly expressed in patients with acute leukemia. Increased expression levels of BAALC are known as poor prognostic factors in adult acute myeloid and lymphoid leukemia. The purpose of the present study was to evaluate the prognostic significance of the BAALC gene expression levels in pediatric acute lymphoblastic leukemia (ALL) and its association with MDR1. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the mRNA expression levels of BAALC and MRD1 were measured in bone marrow samples of 28 new diagnosed childhood ALL patients and 13 children without cancer. Minimal residual disease (MRD) was measured one year after the initiation of the chemotherapy using the RT-qPCR method. The high level expression of BAALC had a significant association with the pre-B-ALL subtype, leukocytosis and positive MRD after one year of treatment in leukemic patients. In addition, a positive correlation between BAALC and MDR1 mRNA expression was shown in this group. In conclusion, to the best of our knowledge, the increase of BAALC expression as a poor prognostic factor for childhood ALL is shown for the first time. Additionally, the correlation between BAALC and MDR1 in mRNA expression levels can aid for an improved understanding of the mechanism through which BAALC may function in ALL and multidrug resistance.

  4. Evaluation of rs62527607 [GT] single nucleotide polymorphism located in BAALC gene in children with acute leukemia using mismatch PCR-RFLP.

    Science.gov (United States)

    Nadimi, Motahareh; Rahgozar, Soheila; Moafi, Alireza; Tavassoli, Manoochehr; Mesrian Tanha, Hamzeh

    2016-01-01

    Acute leukemia is the most common cancer in children and involves several factors that contribute to the development of multidrug resistance and treatment failure. According to our recent studies, the BAALC gene is identified to have high mRNA expression levels in childhood acute lymphoblastic leukemia (ALL) and those with multidrug resistance. Several polymorphisms are associated with the expression of this gene. To date, there has been no study on the rs62527607 [GT] single nucleotide polymorphism (SNP) of BAALC gene and its link with childhood acute lymphoblastic and myeloid leukemia (AML). The purpose of this study is to evaluate the prevalence of this polymorphism in pediatric acute leukemia, as well as its relationship with prognosis. DNA samples were extracted from bone marrow slides of 129 children with ALL and 16 children with AML. The rs62527607 [GT] SNP was evaluated using mismatch polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based analysis. The association between the SNP alleles and patient disease-free survival was then assessed. The prevalence of the T-allele of rs62527607 [GT] SNP in childhood T-ALL and pre-B-ALL was 28.3% and 11.2%, respectively. In the pre-B-ALL patients, 3 year disease free survival was associated with the GG genotype. Results showed a robust association between the rs62527607 SNP and the risk of relapse in ALL, but not AML, patients. T-ALL patients with the GT genotype had an 8.75 fold higher risk of relapse. The current study demonstrates a significant association between the genotype GT and the polymorphic allele G424T, and introduces this SNP as a negative prognostic factor in children with ALL.

  5. PROGNOSTIC VALUE OF BRAIN AND ACUTE LEUKEMIA CYTOPLASMIC GENE EXPRESSION IN EGYPTIAN CHILDREN WITH ACUTE MYELOID LEUKEMIA

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    adel abd elhaleim hagag

    2015-04-01

    Full Text Available Abstract      Background: Acute myeloid leukemia (AML accounts for 25%-35% of the acute leukemia in children. BAALC (Brain and Acute Leukemia, Cytoplasmic gene is a recently identified gene on chromosome 8q22.3 that has prognostic significance in AML.  The aim of this work was to study the impact of BAALC gene expression on prognosis of AML in Egyptian children. Patients and methods: This study was conducted on 40 patients of newly diagnosed AML who were subjected to the following: Full history taking, clinical examination, laboratory investigations including: complete blood count, LDH, bone marrow aspiration, cytochemistry and immunophenotyping, assessment of BAALC Gene by real time PCR in bone marrow aspirate mononuclear cells before the start of chemotherapy. Results: BAALC gene expression showed positive expression in 24 cases (60% and negative expression in 16 cases (40%. Patients who showed positive BAALC gene expression included 10 patients achieved complete remission, 8 patients died and 6 relapsed patients, while patients who showed negative expression include 12 patients achieved complete remission, 1 relapsed patient and 3 patients died. There was significant association between BAALC gene expression and FAB classification of patients of AML patientsas positive BAALC expression is predominantly seen in FAB subtypes M1 and M2 compared with negative BAALC gene expression that was found more in M3 and M4 (8 cases with M1, 12 cases with M2, 1 case with M3 and 3 cases with M4 in positive BAALC expression versus 2 cases with M1, 3 cases with M2, 4 cases with M3 and 7 cases with M4 in BAALC gene negative expression group with significant difference regarding FAB subtypes. As regard age, sex, splenomegaly, lymphadenopathy, pallor, purpura, platelets count, WBCs count, and percentage of blast cells in BM, the present study showed no significant association with BAALC. Conclusion: BAALC expression is an important prognostic factor in AML

  6. Risk stratification of intermediate-risk acute myeloid leukemia: integrative analysis of a multitude of gene mutation and gene expression markers.

    Science.gov (United States)

    Rockova, Veronika; Abbas, Saman; Wouters, Bas J; Erpelinck, Claudia A J; Beverloo, H Berna; Delwel, Ruud; van Putten, Wim L J; Löwenberg, Bob; Valk, Peter J M

    2011-07-28

    Numerous molecular markers have been recently discovered as potential prognostic factors in acute myeloid leukemia (AML). It has become of critical importance to thoroughly evaluate their interrelationships and relative prognostic importance. Gene expression profiling was conducted in a well-characterized cohort of 439 AML patients (age < 60 years) to determine expression levels of EVI1, WT1, BCL2, ABCB1, BAALC, FLT3, CD34, INDO, ERG and MN1. A variety of AML-specific mutations were evaluated, that is, FLT3, NPM1, N-RAS, K-RAS, IDH1, IDH2, and CEBPA(DM/SM) (double/single). Univariable survival analysis shows that (1) patients with FLT3(ITD) mutations have inferior overall survival (OS) and event-free survival (EFS), whereas CEBPA(DM) and NPM1 mutations indicate favorable OS and EFS in intermediate-risk AML, and (2) high transcript levels of BAALC, CD34, MN1, EVl1, and ERG predict inferior OS and EFS. In multivariable survival analysis, CD34, ERG, and CEBPA(DM) remain significant. Using survival tree and regression methodologies, we show that CEBPA(DM), CD34, and IDH2 mutations are capable of separating the intermediate group into 2 AML subgroups with highly distinctive survival characteristics (OS at 60 months: 51.9% vs 14.9%). The integrated statistical approach demonstrates that from the multitude of biomarkers a greatly condensed subset can be selected for improved stratification of intermediate-risk AML.

  7. Highly Expressive Hakka Art

    Institute of Scientific and Technical Information of China (English)

    JENNIFER LIM

    1996-01-01

    SOUTHERN Jiangxi Province was the birthplace of the Hakka ethnic group and has since been the native home and main transfer hub for the spread of the nationality. The highly expressive art of the Hakkas, including folk songs in Xingguo, colored lantern performances in Shicheng, ancient

  8. Cloning, high-level expression, purification and characterization of a ...

    African Journals Online (AJOL)

    Cloning, high-level expression, purification and characterization of a staphylokinase variant, SakøC, ... African Journal of Biotechnology ... Hence in this study, we reported the cloning, high-level expression, purification and characterization of ...

  9. Patterns of transposable element expression and insertion in cancer

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    Evan A Clayton

    2016-11-01

    Full Text Available Human transposable element (TE activity in somatic tissues causes mutations that can contribute to tumorigenesis. Indeed, TE insertion mutations have been implicated in the etiology of a number of different cancer types. Nevertheless, the full extent of somatic TE activity, along with its relationship to tumorigenesis, have yet to be fully explored. Recent developments in bioinformatics software make it possible to analyze TE expression levels and TE insertional activity directly from transcriptome (RNA-seq and whole genome (DNA-seq next-generation sequence data. We applied these new sequence analysis techniques to matched normal and primary tumor patient samples from the Cancer Genome Atlas (TCGA in order to analyze the patterns of TE expression and insertion for three cancer types: breast invasive carcinoma, head and neck squamous cell carcinoma, and lung adenocarcinoma. Our analysis focused on the three most abundant families of active human TEs: Alu, SVA and L1. We found evidence for high levels of somatic TE activity for these three families in normal and cancer samples across diverse tissue types. Abundant transcripts for all three TE families were detected in both normal and cancer tissues along with an average of ~80 unique TE insertions per individual patient/tissue. We observed an increase in L1 transcript expression and L1 insertional activity in primary tumor samples for all three cancer types. Tumor-specific TE insertions are enriched for private mutations, consistent with a potentially causal role in tumorigenesis. We used genome feature analysis to investigate two specific cases of putative cancer-causing TE mutations in further detail. An Alu insertion in an upstream enhancer of the CBL tumor suppressor gene is associated with down-regulation of the gene in a single breast cancer patient, and an L1 insertion in the first exon of the BAALC gene also disrupts its expression in head and neck squamous cell carcinoma. Our results are

  10. Characterization of differentially expressed genes using high-dimensional co-expression networks

    DEFF Research Database (Denmark)

    Coelho Goncalves de Abreu, Gabriel; Labouriau, Rodrigo S.

    2010-01-01

    of spurious information along the network are avoided. The proposed inference procedure is based on the minimization of the Bayesian Information Criterion (BIC) in the class of decomposable graphical models. This class of models can be used to represent complex relationships and has suitable properties...... construct a compact representation of the co-expression network that allows to identify the regions with high concentration of differentially expressed genes. It is argued that differentially expressed genes located in highly interconnected regions of the co-expression network are less informative than...

  11. High-level expression, purification, polyclonal antibody preparation ...

    African Journals Online (AJOL)

    user

    2011-02-14

    Feb 14, 2011 ... Full Length Research Paper. High-level expression ... resistance severely compromises effective therapeutic options. ... In the present study, we first report the expression of the oprD ... databases of National Center for Biotechnology Information (NCBI) ..... assembly of the head of bacteriophage T4. Nature.

  12. Immunoinformatics study on highly expressed Mycobacterium tuberculosis genes during infection.

    Science.gov (United States)

    Nguyen Thi, Le Thuy; Sarmiento, Maria Elena; Calero, Romel; Camacho, Frank; Reyes, Fatima; Hossain, Md Murad; Gonzalez, Gustavo Sierra; Norazmi, Mohd Nor; Acosta, Armando

    2014-09-01

    The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.

  13. High lib mRNA expression in breast carcinomas.

    Science.gov (United States)

    Satoh, Kazuki; Hata, Mitsumi; Yokota, Hiroshi

    2004-06-30

    Lib, first identified as a novel beta-amyloid responsive gene in rat astrocytes, has an extracellular domain of 15 leucine-rich repeats (LRRs) followed by a transmembrane domain and a short cytoplasmic region. It is a distinctly inducible gene and is thought to play a key role in inflammatory states via the LRR extracellular motif, an ideal structural framework for protein-protein and protein-matrix interactions. To evaluate potential roles of Lib, we screened various tumors for Lib expression. Lib mRNA expression was high and uniquely expressed in breast tumor tissues, compared to paired normal breast tissues. Lib mRNA was localized in the ductal carcinoma cells and Lib protein displayed a homophilic association on the surface of cultured cells. These data suggest that Lib may play a role in the progression of breast carcinomas and may be a diagnostic marker for breast tumors.

  14. Multigroup Equivalence Analysis for High-Dimensional Expression Data

    Science.gov (United States)

    Yang, Celeste; Bartolucci, Alfred A.; Cui, Xiangqin

    2015-01-01

    Hypothesis tests of equivalence are typically known for their application in bioequivalence studies and acceptance sampling. Their application to gene expression data, in particular high-dimensional gene expression data, has only recently been studied. In this paper, we examine how two multigroup equivalence tests, the F-test and the range test, perform when applied to microarray expression data. We adapted these tests to a well-known equivalence criterion, the difference ratio. Our simulation results showed that both tests can achieve moderate power while controlling the type I error at nominal level for typical expression microarray studies with the benefit of easy-to-interpret equivalence limits. For the range of parameters simulated in this paper, the F-test is more powerful than the range test. However, for comparing three groups, their powers are similar. Finally, the two multigroup tests were applied to a prostate cancer microarray dataset to identify genes whose expression follows a prespecified trajectory across five prostate cancer stages. PMID:26628859

  15. Multigroup Equivalence Analysis for High-Dimensional Expression Data.

    Science.gov (United States)

    Yang, Celeste; Bartolucci, Alfred A; Cui, Xiangqin

    2015-01-01

    Hypothesis tests of equivalence are typically known for their application in bioequivalence studies and acceptance sampling. Their application to gene expression data, in particular high-dimensional gene expression data, has only recently been studied. In this paper, we examine how two multigroup equivalence tests, the F-test and the range test, perform when applied to microarray expression data. We adapted these tests to a well-known equivalence criterion, the difference ratio. Our simulation results showed that both tests can achieve moderate power while controlling the type I error at nominal level for typical expression microarray studies with the benefit of easy-to-interpret equivalence limits. For the range of parameters simulated in this paper, the F-test is more powerful than the range test. However, for comparing three groups, their powers are similar. Finally, the two multigroup tests were applied to a prostate cancer microarray dataset to identify genes whose expression follows a prespecified trajectory across five prostate cancer stages.

  16. MMSET is highly expressed and associated with aggressiveness in neuroblastoma

    DEFF Research Database (Denmark)

    Hudlebusch, Heidi Rye; Skotte, Julie; Santoni-Rugiu, Eric;

    2011-01-01

    tumor types as well. We have performed immunohistochemical staining of tissue microarrays and found that MMSET protein is frequently and highly expressed in neuroblastoma (MMSET positive in 75% of neuroblastomas, n=164). The expression level of MMSET in neuroblastomas was significantly associated...... with poor survival, negative prognostic factors, and metastatic disease. Moreover, a subset of neuroblastomas for which pre- and post-chemotherapy biopsies were available displayed a strong decrease in MMSET protein levels after chemotherapy. In agreement with neuroblastomas becoming more differentiated...... after treatment, we show that retinoic acid-induced differentiation of human neuroblastoma cells in vitro also leads to a strong decrease in MMSET levels. Furthermore, we demonstrate that the high levels of MMSET in normal neural progenitor cells are strongly downregulated during differentiation...

  17. High SHIP2 Expression Indicates Poor Survival in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Ju Yang

    2014-01-01

    Full Text Available SH2-containing inositol 5′-phosphatase 2 (SHIP2, which generally regulates insulin signaling, cytoskeleton remodeling, and receptor endocytosis, has been suggested to play a significant role in tumor development and progression. However, the associations between SHIP2 expression and the clinical features to evaluate its clinicopathologic significance in colorectal cancer (CRC have not been determined yet. In the present study, one-step quantitative real-time polymerase chain reaction (qPCR test and immunohistochemistry (IHC analysis with CRC tissue microarrays (TMA were employed to evaluate the mRNA and protein expression of SHIP2 in CRC. The results showed that SHIP2 expression in the mRNA and protein levels was significantly higher in CRC tissues than that in corresponding noncancerous tissues (both P<0.05. The expression of SHIP2 protein in CRC was related to lymph node metastasis (P=0.036, distant metastasis (P=0.001, and overall survival (P=0.009. Kaplan-Meier method and Cox multifactor analysis suggested that high SHIP2 protein level (P=0.040 and positive distant metastasis (P=0.048 were critically associated with the unfavorable survival of CRC patients. The findings suggested that SHIP2 may be identified as a useful prognostic marker in CRC and targeting CRC may provide novel strategy for CRC treatment.

  18. Twister ribozymes as highly versatile expression platforms for artificial riboswitches

    Science.gov (United States)

    Felletti, Michele; Stifel, Julia; Wurmthaler, Lena A.; Geiger, Sophie; Hartig, Jörg S.

    2016-01-01

    The utilization of ribozyme-based synthetic switches in biotechnology has many advantages such as an increased robustness due to in cis regulation, small coding space and a high degree of modularity. The report of small endonucleolytic twister ribozymes provides new opportunities for the development of advanced tools for engineering synthetic genetic switches. Here we show that the twister ribozyme is distinguished as an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. Besides important implications in biotechnology and synthetic biology, the observed versatility in artificial genetic control set-ups hints at possible natural roles of this widespread ribozyme class. PMID:27670347

  19. Monoamine oxidase A is highly expressed in classical Hodgkin lymphoma.

    Science.gov (United States)

    Li, Pei Chuan; Siddiqi, Imran N; Mottok, Anja; Loo, Eric Y; Wu, Chieh Hsi; Cozen, Wendy; Steidl, Christian; Shih, Jean Chen

    2017-10-01

    Monoamine oxidase A (MAOA) is a mitochondrial enzyme that catalyzes oxidative deamination of neurotransmitters and dietary amines and produces H2 O2 . It facilitates the progression of gliomas and prostate cancer, but its expression and functional relevance have not been studied in lymphoma. Here, we evaluated MAOA in 427 cases of Hodgkin and non-Hodgkin lymphoma and in a spectrum of reactive lymphoid tissues by immunohistochemistry on formalin-fixed, paraffin-embedded specimens. MAOA was expressed by Hodgkin Reed-Sternberg (HRS) cells in the majority of classical Hodgkin lymphomas (cHLs) (181/241; 75%), with 34.8% showing strong expression. Weak MAOA was also noted in a minority of primary mediastinal large B-cell lymphomas (8/47; 17%) and in a mediastinal gray-zone lymphoma. In contrast, no MAOA was found in non-neoplastic lymphoid tissues, nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL; 0/8) or any other non-Hodgkin lymphomas studied (0/123). MAOA was more common in Epstein-Barr virus (EBV)-negative compared to EBV-positive cHL (p Hodgkin-lymphoma-derived cell lines did not. The MAOA inhibitor clorgyline reduced the growth of L1236 cells and U-HO1 cells, and shRNA knockdown of MAOA reduced the growth of L1236 cells. Conversely, ectopic overexpression of MAOA increased the growth of MAOA-negative HDLM2 cells. Combined treatment with clorgyline and ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) was more effective in reducing cell growth than either regimen alone. In summary, MAOA is highly expressed in cHL and may reflect the distinct biology of this lymphoma. Further studies on the potential utility of MAOA as a diagnostic marker and therapeutic target are warranted. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  20. High pressure-sensitive gene expression in Lactobacillus sanfranciscensis

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    R.F. Vogel

    2005-08-01

    Full Text Available Lactobacillus sanfranciscensis is a Gram-positive lactic acid bacterium used in food biotechnology. It is necessary to investigate many aspects of a model organism to elucidate mechanisms of stress response, to facilitate preparation, application and performance in food fermentation, to understand mechanisms of inactivation, and to identify novel tools for high pressure biotechnology. To investigate the mechanisms of the complex bacterial response to high pressure we have analyzed changes in the proteome and transcriptome by 2-D electrophoresis, and by microarrays and real time PCR, respectively. More than 16 proteins were found to be differentially expressed upon high pressure stress and were compared to those sensitive to other stresses. Except for one apparently high pressure-specific stress protein, no pressure-specific stress proteins were found, and the proteome response to pressure was found to differ from that induced by other stresses. Selected pressure-sensitive proteins were partially sequenced and their genes were identified by reverse genetics. In a transcriptome analysis of a redundancy cleared shot gun library, about 7% of the genes investigated were found to be affected. Most of them appeared to be up-regulated 2- to 4-fold and these results were confirmed by real time PCR. Gene induction was shown for some genes up-regulated at the proteome level (clpL/groEL/rbsK, while the response of others to high hydrostatic pressure at the transcriptome level seemed to differ from that observed at the proteome level. The up-regulation of selected genes supports the view that the cell tries to compensate for pressure-induced impairment of translation and membrane transport.

  1. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  2. High-level expressing YAC vector for transgenic animal bioreactors.

    Science.gov (United States)

    Fujiwara, Y; Miwa, M; Takahashi, R; Kodaira, K; Hirabayashi, M; Suzuki, T; Ueda, M

    1999-04-01

    The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.

  3. Intensity dependence in high-level facial expression adaptation aftereffect.

    Science.gov (United States)

    Hong, Sang Wook; Yoon, K Lira

    2017-06-14

    Perception of a facial expression can be altered or biased by a prolonged viewing of other facial expressions, known as the facial expression adaptation aftereffect (FEAA). Recent studies using antiexpressions have demonstrated a monotonic relation between the magnitude of the FEAA and adaptor extremity, suggesting that facial expressions are opponent coded and represented continuously from one expression to its antiexpression. However, it is unclear whether the opponent-coding scheme can account for the FEAA between two facial expressions. In the current study, we demonstrated that the magnitude of the FEAA between two facial expressions increased monotonically as a function of the intensity of adapting facial expressions, consistent with the predictions based on the opponent-coding model. Further, the monotonic increase in the FEAA occurred even when the intensity of an adapting face was too weak for its expression to be recognized. These results together suggest that multiple facial expressions are encoded and represented by balanced activity of neural populations tuned to different facial expressions.

  4. High expression of markers of apoptosis in Langerhans cell histiocytosis

    DEFF Research Database (Denmark)

    Petersen, Bodil Laub; Lundegaard, Pia Rengtved; Bank, M I;

    2003-01-01

    53 and the number of cells in apoptosis detected with TUNEL. Langerhans cell histiocytosis cells showed strong expression of p53 and in some cases co-expression of Fas and Fas-L. The expression of Fas-L was significantly higher in infiltrates from patients with single-system disease. The actual...... number of pathological Langerhans cells in apoptosis as estimated by TUNEL was low. CONCLUSIONS: The low number of TUNEL-reactive cells can be explained by the rapid turnover of apoptotic cells in the tissue, not leaving the apoptotic cells long enough in the tissue to be detected. The co...

  5. Heritable retroviral transgenes are highly expressed in chickens.

    OpenAIRE

    Briskin, M J; Hsu, R Y; Boggs, T; Schultz, J. A.; Rishell, W; Bosselman, R A

    1991-01-01

    This report describes expression of heritable reticuloendotheliosis virus (REV) vector ME111 in 20 independent lines of transgenic chickens. The results are strikingly different from studies of Moloney virus in transgenic mice, where restricted expression of inherited proviruses has led to their use primarily as insertional mutagens rather than general agents for gene transfer. In contrast, the REV ME111 provirus is actively transcribed in a variety of tissues from transgenic chickens, is exp...

  6. Homologous high-throughput expression and purification of highly conserved E coli proteins

    Directory of Open Access Journals (Sweden)

    Duchmann Rainer

    2007-06-01

    Full Text Available Abstract Background Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD. Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies. Results As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP, N-utilizing substance A (NusA, or glutathione S-transferase (GST to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS. Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500

  7. Expression Analysis of miRNAs and Highly-expressed Small RNAs in Two Rice Subspecies and their Reciprocal Hybrids

    Institute of Scientific and Technical Information of China (English)

    Fangfang Chen; Guangming He; Hang He; Wei Chen; Xiaopeng Zhu; Manzhong Liang; Liangbi Chen; Xing Wang Deng

    2010-01-01

    Heterosis,or hybrid vigor,is the phenomenon whereby progeny of two inbred lines exhibit superior agronomic performance compared with either parent.We analyzed the expression of miRNAs and highly expressed small RNAs(defined according to Solexa sequencing results)in two rice(Oryza sativa)subspecies(japonica cv.Nipponbare and indica cv.93-11)and their reciprocal hybrids using microarrays.We found that of all the 1141 small RNAs tested,140(12%,140 of 1141)and 157(13%,157 of 1141)were identified being significantly differentially expressed in two reciprocal hybrids,respectively.All possible modes of action,including additive,high- and low- parent,above high- and below low-parent modes were exhibited.Both F1 hybrids showed non-additive expression patterns,with downregulation predominating.Interestingly,15 miRNAs displayed stark opposite expression trends relative to midparent in reciprocal hybrids.Computational prediction of targets of differentially expressed miRNAs showed that they participated in multifaceted developmental pathways,and were not distinguishable from the targets of non-differentially expressed miRNAs.Together,our findings reveal that small RNAs play roles in heterosis and add a new layer in the understanding and exploitation of molecular mechanisms of heterosis.

  8. DAG expression: high-throughput gene expression analysis of real-time PCR data using standard curves for relative quantification.

    Directory of Open Access Journals (Sweden)

    María Ballester

    Full Text Available BACKGROUND: Real-time quantitative PCR (qPCR is still the gold-standard technique for gene-expression quantification. Recent technological advances of this method allow for the high-throughput gene-expression analysis, without the limitations of sample space and reagent used. However, non-commercial and user-friendly software for the management and analysis of these data is not available. RESULTS: The recently developed commercial microarrays allow for the drawing of standard curves of multiple assays using the same n-fold diluted samples. Data Analysis Gene (DAG Expression software has been developed to perform high-throughput gene-expression data analysis using standard curves for relative quantification and one or multiple reference genes for sample normalization. We discuss the application of DAG Expression in the analysis of data from an experiment performed with Fluidigm technology, in which 48 genes and 115 samples were measured. Furthermore, the quality of our analysis was tested and compared with other available methods. CONCLUSIONS: DAG Expression is a freely available software that permits the automated analysis and visualization of high-throughput qPCR. A detailed manual and a demo-experiment are provided within the DAG Expression software at http://www.dagexpression.com/dage.zip.

  9. High expression of NPY receptors in the human testis.

    Science.gov (United States)

    Körner, Meike; Waser, Beatriche; Thalmann, George N; Reubii, Jean Claude

    2011-04-30

    NPY receptors represent novel molecular therapeutic targets in cancer and obesity. However, the extent of NPY receptor expression in normal human tissues is poorly investigated. Based on the role of NPY in reproductive functions, the NPY receptor expression was studied in 25 normal human testes and, additionally, 24 testicular tumors using NPY receptor autoradiography. In the normal testis, Leydig cells strongly expressed NPY receptor subtype Y2, and small arterial blood vessels Y1. Y2 receptors were found to be functional with agonist-stimulated [(35)S]GTPγS binding autoradiography. Full functional integrity of the NPY system was further suggested by the immunohistochemical detection of NPY peptide in nerve fibers directly adjacent to Leydig cells and arteries. Germ cell tumors expressed Y1 and Y2 on tumor cells in 33% and Y1 on intratumoral blood vessels in 50%. Based on its strong NPY receptor expression in Leydig cells and blood vessels, the normal human testis represents a potentially important physiological and pharmalogical NPY target.

  10. Recombinant cells that highly express chromosomally-integrated heterologous gene

    Science.gov (United States)

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  11. High-level expression of a chemically synthesized gene for human interferon-gamma using a prokaryotic expression vector.

    OpenAIRE

    1984-01-01

    A chemically synthesized gene for human interferon-gamma has been cloned into a prokaryotic expression vector under the regulation of a synthetic constitutive transcriptional-translational control unit that contains a strong bacteriophage T5 early promoter and a strong ribosome-binding site. Cells harboring the recombinant plasmid express high levels (4 X 10(9) units per liter of culture) of antiviral activity specific for interferon-gamma. Analysis of total cell lysates on NaDodSO4/polyacryl...

  12. Tumor endothelial cells express high pentraxin 3 levels.

    Science.gov (United States)

    Hida, Kyoko; Maishi, Nako; Kawamoto, Taisuke; Akiyama, Kosuke; Ohga, Noritaka; Hida, Yasuhiro; Yamada, Kenji; Hojo, Takayuki; Kikuchi, Hiroshi; Sato, Masumi; Torii, Chisaho; Shinohara, Nobuo; Shindoh, Masanobu

    2016-12-01

    It has been described that tumor progression has many similarities to inflammation and wound healing in terms of the signaling processes involved. Among biological responses, angiogenesis, which is necessary for tumor progression and metastasis, is a common hallmark; therefore, tumor blood vessels have been considered as important therapeutic targets in anticancer therapy. We focused on pentraxin 3 (PTX3), which is a marker of cancer-related inflammation, but we found no reports on its expression and function in tumor blood vessels. Here we showed that PTX3 is expressed in mouse and human tumor blood vessels based on immunohistochemical analysis. We found that PTX3 is upregulated in primary mouse and human tumor endothelial cells compared to normal endothelial cells. We also showed that PTX3 plays an important role in the proliferation of the tumor endothelial cells. These results suggest that PTX3 is an important target for antiangiogenic therapy.

  13. Skp2 expression is associated with high risk and elevated Ki67 expression in gastrointestinal stromal tumours

    Directory of Open Access Journals (Sweden)

    Terracciano Luigi

    2008-05-01

    Full Text Available Abstract Background Gastrointestinal stromal tumors (GIST exhibit an unpredictable clinical course and can rapidly progress to lethality. Predictions about the biological behavior of GIST are based on a number of canonical clinical and pathologic parameters whose validity in distinguishing between a benign and a malignant tumour is still imperfect. The aim of our study was to investigate the role of morphologic parameters and expression of cells cycle regulators as prognosticators in GIST. Methods We performed an immunohistochemical analysis for Ki67, p27Kip1, Jab1, and Skp2, on a Tissue Microarray (TMA containing 94 GIST. Expression of the above proteins was correlated to classically used prognosticators, as well as to risk groups. Clinical significance of histologic and immunohistochemical features were evaluated in 59 patients for whom follow-up information was available. Results Overexpression of Ki67 and Skp2, and p27Kip1 loss directly correlated with the high risk group (p = 0.03 for Ki67 and Skp2, p = 0.05 for p27Kip1. Jab1 expression did not exhibit correlation with risk. In 59 cases provided with clinical follow-up, high cellularity, presence of necrosis, and Ki67 overexpression were predictive of a reduced overall survival in a univariate model. The same parameters, as well as mitotic rate, tumour size, and p27Kip1 loss were indicative of a shortened relapse free survival interval. High cellularity, and high mitotic rate retained their prognostic significance by multivariate analysis. Conclusion Our data suggest that a number of histologic parameters in combination with immunohistochemical expression of cell cycle regulators can facilitate risk categorization and predict biologic behavior in GIST. Importantly this study demonstrates, for the first time, that Skp2 expression correlates with Ki67 expression and high risk in GIST.

  14. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

    Directory of Open Access Journals (Sweden)

    Yoichiro Ito

    Full Text Available Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

  15. High Frequency Electromagnetic Field Induces Lipocalin 2 Expression in

    Directory of Open Access Journals (Sweden)

    Amaneh Mohammadi Roushandeh

    2010-06-01

    Full Text Available Objective(sNeutrophil gelatinase-associated lipocalin (NGAL/Lcn2, comprise a group of small extracellular proteins with a common β-sheet-dominated 3-dimensional structure. In the past, it was assumed that the predominant role of lipocalin was acting as transport proteins. Recently it has been found that oxidative stress induces Lcn2 expression. It has been also proved that electromagnetic field (EMF produces reactive oxygen species (ROS in different tissues. Expression of Lcn2 following exposure to electromagnetic field has been investigated in this study. Materials and MethodsBalb/c mice (8 weeks old were exposed to 3 mT, 50 HZ EMF for 2 months, 4 hr/day. Afterwards, the mice were sacrificed by cervical dislocation and livers were removed. The liver specimens were stained with Haematoxylin- Eosin (H&E and analyzed under an optical microscope. Total RNA was extracted from liver and reverse transcription was performed by SuperScript III reverse transcriptase with 1 µg of total RNA. Assessment of Lcn2 expression was performed by semiquantitative and real time- PCR.ResultsThe light microscopic studies revealed that the number of lymphocyte cells was increased compared to control and dilation of sinosoids was observed in the liver. Lcn2 was up-regulated in the mice exposed to EMF both in mRNA and protein levels.ConclusionTo the extent of our knowledge, this is the first report dealing with up-regulation of Lcn2 in liver after exposure to EMF. The up-regulation might be a compensatory response that involves cell defense pathways and protective effects against ROS. However, further and complementary studies are required in this regards.

  16. Interruptions in gene expression drive highly expressed operons to the leading strand of DNA replication.

    Science.gov (United States)

    Price, Morgan N; Alm, Eric J; Arkin, Adam P

    2005-01-01

    In bacteria, most genes are on the leading strand of replication, a phenomenon attributed to collisions between the DNA and RNA polymerases. In Escherichia coli, these collisions slow the movement of the replication fork through actively transcribed genes only if they are coded on the lagging strand. For genes on both strands, however, these collisions sever nascent transcripts and interrupt gene expression. Based on these observations, we propose a new theory to explain strand bias: genes whose expression is important for fitness are selected to the leading strand because this reduces the duration of these interruptions. Our theory predicts that multi-gene operons, which are subject to longer interruptions, should be more strongly selected to the leading strand than singleton transcripts. We show that this is true even after controlling for the tendency for essential genes, which are strongly biased to the leading strand, to occur in operons. Our theory also predicts that other factors that are associated with strand bias should have stronger effects for genes that are in operons. We find that expression level and phylogenetic ubiquity are correlated with strand bias for both essential and non-essential genes, but only for genes in operons.

  17. Telomeric expression sites are highly conserved in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Christiane Hertz-Fowler

    Full Text Available Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs. The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

  18. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  19. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning

  20. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EF

  1. Neuroendocrine gastric carcinoma expressing somatostatin: A highly malignant, rare tumor

    Institute of Scientific and Technical Information of China (English)

    Jaques Waisberg; Leandro Luongo de Matos; Ana Maria do Amaral Antonio Mader; Sérgio Pezzolo; Esmeralda Miristene Eher; Vera Luiza Capelozzi; Manlio Basilio Speranzini

    2006-01-01

    Poorly differentiated gastric neuroendocrine carcinomas,although rare, deserve particular attention, as they are aggressive and have an extremely poor prognosis. In this report we describe a gastric neuroendocrine carcinoma with rapidly fatal outcome. Immunohistological staining of the resected specimens revealed that the tumor was an endocrine carcinoma. The tumor disclosed intense immunoreactivity to pan-neuroendocrine markers and diffuse somatostatin immunoreactivity. There were no psammoma bodies and no demonstrable association with von Recklinghausen's neurofibromatosis. In the gastrointestinal tract, neuroendocrine tumors producing predominantly somatostatin have been described only in the duodenum. To the best of our knowledge, the present report is the second case report of a neuroendocrine gastric carcinoma expressing diffusely somatostatin as the only neuroendocrine regulatory peptide.

  2. Parallel Gene Expression Differences between Low and High Latitude Populations of Drosophila melanogaster and D. simulans.

    Science.gov (United States)

    Zhao, Li; Wit, Janneke; Svetec, Nicolas; Begun, David J

    2015-05-01

    Gene expression variation within species is relatively common, however, the role of natural selection in the maintenance of this variation is poorly understood. Here we investigate low and high latitude populations of Drosophila melanogaster and its sister species, D. simulans, to determine whether the two species show similar patterns of population differentiation, consistent with a role for spatially varying selection in maintaining gene expression variation. We compared at two temperatures the whole male transcriptome of D. melanogaster and D. simulans sampled from Panama City (Panama) and Maine (USA). We observed a significant excess of genes exhibiting differential expression in both species, consistent with parallel adaptation to heterogeneous environments. Moreover, the majority of genes showing parallel expression differentiation showed the same direction of differential expression in the two species and the magnitudes of expression differences between high and low latitude populations were correlated across species, further bolstering the conclusion that parallelism for expression phenotypes results from spatially varying selection. However, the species also exhibited important differences in expression phenotypes. For example, the genomic extent of genotype × environment interaction was much more common in D. melanogaster. Highly differentiated SNPs between low and high latitudes were enriched in the 3' UTRs and CDS of the geographically differently expressed genes in both species, consistent with an important role for cis-acting variants in driving local adaptation for expression-related phenotypes.

  3. High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains.

    Science.gov (United States)

    Yang, Hu; Zhai, Chao; Yu, Xianhong; Li, Zhezhe; Tang, Wei; Liu, Yunyun; Ma, Xiaojian; Zhong, Xing; Li, Guolong; Wu, Di; Ma, Lixin

    2016-06-01

    Proteinase K is widely used in scientific research and industries. This report was aimed to achieve high-level expression of proteinase K using Pichia pastoris GS115 as the host strain. The coding sequence of a variant of proteinase K that has higher activity than the wild type protein was chosen and optimized based on the codon usage preference of P. pastoris. The novel open reading frame was synthesized and a series of multi-copy expression vectors were constructed based on the pHBM905BDM plasmid, allowing for the tandem integration of multiple copies of the target gene into the genome of P. pastoris with a single recombination. These strains were used to study the correlation between the gene copy number and the expression level of proteinase K. The results of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the host genome stably. Meanwhile, the results of qPCR and enzyme activity assays indicated that the mRNA and protein expression levels of the target gene increased as the gene copy number increased. Moreover, the effect of gene dosage on the expression level of the recombinant protein was more obvious using high-density fermentation. The maximum expression level and enzyme activity of proteinase K, which were obtained from the recombinant yeast strain bearing 5 copies of the target gene after an 84-h induction, were approximately 8.069 mg/mL and 108,295 U/mL, respectively. The recombinant proteinase was purified and characterized. The optimum pH and temperature for the activity of this protease were approximately pH 11 and 55 °C, respectively.

  4. NLRP3 Inflammasome Expression and Signaling in Human Diabetic Wounds and in High Glucose Induced Macrophages

    Directory of Open Access Journals (Sweden)

    Xiaotian Zhang

    2017-01-01

    Full Text Available Introduction. To investigate the contribution and mechanism of NLRP3 inflammasome expression in human wounds in diabetes mellitus and in high glucose induced macrophages. Methods. In the present study, we compared the expression of NLRP3 inflammasome in debridement wound tissue from diabetic and nondiabetic patients. We also examined whether high glucose induces NLRP3 inflammasome expression in cultures THP-1-derived macrophages and the influence on IL-1β expression. Results. The expressions of NLRP3, caspase1, and IL-1β, at both the mRNA and protein level, were significantly higher in wounds of diabetic patients compared with nondiabetic wounds (P<0.05. High glucose induced a significant increase in NLRP3 inflammasome and IL-1β expression in THP-1-derived macrophages. M1 macrophage surface marker with CCR7 was significantly upregulated after high glucose stimulation. SiRNA-mediated silencing of NLRP3 expression downregulates the expression of IL-1β. Conclusion. The higher expression of NLRP3, caspase1, and secretion of IL-1β, signaling, and activation might contribute to the hyperinflammation in the human diabetic wound and in high glucose induced macrophages. It may be a novel target to treat the DM patients with chronic wound.

  5. Prediction of highly expressed genes in microbes based on chromatin accessibility

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Ussery, David

    2007-01-01

    BACKGROUND: It is well known that gene expression is dependent on chromatin structure in eukaryotes and it is likely that chromatin can play a role in bacterial gene expression as well. Here, we use a nucleosomal position preference measure of anisotropic DNA flexibility to predict highly expressed...... and ribosomal RNA are encoded by DNA having significantly lower position preference values than other genes in fast-replicating microbes. CONCLUSION: This insight into DNA structure-dependent gene expression in microbes may be exploited for predicting the expression of non-translated genes such as non...

  6. Venus Express bistatic radar: High-elevation anomalous reflectivity

    Science.gov (United States)

    Simpson, Richard A.; Tyler, G. Leonard; Häusler, Bernd; Mattei, Riccardo; Pätzold, Martin

    2009-06-01

    Magellan (MGN) bistatic radar observations in 1994 confirmed earlier Pioneer Venus reports of unusual Venus surface reflectivity and emissivity at elevations above 6054 km radius. They also revealed that the anomalous values of surface dielectric constant $\\varepsilon$ near Cleopatra Patera included a large imaginary component ($\\varepsilon$ ≈ -i 100) at 13 cm wavelength, consistent with a semiconducting surface material. The MGN observations were conducted using a linearly polarized wave, canted at 45° with respect to the plane of incidence and radiated by the MGN synthetic aperture radar antenna toward the specularly reflecting region of the mean planetary surface. In 2006 similar experiments were conducted using 13 cm circularly polarized transmissions from Venus Express (VEX). The VEX signal-to-noise ratio (SNR) was lower than that of MGN, but elevated ∣$\\varepsilon$∣ has been inferred broadly over Maxwell Montes. A quasi-specular echo was detected near Cleopatra but with insufficient SNR to address the question of conductivity. An early failure of the VEX 13 cm radio system precludes further measurements with VEX.

  7. A 4-gene expression score associated with high levels of Wilms Tumor-1 (WT1) expression is an adverse prognostic factor in acute myeloid leukaemia

    NARCIS (Netherlands)

    Niavarani, A. (Ahmadreza); Herold, T. (Tobias); Y. Reyal (Yasmin); M.C. Sauerland (Maria); T. Büchner (Thomas); W. Hiddemann (Wolfgang); S.K. Bohlander (Stefan); P.J.M. Valk (Peter); Bonnet, D. (Dominique)

    2016-01-01

    textabstractWilms Tumor-1 (WT1) expression level is implicated in the prognosis of acute myeloid leukaemia (AML). We hypothesized that a gene expression profile associated with WT1 expression levels might be a good surrogate marker. We identified high WT1 gene sets by comparing the gene expression p

  8. High-throughput cloning and expression in recalcitrant bacteria

    NARCIS (Netherlands)

    Geertsma, Eric R.; Poolman, Bert

    2007-01-01

    We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism

  9. Yeast expression proteomics by high-resolution mass spectrometry

    DEFF Research Database (Denmark)

    Walther, Tobias C; Olsen, Jesper Velgaard; Mann, Matthias

    2010-01-01

    -translational controls contribute majorly to regulation of protein abundance, for example in heat shock stress response. The development of new sample preparation methods, high-resolution mass spectrometry and novel bioinfomatic tools close this gap and allow the global quantitation of the yeast proteome under different...... conditions. Here, we provide background information on proteomics by mass-spectrometry and describe the practice of a comprehensive yeast proteome analysis....

  10. Differential expression of apolipoprotein D in male reproductive system of rats by high-fat diet.

    Science.gov (United States)

    Lim, W; Bae, H; Song, G

    2016-11-01

    Apolipoprotein D, a 29-kDa secreted glycoprotein that belongs to the lipocalin superfamily, is widely expressed in various tissues and associated with lipid metabolism as a component of high-density lipoproteins. Although Apolipoprotein D binds to small hydrophobic ligands including cholesterol, little is known about effects of high-fat diet with cholesterol on expression of Apolipoprotein D in the male reproductive tract. Therefore, we investigated Apod expression in penises, prostate glands, and testes from rats fed a high-fat diet including a high amount of cholesterol. Our previous research indicated that a high-fat diet induces dyslipidemia leading to histological changes and dysfunction of male reproduction in rats. Consistent with these results, Apod mRNA expression was significantly (p high-fat diet as compared with normal diet. In addition, Apod mRNA and protein were detected predominantly in urethral epithelium and penile follicle from rats. Moreover, changes in expression of specific microRNAs (miR-229b-3p, miR-423-3p, and miR-490-3p) regulating Apod in the penises and prostate glands were negatively associated with Apod expression. Collectively, results of this study suggest that Apod is a novel regulatory gene in the male reproductive system, especially in penises of rats fed a high-cholesterol diet, and that expression of Apod is regulated at the posttranscriptional level by target microRNAs.

  11. Apolipoprotein E expression and behavioral toxicity of high charge, high energy (HZE) particle radiation

    Energy Technology Data Exchange (ETDEWEB)

    Higuchi, Yoshinori; Nelson, G.A.; Slater, J.M.; Pearlstein, R.D. [Loma Linda Univ., CA (United States). Medical Center; Vazquez, M. [Brookhaven National Lab., Upton, NY (United States); Laskowitz, D.T. [Duke Univ., Durham, NC (United States). Medical Center

    2002-12-01

    Apolipoprotein E (apoE) is a lipid binding protein that plays an important role in tissue repair following brain injury. In the present studies, we have investigated whether apoE affects the behavioral toxicity of high charge, high energy (HZE) particle radiation. Sixteen male apoE knockout (KO) mice and sixteen genetically matched wild-type (WT) C57BL mice were used in this experiment. Half of the KO and half of the WT animals were irradiated with 600 MeV/amu iron particles (2 Gy whole body). The effect of irradiation on motor coordination and stamina (Rotarod test), exploratory behavior (open field test), and spatial working and reference memory (Morris water maze) was assessed. Rotarod test: Performance was adversely affected by radiation exposure in both KO and WT groups at 30 d after irradiation. By 60 d after radiation, the radiation effect was lost in WT, but still apparent in irradiated KO mice. Open field test: Radiation reduced open field exploratory activity 14, 28, 56, 84, and 168 d after irradiation of KO mice, but had no effect on WT mice. Morris water maze: Radiation adversely affected spatial working memory in the KO mice, but had no discernible effect in the WT mice as assessed 180 d after irradiation. In contrast, irradiated WT mice showed marked impairment of spatial reference memory in comparison to non-irradiated mice, while no effect of radiation was observed in KO mice. These studies show that apoE expression influences the behavioral toxicity of HZE particle radiation and suggest that apoE plays a role in the repair/recovery from radiation injury of the central nervous system (CNS). ApoE deficiency may exacerbate the previously reported effects of HZE particle radiation in accelerating the brain aging process. (author)

  12. High-level expression of the native barley alpha-amylase/subtilisin inhibitor in Pichia pastoris

    DEFF Research Database (Denmark)

    Micheelsen, Pernille Ollendorff; Ostergaard, Peter Rahbek; Lange, Lene

    2008-01-01

    An expression system for high-level expression of the native Hordeum vulgare alpha-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been design...... and characterized by Edman degradation, liquid chromatography mass spectrometry and insoluble blue starch assay, and was shown to possess the same characteristics as wild-type protein purified from barley grains....

  13. Freedom of Expression Laws and the College Press: Lessons Learned from the High Schools.

    Science.gov (United States)

    Paxton, Mark

    This paper examines two recent attempts to enact state freedom of expression laws for public college and university students and discusses the prospects for such laws in the context of state scholastic freedom of expression laws covering high school journalists in six states. It examines the case of Kincaid v. Gibson, which decided that…

  14. NLRP3 Inflammasome Expression and Signaling in Human Diabetic Wounds and in High Glucose Induced Macrophages

    Science.gov (United States)

    Zhang, Xiaotian; Dai, Jiezhi; Li, Li

    2017-01-01

    Introduction. To investigate the contribution and mechanism of NLRP3 inflammasome expression in human wounds in diabetes mellitus and in high glucose induced macrophages. Methods. In the present study, we compared the expression of NLRP3 inflammasome in debridement wound tissue from diabetic and nondiabetic patients. We also examined whether high glucose induces NLRP3 inflammasome expression in cultures THP-1-derived macrophages and the influence on IL-1β expression. Results. The expressions of NLRP3, caspase1, and IL-1β, at both the mRNA and protein level, were significantly higher in wounds of diabetic patients compared with nondiabetic wounds (P CCR7 was significantly upregulated after high glucose stimulation. SiRNA-mediated silencing of NLRP3 expression downregulates the expression of IL-1β. Conclusion. The higher expression of NLRP3, caspase1, and secretion of IL-1β, signaling, and activation might contribute to the hyperinflammation in the human diabetic wound and in high glucose induced macrophages. It may be a novel target to treat the DM patients with chronic wound. PMID:28164132

  15. Highly expressed genes are associated with inverse antisense transcription in mouse

    Indian Academy of Sciences (India)

    Andras Györffy; Pawel Surowiak; Zsolt Tulassay; Balazs Györffy

    2007-08-01

    There is a growing evidence, that antisense transcription might have a key role in a range of human diseases. Although predefined sense–antisense pairs were extensively studied, the antisense expression of the known sense genes is rarely investigated. We retrieved and correlated the expression of sense and antisense sequences of 1182 mouse transcripts to assess the prevalence and to find the characteristic pattern of antisense transcription. We contrasted three Affymetrix MGU74A version 1 mouse genome chips to six MGU74A version 2 chips. For these 1182 transcripts, the version 1 chips contain the antisense sequences of the transcripts presented on the version 2 chips. The original data was taken from the GEO database (GDS431 and GDS432). As the Affymetrix data are semiquantitative, the relative expression levels of antisense partners were analysed. We detected antisense transcription, although the average antisense expression is shifted towards smaller expression values (MGU74A version 1, 516; version 2, 1688). An inverse direct correlation between sense and antisense expression values could be observed at high expression values. At a very high relative expression—above 40,000—the Pearson correlation coefficient is getting closer to −1. Transcripts with high inverse expression ratio may be correlated to the investigated gene (major histocompatibility complex class II trans activator). The ratio of sense to antisense transcripts varied among different chromosomes; on chromosomes 14 and 1 the level of antisense expression was higher than that of sense. We conclude that antisense transcription is a common phenomenon in the mouse genome. The hypothesis of regulatory role of antisense transcripts is supported by the inverse antisense gene expression of highly expressed genes.

  16. SAMSN1 is highly expressed and associated with a poor survival in glioblastoma multiforme.

    Directory of Open Access Journals (Sweden)

    Yong Yan

    Full Text Available OBJECTIVES: To study the expression pattern and prognostic significance of SAMSN1 in glioma. METHODS: Affymetrix and Arrystar gene microarray data in the setting of glioma was analyzed to preliminarily study the expression pattern of SAMSN1 in glioma tissues, and Hieratical clustering of gene microarray data was performed to filter out genes that have prognostic value in malignant glioma. Survival analysis by Kaplan-Meier estimates stratified by SAMSN1 expression was then made based on the data of more than 500 GBM cases provided by The Cancer Genome Atlas (TCGA project. At last, we detected the expression of SAMSN1 in large numbers of glioma and normal brain tissue samples using Tissue Microarray (TMA. Survival analysis by Kaplan-Meier estimates in each grade of glioma was stratified by SAMSN1 expression. Multivariate survival analysis was made by Cox proportional hazards regression models in corresponding groups of glioma. RESULTS: With the expression data of SAMSN1 and 68 other genes, high-grade glioma could be classified into two groups with clearly different prognoses. Gene and large sample tissue microarrays showed high expression of SAMSN1 in glioma particularly in GBM. Survival analysis based on the TCGA GBM data matrix and TMA multi-grade glioma dataset found that SAMSN1 expression was closely related to the prognosis of GBM, either PFS or OS (P<0.05. Multivariate survival analysis with Cox proportional hazards regression models confirmed that high expression of SAMSN1 was a strong risk factor for PFS and OS of GBM patients. CONCLUSION: SAMSN1 is over-expressed in glioma as compared with that found in normal brains, especially in GBM. High expression of SAMSN1 is a significant risk factor for the progression free and overall survival of GBM.

  17. Evaluation of P-glycoprotein (Pgp) expression in human osteosarcoma by high-throughput tissue microarray.

    Science.gov (United States)

    Gao, Yan; Liao, Yunfei; Shen, Jacson K; Feng, Yong; Choy, Edwin; Cote, Gregory; Harmon, David; Mankin, Henry J; Hornicek, Francis J; Duan, Zhenfeng

    2016-09-01

    Survival of osteosarcoma patients is currently limited by the development of metastases and multidrug resistance (MDR). A well-established cause of MDR involves overexpression of P-glycoprotein (Pgp) in tumor cells. However, some discrepancies still exist as to the clinical significance of Pgp in osteosarcoma. We sought to elucidate further whether the Pgp expression correlated with clinical behavior in a series of patients with osteosarcoma via high-throughput tissue microarray (TMA) analysis. Immunohistochemical analysis of Pgp expression in a TMA of 114 specimens with a retrospective review of 70 osteosarcoma patients admitted to the Massachusetts General Hospital (MGH) was performed. High Pgp expression was correlated with metastasis development and poor response to pre-operative chemotherapy in osteosarcoma patients. Eighteen of the fifty-seven patients initially admitted with primary osteosarcoma showed high Pgp expression. Among these 18 patients with high Pgp expression, 13 of 18 (72%) patients eventually developed metastases. There was no significant clinical relevance between Pgp expression and osteosarcoma survival. These results support that high expression of Pgp is important, but cannot be assigned as, an individual predictor in the development of human osteosarcoma. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1606-1612, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  18. High Immunoglobulin A Levels Mediate the Association Between High Anger Expression and Low Somatic Symptoms in Intimate Partner Violence Perpetrators.

    Science.gov (United States)

    Romero-Martínez, A; Lila, M; Vitoria-Estruch, S; Moya-Albiol, L

    2016-02-01

    It has been hypothesized that anger expression may be associated with increased salivary immunoglobulin A (sIgA) levels, which is associated with decreased somatic symptoms, and therefore anger expression may be associated with reduced somatic symptoms in intimate partner violence (IPV) perpetrators. This study tested the potential mediating effect of sIgA levels on the relationship between anger expression and respiratory and gastrointestinal symptoms in IPV perpetrators and non-violent controls. The sample consisted of IPV perpetrators (n = 19) and controls (n = 21). Saliva samples were collected for assessing sIgA levels. The State-Trait Anger Expression Inventory-2 was used to assess anger expression and the Revised version of the Somatic Symptoms Scale developed by Sandín and Chorot to measure somatic symptoms. High anger expression was associated with low levels of respiratory and gastrointestinal symptoms in IPV perpetrators mediated through high sIgA levels but the same was not true for non-violent controls. This finding supports the hypothesis that for IPV perpetrators, anger expression may be physiologically and psychologically rewarding. Future research examining other immunological parameters is needed to further test this hypothesis. Such effort may illuminate why some IPV perpetrators continue to use violence against their partners.

  19. Ezrin is highly expressed in early thymocytes, but dispensable for T cell development in mice.

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    Meredith H Shaffer

    Full Text Available BACKGROUND: Ezrin/radixin/moesin (ERM proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin(-/- mice likely arise as a consequence of nutritional stress. CONCLUSIONS/SIGNIFICANCE: We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.

  20. Evidence against the energetic cost hypothesis for the short introns in highly expressed genes

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    Niu Deng-Ke

    2008-05-01

    Full Text Available Abstract Background In animals, the moss Physcomitrella patens and the pollen of Arabidopsis thaliana, highly expressed genes have shorter introns than weakly expressed genes. A popular explanation for this is selection for transcription efficiency, which includes two sub-hypotheses: to minimize the energetic cost or to minimize the time cost. Results In an individual human, different organs may differ up to hundreds of times in cell number (for example, a liver versus a hypothalamus. Considered at the individual level, a gene specifically expressed in a large organ is actually transcribed tens or hundreds of times more than a gene with a similar expression level (a measure of mRNA abundance per cell specifically expressed in a small organ. According to the energetic cost hypothesis, the former should have shorter introns than the latter. However, in humans and mice we have not found significant differences in intron length between large-tissue/organ-specific genes and small-tissue/organ-specific genes with similar expression levels. Qualitative estimation shows that the deleterious effect (that is, the energetic burden of long introns in highly expressed genes is too negligible to be efficiently selected against in mammals. Conclusion The short introns in highly expressed genes should not be attributed to energy constraint. We evaluated evidence for the time cost hypothesis and other alternatives.

  1. High chondroitin sulfate proteoglycan 4 expression correlates with poor outcome in patients with breast cancer.

    Science.gov (United States)

    Hsu, Nicholas C; Nien, Pei-Yung; Yokoyama, Kazunari K; Chu, Pei-Yi; Hou, Ming-Feng

    2013-11-15

    Chondroitin sulfate proteoglycan 4 (CSPG4), a transmembrane proteoglycan originally identified in melanoma cells, has been reported to be expressed in breast cancer cells. This study was performed to examine the expression and significance of CSPG4 in a cohort of breast cancer patients. Immunohistochemical analysis of CSPG4 was performed on tissue microarrays constructed from tissue specimens from 240 breast cancer patients. CSPG4 staining was correlated with clinical and pathological characteristics, overall survival (OS), and disease recurrence. Contradicting to a previous report, our results showed that high CSPG4 expression was not related to triple-negative status of breast cancer patients. The Kaplan-Meier method showed that high CSPG4 expression was significantly associated with shorter time to recurrence (TTR). Patients with high CSPG4 expression had poorer OS and shorter TTR in a multivariate survival analysis after adjustment for stage, tumor grade, expression of estrogen receptor and progesterone receptor, and HER2 overexpression. This study showed that high CSPG4 expression correlates with disease recurrence and OS in breast cancers.

  2. Global gene expression profiling reveals genes expressed differentially in cattle with high and low residual feed intake.

    Science.gov (United States)

    Chen, Y; Gondro, C; Quinn, K; Herd, R M; Parnell, P F; Vanselow, B

    2011-10-01

    Feed efficiency is an economically important trait in beef production. It can be measured as residual feed intake. This is the difference between actual feed intake recorded over a test period and the expected feed intake of an animal based on its size and growth rate. DNA-based marker-assisted selection would help beef breeders to accelerate genetic improvement for feed efficiency by reducing the generation interval and would obviate the high cost of measuring residual feed intake. Although numbers of quantitative trait loci and candidate genes have been identified with the advance of molecular genetics, our understanding of the physiological mechanisms and the nature of genes underlying residual feed intake is limited. The aim of the study was to use global gene expression profiling by microarray to identify genes that are differentially expressed in cattle, using lines genetically selected for low and high residual feed intake, and to uncover candidate genes for residual feed intake. A long-oligo microarray with 24 000 probes was used to profile the liver transcriptome of 44 cattle selected for high or low residual feed intake. One hundred and sixty-one unique genes were identified as being differentially expressed between animals with high and low residual feed intake. These genes were involved in seven gene networks affecting cellular growth and proliferation, cellular assembly and organization, cell signalling, drug metabolism, protein synthesis, lipid metabolism, and carbohydrate metabolism. Analysis of functional data using a transcriptional approach allows a better understanding of the underlying biological processes involved in residual feed intake and also allows the identification of candidate genes for marker-assisted selection. © 2011 The Authors, Animal Genetics © 2011 Stichting International Foundation for Animal Genetics.

  3. A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

    Directory of Open Access Journals (Sweden)

    Bottomley Stephen P

    2006-03-01

    Full Text Available Abstract Background In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. Results The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. Conclusion The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high

  4. The influence of bovine milk high or low in isoflavones on hepatic gene expression in mice

    DEFF Research Database (Denmark)

    Skaanild, Mette Tingleff; Nielsen, Tina Skau

    2012-01-01

    in hepatic gene expression after dietary intake of milk high and low in isoflavones. In addition to pelleted feed female NMRI mice were offered water, water added either 17β-estradiol, equol, Tween 80, and milk high and low in isoflavone content for a week. Gene expression was analyzed using an array q......Isoflavones have generated much attention due to their potential positive effects in various diseases. Phytoestrogens especially equol can be found in bovine milk, as feed ration for dairy cows is comprised of plants containing phytoestrogens. The aim of this study was to analyze the changes......PCR kit. It was revealed that Tween 80 and 17β-estradiol upregulated both phase I and phase II genes to the same extent whereas equol alone, high and low isoflavone milk did not alter the expression of phase I genes but decreased the expression of phase II genes. This study shows that dietary isoflavones...

  5. Selection for the compactness of highly expressed genes in Gallus gallus

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    Zhou Ming

    2010-05-01

    (n = 1105, and compared the first intron length and the average intron length between highly expressed genes (top 5% expressed genes and weakly expressed genes (bottom 5% expressed genes. We found that the first intron length and the average intron length in highly expressed genes are not different from that in weakly expressed genes. We also made a comparison between ubiquitously expressed genes and narrowly expressed somatic genes with similar expression levels. Our data demonstrated that ubiquitously expressed genes are less compact than narrowly expressed genes with the similar expression levels. Obviously, these observations can not be explained by mutational bias hypotheses either. We also found that the significant trend between genes' compactness and expression level could not be affected by local mutational biases. We argued that the selection of economy model is most likely one to explain the relationship between gene expression and gene characteristics in chicken genome. Conclusion Natural selection appears to favor the compactness of highly expressed genes in chicken genome. This observation can be explained by the selection of economy model. Reviewers This article was reviewed by Dr. Gavin Huttley, Dr. Liran Carmel (nominated by Dr. Eugene V. Koonin and Dr. Araxi Urrutia (nominated by Dr. Laurence D. Hurst.

  6. High expression of testes-specific protease 50 is associated with poor prognosis in colorectal carcinoma.

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    Lei Zheng

    Full Text Available BACKGROUND: Testes-specific protease 50 (TSP50 is normally expressed in testes and abnormally expressed in breast cancer, but whether TSP50 is expressed in colorectal carcinoma (CRC and its clinical significance is unclear. We aimed to detect TSP50 expression in CRC, correlate it with clinicopathological factors, and assess its potential diagnostic and prognostic value. METHODOLOGY/PRINCIPAL FINDINGS: TSP50 mRNAs and proteins were detected in 7 CRC cell lines and 8 CRC specimens via RT-PCR and Western blot analysis. Immunohistochemical analysis of TSP50, p53 and carcinoembryonic antigen (CEA with tissue microarrays composed of 95 CRCs, 20 colorectal adenomas and 20 normal colorectal tissues were carried out and correlated with clinicopathological characteristics and disease-specific survival for CRC patients. There was no significant correlation between the expression levels of TSP50 and p53 (P = 0.751 or CEA (P = 0.663. Abundant expression of TSP50 protein was found in CRCs (68.4% while it was poorly expressed in colorectal adenomas and normal tissues (P<0.0001. Thus, CRCs can be distinguished from them with high specificity (92.5% and positive predictive value (PPV, 95.6%. The survival of CRC patients with high TSP50 expression was significantly shorter than that of the patients with low TSP50 expression (P = 0.010, specifically in patients who had early-stage tumors (stage I and II; P = 0.004. Multivariate Cox regression analysis indicated that high TSP50 expression was a statistically significant independent risk factor (hazard ratio  = 2.205, 95% CI = 1.214-4.004, P = 0.009. CONCLUSION: Our data demonstrate that TSP50 is a potential effective indicator of poor survival for CRC patients, especially for those with early-stage tumors.

  7. High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Fei Zhong; Zhen-Yu Zhong; Shuang Liang; Xiu-Jin Li

    2006-01-01

    AIM: To develop a highly efficacious method for preparation of soluble SARS S-protein using adenovirus vector to meet the requirement for S-protein investigation.METHODS: The human adenovirus vector was used to express the soluble S-protein (corresponding to 1~1190 amino acids) fused with Myc/His tag using codon-optimized gene construct in HEK239 cells. The recombinant adenovirus bearing S-protein gene was generated by ligation method. The expressed S-protein with Myc/His tag was purified from culture medium with Ni-NTA agarose beads followed by dialysis. The S-protein was detected by Western blot and its biologic activity was analyzed by binding to Vero cells.RESULTS: Under the conditions of infection dose (MOI of 50) and expression time (48 h), the high-level expression of S-protein was obtained. The expression level was determined to be approximately 75 μg/106cells after purification. Purified soluble S-protein was readily detected by Western blot with anti-Myc antibody and showed the ability to bind to surface of Vero cells,demonstrating that the soluble S-protein could remain the biologic activity in the native molecule.CONCLUSION: The high-level expression of S-protein in HEK293 cells mediated by adenovirus can be achieved under the optimized expression conditions. The proteins possess the biologic activity, which lays a foundation for further investigation of S-protein biological function.

  8. High KRT8 expression promotes tumor progression and metastasis of gastric cancer.

    Science.gov (United States)

    Fang, Jian; Wang, Hao; Liu, Yun; Ding, Fangfang; Ni, Ying; Shao, Shihe

    2017-02-01

    Keratin8 (KRT8) is the major component of the intermediate filament cytoskeleton and predominantly expressed in simple epithelial tissues. Aberrant expression of KRT8 is associated with multiple tumor progression and metastasis. However, the role of KRT8 in gastric cancer (GC) remains unclear. In this study, KRT8 expression was investigated and it was found to be upregulated along with human GC progression and metastasis at both mRNA and protein levels in human gastric cancer tissues. In addition, KRT8 overexpression enhanced the proliferation and migration of human gastric cancer cells, whereas the knock-down of KRT8 by siRNA only inhibited migration of human gastric cancer cells. Integrinβ1-FAK-induced epithelial-mesenchymal-transition (EMT) only existed in the high KRT8 cells. Furthermore, KRT8 overexpression led to increase in p-smad2/3 levels and TGFβ dependent signaling events. KRT8 expression in GC was related to tumor clinical stage and worse survival. Kaplan-Meier analysis proved that KRT8 was associated with overall survival of patients with GC that patients with high KRT8 expression tend to have unfavorable outcome. Moreover, Cox's proportional hazards analysis showed that high KRT8 expression was a prognostic marker of poor outcome. These results provided that KRT8 expression may therefore be a biomarker or potential therapeutic target to identify patients with worse survival.

  9. High CD133 Expression Is Associated with Worse Prognosis in Patients with Glioblastoma.

    Science.gov (United States)

    Zhang, Wei; Chen, Huanran; Lv, Shengqing; Yang, Hui

    2016-05-01

    The CD133 antigen has been identified as a putative stem cell marker in gliomas. However, the prognostic significance of CD133 expression in glioblastoma patients remained controversial. A meta-analysis of published data was performed to comprehensively assess the prognostic role of CD133 expression in glioblastoma patients. Publications assessing the prognostic significance of CD133 expression in glioblastoma patients were identified in PubMed, Embase, and Web of Science up to November 2014. The pooled hazard ratio (HR) with 95% confidence interval (95% CI) was calculated using meta-analysis to evaluate the prognostic significance of CD133 expression in glioblastoma. Ten studies with a total of 715 glioblastoma patients were included into the meta-analysis. Overall, high CD133 expression was associated with poorer overall survival in patients with glioblastoma (HR = 1.96, 95% CI 1.46-2.64, P glioblastoma (HR = 2.03, 95% CI 1.43-2.88, P glioblastoma. Meta-analysis of studies with adjusted estimates further showed that high CD133 expression was an independent prognostic factor of glioblastoma. High CD133 expression is associated with worse prognosis in patients with glioblastoma. More prospective studies with well-design are needed to confirm this finding.

  10. High expression of p300 is linked to aggressive features and poor prognosis of Nasopharyngeal Carcinoma

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    Liao Zhi-Wei

    2012-05-01

    Full Text Available Abstract Background Increased expression of transcriptional coactivator p300 has been observed in a variety of human cancers. However, the expression status of p300 protein/mRNA in nasopharyngeal carcinoma (NPC tissues and its clinicopathologic/prognostic implication are poorly understood. Methods In our study, mRNA and protein expression levels of p300 was explored by reverse transcription-polymerase chain reaction (RT-PCR, Western blotting (WB and immunohistochemistry (IHC in nasopharyngeal mucosal and NPC tissues. The data were analyzed by receiver operating characteristic (ROC curve analysis, spearman’s rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model. Results Up-regulated expression of p300 mRNA/p300 protein was detected in NPC tissues by RT-PCR and WB, when compared to nasopharyngeal mucosal tissues. Based on ROC curve analysis, the cutoff score for p300 high expression was defined when more than 35% of the tumor cells were positively stained. High expression of p300 was observed in 127/209 (60.7% of NPCs. In NPCs, high expression of p300 was positively associated with later T classification, later N classification, distant metastasis and later clinical stage (P P = 0.002 and overall survival (P = 0.001 in NPCs. More importantly, p300 expression was evaluated as an independent prognostic factor for NPC in multivariate analysis (P = 0.036. Conclusions Our findings support that high expression of p300 protein might be important in conferring a more aggressive behavior, and is an independent molecular marker for shortened survival time of patients with NPC.

  11. Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells.

    Science.gov (United States)

    Page, Andrew F; Minocha, Rakesh; Minocha, Subhash C

    2012-01-01

    Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the

  12. Seizure-related 6,a brain-specific expression gene,is highly expressed in the human cerebellum

    Institute of Scientific and Technical Information of China (English)

    Jianming Jiang; Long Yu; Yangtai Guan; Zhiliang Yu; Xinghua Huang; Xiaosong Chen; Lisha Tang; Xianning Zhang

    2010-01-01

    Epilepsy is a complex,Mendelian disease,and most cases are sporadic.Genomic comparisons of tissue from identified monogenic epilepsies with multigenic and acquired syndromes could ultimately reveal crucial molecular neuropathology for an epileptic phenotype.In the present study,a novel gene,human seizure-related(hSEZ)-6,was isolated from a human brain cDNA library.hSEZ-6 comprises 17 exons and spans a region of at least 55.6 kb,which was localized to 17q12 by radiation hybridization,hSEZ-6 exhibits two isoform types,hSEZ-6A and hSEZ-6B,which encode996 and 995 amino acids,respectively.The two putative hSEZ-6 proteins contain similar motifs and share 82% and 84% identity with mouse SEZ-6A protein,whose expression level increased in mouse cerebral cortex-derived cells treated with a convulsant drug,pentylentetrazole.Northern blot analysis demonstrated that hSEZ-6 is expressed highly in the cerebellum and in nucleus of the extrapyramidal system,such as the caudate nucleus and putamen.Reverse transcription polymerase chain reaction revealed that hSEZ-6 is expressed in neurons rather than gliocytes,which suggests that hSEZ-6 is a seizure-related gene.

  13. Reduced Popdc3 expression correlates with high risk and poor survival in patients with gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Deng Luo; Ming-Liang Lu; Gong-Fang Zhao; Hua Huang; Meng-Yao Zheng; Jiang Chang; Lin Lv

    2012-01-01

    AIM:To investigate the expression of Popeye domain containing 3 (Popdc3) and its correlation with clinicopathological features and prognosis of gastric cancer.METHODS:The method of immunohistochemistry was used to investigate the expression of Popdc3 in 306 cases of human gastric cancer and 84 noncancerous gastric tissues.Simultaneously,the relationship between Popdc3 expression and the survival of the patients was retrospectively analyzed.RESULTS:Popdc3 was detected in 72 (85.71%) of 84 human nontumor mucosa.High expression of Popdc3 protein was detected in 78 (25.49%) of 306 human gastric cancer cases,and low expression was detected in 228 (74.51%).Low expression of Popdc3 correlated with depth of invasion (P < 0.0001),regional lymph nodes (P < 0.0001) and distant metastasis (P =0.02),and tumor,nodes,metastasis (TNM) stages (P< 0.0001).On multivariate analysis,only the patient's gender,regional lymph node metastasis,distant metastasis,TNM stages,and the expression of Popdc3 were independent prognostic factors in patients with gastric cancer.The Kaplan-Meier plot showed that low Popdc3 expression had a much more significant effect on the survival of those patients with early-stage tumors (x2 =104.741,P < 0.0001),with a > 51.9% reduction in the three-year survival compared with high Popdc3 expression.In late stages,the difference was also significant (x2 =5.930,P =0.015),with a 32.6% reduction in the three-year survival.CONCLUSION:Reduced expression of Popdc3 may play a significant role in the carcinogenesis and progression of gastric cancer.Popdc3 may be an independent prognostic factor.

  14. Expression of NDRG2 is down-regulated in high-risk adenomas and colorectal carcinoma

    DEFF Research Database (Denmark)

    Lorentzen, Anders; Vogel, Lotte K.; Lewinsky, Rikke H;

    2007-01-01

    BACKGROUND: It has recently been shown that NDRG2 mRNA is down-regulated or undetectable in several human cancers and cancer cell-lines. Although the function of NDRG2 is unknown, high NDRG2 expression correlates with improved prognosis in high-grade gliomas. The aim of this study has been to exa...

  15. Endothelial lipase is highly expressed in macrophages in advanced human atherosclerotic lesions

    DEFF Research Database (Denmark)

    Bartels, Emil D; Nielsen, John E; Lindegaard, Marie Louise Skakkebæk

    2007-01-01

    RNA expression increased markedly when either type of monocytes was differentiated into macrophages. Upon further differentiation into foam cells EL mRNA decreased whereas protein levels remained high compared to monocytes. In conclusion, macrophages in advanced human atherosclerotic lesions display high levels...

  16. High ubiquitous mitochondrial creatine kinase expression in hepatocellular carcinoma denotes a poor prognosis with highly malignant potential.

    Science.gov (United States)

    Uranbileg, Baasanjav; Enooku, Kenichiro; Soroida, Yoko; Ohkawa, Ryunosuke; Kudo, Yotaro; Nakagawa, Hayato; Tateishi, Ryosuke; Yoshida, Haruhiko; Shinzawa, Seiko; Moriya, Kyoji; Ohtomo, Natsuko; Nishikawa, Takako; Inoue, Yukiko; Tomiya, Tomoaki; Kojima, Soichi; Matsuura, Tomokazu; Koike, Kazuhiko; Yatomi, Yutaka; Ikeda, Hitoshi

    2014-05-01

    We previously reported the increased serum mitochondrial creatine kinase (MtCK) activity in patients with hepatocellular carcinoma (HCC), mostly due to the increase in ubiquitous MtCK (uMtCK), and high uMtCK mRNA expression in HCC cell lines. We explored the mechanism(s) and the relevance of high uMtCK expression in HCC. In hepatitis C virus core gene transgenic mice, known to lose mitochondrial integrity in liver and subsequently develop HCC, uMtCK mRNA and protein levels were increased in HCC tissues but not in non-tumorous liver tissues. Transient overexpression of ankyrin repeat and suppressor of cytokine signaling box protein 9 (ASB9) reduced uMtCK protein levels in HCC cells, suggesting that increased uMtCK levels in HCC cells may be caused by increased gene expression and decreased protein degradation due to reduced ASB9 expression. The reduction of uMtCK expression by siRNA led to increased cell death, and reduced proliferation, migration and invasion in HCC cell lines. Then, consecutive 105 HCC patients, who underwent radiofrequency ablation with curative intent, were enrolled to analyze their prognosis. The patients with serum MtCK activity >19.4 U/L prior to the treatment had significantly shorter survival time than those with serum MtCK activity ≤ 19.4 U/L, where higher serum MtCK activity was retained as an independent risk for HCC-related death on multivariate analysis. In conclusion, high uMtCK expression in HCC may be caused by hepatocarcinogenesis per se but not by loss of mitochondrial integrity, of which ASB9 could be a negative regulator, and associated with highly malignant potential to suggest a poor prognosis.

  17. A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker

    Directory of Open Access Journals (Sweden)

    Mueller-Roeber Bernd

    2010-05-01

    . The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources.

  18. Establishment of Stable High Expression Cell Line with Green Fluorescent Protein and Resistance Genes

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shengtao; LIU Wenli; HE Peigen; GONG Feili; YANG Dongliang

    2006-01-01

    In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

  19. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    Directory of Open Access Journals (Sweden)

    Meili Gao

    2012-01-01

    Full Text Available Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed.

  20. High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

    DEFF Research Database (Denmark)

    Shin, Yun-Ji; Hong, Shin-Young; Kwon, Tae-Ho;

    2003-01-01

    this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield...

  1. LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2.

    Directory of Open Access Journals (Sweden)

    Maryem A Hussein

    Full Text Available High cholesterol and diabetes are major risk factors for atherosclerosis. Regression of atherosclerosis is mediated in part by the Liver X Receptor (LXR through the induction of genes involved in cholesterol transport and efflux. In the context of diabetes, regression of atherosclerosis is impaired. We proposed that changes in glucose levels modulate LXR-dependent gene expression. Using a mouse macrophage cell line (RAW 264.7 and primary bone marrow derived macrophages (BMDMs cultured in normal or diabetes relevant high glucose conditions we found that high glucose inhibits the LXR-dependent expression of ATP-binding cassette transporter A1 (ABCA1, but not ABCG1. To probe for this mechanism, we surveyed the expression of a host of chromatin-modifying enzymes and found that Protein Arginine Methyltransferase 2 (PRMT2 was reduced in high compared to normal glucose conditions. Importantly, ABCA1 expression and ABCA1-mediated cholesterol efflux were reduced in Prmt2-/- compared to wild type BMDMs. Monocytes from diabetic mice also showed decreased expression of Prmt2 compared to non-diabetic counterparts. Thus, PRMT2 represents a glucose-sensitive factor that plays a role in LXR-mediated ABCA1-dependent cholesterol efflux and lends insight to the presence of increased atherosclerosis in diabetic patients.

  2. Expression and Effects of High-Mobility Group Box 1 in Cervical Cancer

    Directory of Open Access Journals (Sweden)

    Xiaoao Pang

    2014-05-01

    Full Text Available We investigated the significance of high- mobility group box1 (HMGB1 and T-cell-mediated immunity and prognostic value in cervical cancer. HMGB1, forkhead/winged helix transcription factor p3 (Foxp3, IL-2, and IL-10 protein expression was analyzed in 100 cervical tissue samples including cervical cancer, cervical intraepithelial neoplasia (CIN, and healthy control samples using immunohistochemistry. Serum squamous cell carcinoma antigen (SCC-Ag was immunoradiometrically measured in 32 serum samples from 37 cases of squamous cervical cancer. HMGB1 and SCC-Ag were then correlated to clinicopathological characteristics. HMGB1 expression tends to increase as cervical cancer progresses and it was found to be significantly correlated to FIGO stage and lymph node metastasis. These findings suggest that HMGB1 may be a useful prognostic indicator of cervical carcinoma. In addition, there were significant positive relationships between HMGB1 and FOXP3 or IL-10 expression (both p < 0.05. In contrast, HMGB1 and IL-2 expression was negatively correlated (p < 0.05. HMGB1 expression may activate Tregs or facilitate Th2 polarization to promote immune evasion of cervical cancer. Elevated HMGB1 protein in cervical carcinoma samples was associated with a high recurrence of HPV infection in univariate analysis (p < 0.05. HMGB1 expression and levels of SCC-Ag were directly correlated in SCC (p < 0.05. Thus, HMGB1 may be a useful biomarker for patient prognosis and cervical cancer prediction and treatment.

  3. Genomic Imprinting of IGF2 Is Maintained in Infantile Hemangioma despite its High Level of Expression

    Science.gov (United States)

    Yu, Ying; Wylie-Sears, Jill; Boscolo, Elisa; Mulliken, John B; Bischoff, Joyce

    2004-01-01

    Hemangioma, the most common tumor of infancy, is characterized by rapid growth and slow regression. Increased mRNA expression of insulin-like growth factor 2 (IGF2) has been detected in the proliferating phase by cDNA microarray analysis, but the underlying mechanism causing the increase remains unknown. Here, using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry, we show that IGF2 is highly expressed in both proliferating and involuting phase hemangioma, but is not detectable in other vascular lesions such as pyogenic granuloma, venous malformation, lymphatic malformation, or in normal infant skin. Loss of imprinting of the Igf2 gene has been associated with IGF2 overexpression in a variety of childhood tumors. To determine if loss of imprinting and consequent bi-allelic expression might contribute to the increased expression of IGF2, we examined the genomic imprinting status of Igf2 in 48 individual hemangiomas. We determined allele-specific Igf2 expression using reverse transcriptase–PCR combined with analysis of an Apa I–sensitive restriction fragment length polymorphism. Similar to heterozygous normal skin controls, all 15 informative hemangiomas showed uniform mono-allelic expression of Igf2. Therefore, loss of imprinting is not involved in the increased expression of IGF2 in infantile hemangioma. PMID:15706404

  4. Neoadjuvant multidrug chemotherapy including High-Dose Methotrexate modifies VEGF expression in Osteosarcoma: an immunohistochemical analysis

    Directory of Open Access Journals (Sweden)

    Rosa Michele A

    2010-02-01

    Full Text Available Abstract Background Angiogenesis plays a role in the progression of osteosarcoma, as well as in other mesenchymal tumors and carcinomas, and it is most commonly assessed by vascular endothelial growth factor (VEGF expression or tumor CD31-positive microvessel density (MVD. Tumor VEGF expression is predictive of poor prognosis, and chemotherapy can affect the selection of angiogenic pattern. The aim of the study was to investigate the clinical and prognostic significance of VEGF and CD31 in osteosarcoma, both at diagnosis and after neoadjuvant chemotherapy, in order to identify a potential role of chemotherapy in angiogenic phenotype. Methods A retrospective analysis was performed on 16 patients with high grade osteosarcoma. In each case archival pre-treatment biopsy tissue and post-chemotherapy tumor specimens were immunohistochemically stained against CD31 and VEGF, as markers of angiogenic proliferation both in newly diagnosed primary osteosarcoma and after multidrug chemotherapy including high-dose methotrexate (HDMTX. The correlation between clinicopathological parameters and the degree of tumor VEGF and CD31 expression was statistically assessed using the χ2 test verified with Yates' test for comparison of two groups. Significance was set at p Results Expression of VEGF was positive in 11 cases/16 of cases at diagnosis. Moreover, 8 cases/16 untreated osteosarcomas were CD31-negative, but the other 8 showed an high expression of CD31. VEGF expression in viable tumor cells after neoadjuvant chemotherapy was observed in all cases; in particular, there was an increased VEGF expression (post-chemotherapy VEGF - biopsy VEGF in 11 cases/16. CD31 expression increased in 11 cases/16 and decreased in 3 cases after chemotherapy. The data relating to the change in staining following chemotherapy appear statistically significant for VEGF expression (p p > 0,05. Conclusions Even if the study included few patients, these results confirm that VEGF and CD

  5. Analysis of DNA strand-specific differential expression with high density tiling microarrays

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    Antequera Francisco

    2010-03-01

    Full Text Available Abstract Background DNA microarray technology allows the analysis of genome structure and dynamics at genome-wide scale. Expression microarrays (EMA contain probes for annotated open reading frames (ORF and are widely used for the analysis of differential gene expression. By contrast, tiling microarrays (TMA have a much higher probe density and provide unbiased genome-wide coverage. The purpose of this study was to develop a protocol to exploit the high resolution of TMAs for quantitative measurement of DNA strand-specific differential expression of annotated and non-annotated transcripts. Results We extensively filtered probes present in Affymetrix Genechip Yeast Genome 2.0 expression and GeneChip S. pombe 1.0FR tiling microarrays to generate custom Chip Description Files (CDF in order to compare their efficiency. We experimentally tested the potential of our approach by measuring the differential expression of 4904 genes in the yeast Schizosaccharomyces pombe growing under conditions of oxidative stress. The results showed a Pearson correlation coefficient of 0.943 between both platforms, indicating that TMAs are as reliable as EMAs for quantitative expression analysis. A significant advantage of TMAs over EMAs is the possibility of detecting non-annotated transcripts generated only under specific physiological conditions. To take full advantage of this property, we have used a target-labelling protocol that preserves the original polarity of the transcripts and, therefore, allows the strand-specific differential expression of non-annotated transcripts to be determined. By using a segmentation algorithm prior to generating the corresponding custom CDFs, we identified and quantitatively measured the expression of 510 transcripts longer than 180 nucleotides and not overlapping previously annotated ORFs that were differentially expressed at least 2-fold under oxidative stress. Conclusions We show that the information derived from TMA

  6. Highly expressed genes in human high grade gliomas: immunohistochemical analysis of data from the Human Protein Atlas

    Directory of Open Access Journals (Sweden)

    Michael A. Meyer

    2014-06-01

    Full Text Available Gene expression within human glioblastomas were analyzed from data on 20,083 genes entered into the on-line Human Protein Atlas. In selecting genes that are strongly expressed within normal human brain tissue, 58 genes were identified from a search of the 20,083 entries that were rated as showing 90% or greater intensity of expression within normal brain tissues. Of these 58, a subset of 48 genes was identified that not only had expression data for human glioblastomas but also for the human glioblastoma cell line U-251. Four of these 48 selected genes were found to be strongly expressed within the cytoplasm when assessed by both histologic sampling of high grade glioma patient cases as well as U-251 glioblastoma cell line immunofluoresence analysis. These four human genes are: AGBL2 (ATP/GTP binding protein-like 2, BLOC1S6 (biogenesis of lysosomal organelles complex-1, subunit 6, MAP1A (microtubule-associated protein 1A and ZSWIM5 (zinc finger, SWIM-type containing 5, also known as KIAA1511. Further research is advocated to investigate the role of ZSWIM5 and AGBL2 in glioma cell biology.

  7. Analysis on differential expressed genes of ovarian tissue between high- and low-yield laying hen.

    Science.gov (United States)

    Chen, Wei; Song, Ling-Jun; Zeng, Yong-Qing; Yang, Yun; Wang, Hui

    2013-01-01

    In order to elucidate molecular genetic mechanism of laying hen reproduction at the transcriptional level and the structure of significantly differential genes, the mRNA differential display and reverse northern dot-blot were used to detect the differential expression of genes in the ovary tissue of low-yield laying hens and high-yield laying hens in the present study. Sixteen 32-week-old CAU-pink laying hens divided into two groups were used and the laying performance was measured. The results showed that only the egg numbers were significantly different between the two groups; and from 15 primer pairs, a total of 336 bands were displayed of which 59 cDNA bands were found to be differentially expressed in both high-yield and low-yield laying hen. The sequence analysis indicated that the expression of such bands as H-AP5, H-P5, and H-P4 was significantly potentiated in high-yield laying hen using primer pairs AP5/HT11G, P5/HT11G and P4/HT11G and these transcripts had high homology (98%) to HoxDb, HoxCa, and HoxBa, respectively. The differentially expressed gene fragments may be relevant to the progression of the high-yield hens to the egg-laying stage. And further study is required to elucidate the molecular function to improve the productivity of laying hens.

  8. High expression of interleukin-22 and its receptor predicts poor prognosis in pancreatic ductal adenocarcinoma.

    Science.gov (United States)

    Wen, Zhang; Liao, Quan; Zhao, Jianguo; Hu, Ya; You, Lei; Lu, Zhaohui; Jia, Congwei; Wei, Yingxin; Zhao, Yupei

    2014-01-01

    The cytokine interleukin-22 (IL-22) and its receptor are present in the tumor microenvironment. Their function in pancreatic ductal adenocarcinoma (PDAC) remains largely unknown. The goal of the present study was to measure the expression of IL-22 and IL-22R in PDAC and assess their relationship with clinicopathological features and prognosis. The expression of IL-22 and IL-22R was evaluated by immunohistochemistry in PDAC tissues from 57 patients and by Western blotting in six tumors and adjacent nontumor tissues. A statistical analysis was conducted to assess the relationship between levels of expression, clinicopathological factors, and overall survival. In addition, the relationship between the expression of IL-22 and IL-22R and invasion was assessed by Western blotting and transwell assay with the PDAC cell lines PANC1 and BxPC3. Positive IL-22 staining was detected in PDAC tissues and adjacent nontumor tissues. Positive IL-22R staining was detected in PDAC cells. High expression of IL-22 and IL-22R correlated significantly with lymph node involvement. IL-22 increased the phosphorylation of signal transducer and activator of transcription3, the expression of matrix metalloproteinase 9, and the invasion in PANC1 and BxPC3 cells in vitro while silencing of IL-22R RNA caused opposite effects. Most importantly, overall survival was significantly poorer in patients with high expression of IL-22 and IL-22R than in those with low expression. These findings reveal the positive role of IL-22 and IL-22R in invasion and metastasis in human PDAC. IL-22 and IL-22R may be suitable independent prognostic markers in PDAC.

  9. Effects of BST and high energy diet on gene expression in mammary parenchyma of dairy heifers

    Directory of Open Access Journals (Sweden)

    Betina Joyce Lew

    2013-07-01

    Full Text Available The objective of this study was to determine the effects of dietary energy and recombinant bovine somatotropin (bST injection to identify genes that might control mammogenesis. Total RNA was extracted from the parenchymal tissue of 32 heifers randomly assigned to one of four treatments: two diets (a standard diet and a high energy, high protein diet, each with or without bST. To perform microarray experiments, RNA samples were pooled (2 animals/pool before reverse transcription and labeling with Cy3 or Cy5. A 4-node loop design was used to examine the differential gene expression among treatments using a bovine-specific cDNA microarray (National Bovine Functional Genomics Consortium Library, NBFGC containing 18,263 unique expressed sequence tags (EST. Significance levels of differential gene expression among treatments were assessed using a mixed model approach. Injection of bST altered the expression of 12 % of the genes on NBFGC slide related to tissue development, whereas 6% were altered by diet. Administration of bST increases the expression of genes positively related to cell proliferation and mammary parenchyma to a greater extent than a high energy diet.

  10. Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase.

    Science.gov (United States)

    Romero, Lucía Virginia; Targovnik, Alexandra Marisa; Wolman, Federico Javier; Cascone, Osvaldo; Miranda, María Victoria

    2011-05-01

    A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27 °C instead of at 24 °C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V(max) and K(m) values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots.

  11. Screening differentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Nan Cui; Jian-Wu Tang; Li Hou; Bo Song; Li Li; Ji-Wei Liu

    2005-01-01

    AIM: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis.METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed.RESULTS: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes.CONCLUSION: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.

  12. Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut.

    Science.gov (United States)

    Allen, Aron; Islamovic, Emir; Kaur, Jagdeep; Gold, Scott; Shah, Dilip; Smith, Thomas J

    2011-10-01

    The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins.

  13. High Glucose Represses hERG K+ Channel Expression through Trafficking Inhibition

    Directory of Open Access Journals (Sweden)

    Yuan-Qi Shi

    2015-08-01

    Full Text Available Background/Aims: Abnormal QT prolongation is the most prominent cardiac electrical disturbance in patients with diabetes mellitus (DM. It is well known that the human ether-ago-go-related gene (hERG controls the rapid delayed rectifier K+ current (IKr in cardiac cells. The expression of the hERG channel is severely down-regulated in diabetic hearts, and this down-regulation is a critical contributor to the slowing of repolarization and QT prolongation. However, the intracellular mechanisms underlying the diabetes-induced hERG deficiency remain unknown. Methods: The expression of the hERG channel was assessed via western blot analysis, and the hERG current was detected with a patch-clamp technique. Results: The results of our study revealed that the expression of the hERG protein and the hERG current were substantially decreased in high-glucose-treated hERG-HEK cells. Moreover, we demonstrated that the high-glucose-mediated damage to the hERG channel depended on the down-regulation of protein levels but not the alteration of channel kinetics. These discoveries indicated that high glucose likely disrupted hERG channel trafficking. From the western blot and immunoprecipitation analyses, we found that high glucose induced trafficking inhibition through an effect on the expression of Hsp90 and its interaction with hERG. Furthermore, the high-glucose-induced inhibition of hERG channel trafficking could activate the unfolded protein response (UPR by up-regulating the expression levels of activating transcription factor-6 (ATF-6 and the ER chaperone protein calnexin. In addition, we demonstrated that 100 nM insulin up-regulated the expression of the hERG channel and rescued the hERG channel repression caused by high glucose. Conclusion: The results of our study provide the first evidence of a high-glucose-induced hERG channel deficiency resulting from the inhibition of channel trafficking. Furthermore, insulin promotes the expression of the hERG channel

  14. Differential gene expression in patients with anal fistula reveals high levels of prolactin recepetor

    Directory of Open Access Journals (Sweden)

    Song Yi-Huan

    2017-01-01

    Full Text Available Background/Aim. There are limited data examining variations in the local expression of inflammatory mediators in anal fistulas where it is anticipated that an improved understanding of the inflammatory milieu might lead to the potential therapeutic option of instillation therapy in complicated cases. The aim of the present study was to examine prolactin receptors (PRLR as inflammatory markers and to correlate their expression with both the complexity of anal fistulas and the likelihood of fistula recurrence. Methods. Microarray was used to screen the differentially expressed gene profile of anal fistula using anal mucosa samples with hemorrhoids with ageand sex-matched patients as controls and then a prospective analysis of 65 patients was conducted with anal fistulas. PRLR immunohistochemistry was performed to define expression in simple, complex and recurrent anal fistula cases. The quantitative image comparison was performed combining staining intensity with cellular distribution in order to create high and low score PRLR immunohistochemical groupings. Results. A differential expression profile of 190 genes was found. PRLR expression was 2.91 times lower in anal fistula compared with control. Sixty-five patients were assessed (35 simple, 30 complex cases. Simple fistulas showed significantly higher PRLR expression than complex cases with recurrent fistulae showing overall lower PRLR expression than de novo cases (p = 0.001. These findings were reflected in measurable integrated optical density for complex and recurrent cases (complex cases, 8.31 ± 4.91 x 104 vs simple cases, 12.30 ± 6.91 x 104; p < 0.01; recurrent cases, 7.21 ± 3.51 x 104 vs primarily healing cases, 8.31 ± 4.91 x 104; p < 0.05. In univariate regression analysis, low PRLR expression correlated with fistula complexity; a significant independent effect maintained in multivariate analysis odds ratio [(OR low to high PRLR expression = 9.52; p = 0.001]. Conclusion. PRLR

  15. Propofol inhibits high glucose-induced PP2A expression in human umbilical vein endothelial cells.

    Science.gov (United States)

    Wu, Qichao; Zhao, Yanjun; Duan, Wenming; Liu, Yi; Chen, Xiangyuan; Zhu, Minmin

    2017-04-01

    Perioperative hyperglycemia is a common clinical metabolic disorder. Hyperglycemia could induce endothelial apoptosis, dysfunction and inflammation, resulting in endothelial injury. Propofol is a widely used anesthetic drug in clinical settings. Our previous studies indicated that propofol, via inhibiting high glucose-induced phosphatase A2 (PP2A) expression, attenuated high glucose-induced reactive oxygen species (ROS) accumulation, thus improving endothelial apoptosis, dysfunction and inflammation. However, the mechanisms by which propofol attenuated high glucose-induced PP2A expression is still obscure. In the present study, we examined how propofol attenuates high glucose-induced endothelial PP2A expression. Compared with 5mM glucose treatment, 15mM glucose up-regulated expression and activity of PP2A, increased cAMP response element binding protein (CREB), Ca(2+)-calmodulin dependent kinase II (CaMK II) phosphorylation and Ca(2+) accumulation. More importantly, propofol decreased PP2A expression and activity, attenuated CREB, CaMK II phosphorylation and Ca(2+) accumulation in a concentration-dependent manner. Moreover, we demonstrated that the effect of propofol was similar to that of MK801, an inhibitor of NMDA receptor. In contrast, rapastinel, an activator of NMDA receptor, antagonized the effect of propofol. Also, the effect of KN93, an inhibitor of CaMK II, was similar to that of propofol, except KN93 had no effect on 15mM glucose-mediated Ca(2+) accumulation. Our data indicated that propofol, via inhibiting NMDA receptor, attenuated 15mM glucose-induced Ca(2+) accumulation, CaMK II and CREB phosphorylation, thus inhibiting PP2A expression and improving 15mM glucose-induced endothelial dysfunction and inflammation. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. RNA-Seq analysis of high NaCl-induced gene expression.

    Science.gov (United States)

    Izumi, Yuichiro; Yang, Wenjing; Zhu, Jun; Burg, Maurice B; Ferraris, Joan D

    2015-10-01

    High extracellular NaCl is known to change expression of numerous genes, many of which are regulated by the osmoprotective transcription factor nuclear factor of activated T cells-5 (NFAT5). In the present study we employed RNA-Seq to provide a comprehensive, unbiased account of genes regulated by high NaCl in mouse embryonic fibroblast cells (MEFs). To identify genes regulated by NFAT5 we compared wild-type MEFs (WT-MEFs) to MEFs in which mutation of the NFAT5 gene inhibits its transcriptional activity (Null-MEFs). In WT-MEFs adding NaCl to raise osmolality from 300 to 500 mosmol/kg for 24 h increases expression of 167 genes and reduces expression of 412. Raising osmolality through multiple passages (adapted cells) increases expression of 196 genes and reduces expression of 528. In Null-MEFs, after 24 h of high NaCl, expression of 217 genes increase and 428 decrease, while in adapted Null-MEFs 143 increase and 622 decrease. Fewer than 10% of genes are regulated in common between WT- and null-MEFs, indicating a profound difference in regulation of high-NaCl induced genes induced by NFAT5 compared with those induced in the absence of NFAT5. Based on our findings we suggest a mechanism for this phenomenon, which had previously been unexplained. The NFAT5 DNA-binding motif (osmotic response element) is overrepresented in the vicinity of genes that NFAT5 upregulates, but not genes that it downregulates. We used Gene Ontology and manual curation to determine the function of the genes targeted by NFAT5, revealing many novel consequences of NFAT5 transcriptional activity.

  17. Optimized over-expression of [FeFe] hydrogenases with high specific activity in Clostridium acetobutylicum

    Energy Technology Data Exchange (ETDEWEB)

    von Abendroth, Gregory; Stripp, Sven; Happe, Thomas [Ruhr-Universitaet Bochum, Lehrstuhl fuer Biochemie der Pflanzen, AG Photobiotechnologie, 44780 Bochum (Germany); Silakov, Alexey [Max-Planck-Institut fuer Bioanorganische Chemie, 45470 Muelheim an der Ruhr (Germany); Croux, Christian; Soucaille, Philippe; Girbal, Laurence [UMR5504, UMR792 Ingenierie des Systemes Biologiques et des Procedes, CNRS, INRA, INSA, 31400 Toulouse (France)

    2008-11-15

    It was previously shown that Clostridium acetobutylicum is capable to over-express various [FeFe] hydrogenases although the protein yield was low. In this study we report on doubling the yield of the clostridial hydrogenase by replacing the native gene hydA1{sub Ca} with a recombinant one via homologous recombination. The purified protein HydA1{sub Ca} shows an unexpected high specific activity (up to 2257 {mu}mol H{sub 2} min{sup -1} mg{sup -1}) for hydrogen evolution. Furthermore, the highly active green algal hydrogenase HydA1{sub Cr} from Chlamydomonas reinhardtii was heterologously expressed in C. acetobutylicum, and purified with increased yield (1 mg protein per liter of cells) and high activity (625 {mu}mol H{sub 2} min{sup -1} mg{sup -1}). EPR studies demonstrate intact H-clusters for homologously and heterologously expressed [FeFe] hydrogenases in the CO-inhibited oxidized redox state, and prove the high efficiency of the C. acetobutylicum expression system. (author)

  18. High glucose-induced oxidative stress increases transient receptor potential channel expression in human monocytes

    DEFF Research Database (Denmark)

    Wuensch, Tilo; Thilo, Florian; Krueger, Katharina;

    2010-01-01

    Transient receptor potential (TRP) channel-induced cation influx activates human monocytes, which play an important role in the pathogenesis of atherosclerosis. In the present study, we investigated the effects of high glucose-induced oxidative stress on TRP channel expression in human monocytes....

  19. Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

    DEFF Research Database (Denmark)

    Moe, Maren; Meuwissen, Theo; Lien, Sigbjørn

    2007-01-01

    Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low...

  20. Expression of interferon gamma by a highly virulent Newcastle disease virus decreases its pathogenicity in chickens

    Science.gov (United States)

    Infection of chickens with highly virulent NDV results in rapid death, which is preceded by increased expression of interferon gamma (IFN-g) in target tissues. IFN-g is a cytokine that has pleiotropic biological effects including intrinsic antiviral activity and immunomodulatory effects. Here we a...

  1. Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.

    NARCIS (Netherlands)

    Takano, Eriko; White, Janet; Thompson, Charles J.; Bibb, Mervyn J.

    1995-01-01

    A high-copy-number plasmid expression vector (pIJ6021) was constructed that contains a thiostrepton-inducible promoter, PtipA, from Streptomyces lividans 66. The promoter and ribosome-binding site of tipA lie immediately upstream from a multiple cloning site (MCS) which begins with a NdeI site (5'-C

  2. Changes and significance of SIRT3 expression in cellular senescence induced by high glucose

    Directory of Open Access Journals (Sweden)

    Bin ZHANG

    2011-09-01

    Full Text Available Objective To investigate the role of the silent information regulator 3(SIRT3 in the decrepitude process of human diploid fibroblasts(WI-38 induced by high glucose.Methods The WI-38 cells [population doublings(PDs,20-32] were cultured in media containing different concentrations of glucose as follows: low glucose(3.34mmol/L,LG,normal glucose(5.56mmol/L,NG,and high glucose(25mmol/L,HG.The protein expression levels of p21,p27,catalase,MnSOD,and SIRT3 were evaluated through Western blot.The double-label immunofluorescence assay was used to detect the location and expression of SIRT3,MnSOD,and senescence-associated heterochromatin foci(SAHF in the WI-38 cells.The ROS level was determined with fluorescent probe.Results The results from the Western blot showed that the protein expression of SIRT3,catalase,and MnSOD decreased significantly in the HG group compared with the LG and NG groups(P 0.05.SIRT3 and MnSOD were highly expressed in the cytoplasm.The ROS levels in the HG group were elevated compared with those in the LG and NG groups.Conclusion SIRT3 may play an important role in cellular senescence induced by high glucose in human diploid fibroblasts.

  3. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Science.gov (United States)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  4. The R package FANet: sparse factor analysis model for high dimensional gene co-expression networks

    OpenAIRE

    Blum, Anne; Houee-Bigot, Magalie; Lagarrigue, Sandrine; Causeur, David

    2014-01-01

    Inference on gene regulatory networks from high-throughput expression data turns out to be one of the main current challenges in systems biology. Such interaction networks are very insightful for the deep understanding of biological relationships between genes. In particular, a functional characterization of gene modules of highly interacting genes enables the identification of biological processes underlying complex traits as diseases. Inference on this dependence structure shall...

  5. Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors

    OpenAIRE

    Giritch, Anatoli; Marillonnet, Sylvestre; Engler, Carola; van Eldik, Gerben; Botterman, Johan; Klimyuk, Victor; Gleba, Yuri

    2006-01-01

    Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses tha...

  6. Pulmonary Large Cell Carcinoma Displays High Expression of EMMPRIN and VEGF

    Institute of Scientific and Technical Information of China (English)

    Yushuang Zheng; Miao Yu; Huachuan Zheng; Yifu Guan; Yasuo Takano

    2008-01-01

    OBJECTIVE To investigate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and vascular endothelial growth factor (VEGF) in lung carcinomas,and to clarify their roles in carcinoma progression.METHODS Expression of EMMPRIN and VEGF was examined with tissue microarrays (TMAs) of lung carcinomas (n = 181),and their suppression in adjacent normal lung samples (n = 40) were determined by immunohistochemistry.The results were compared with clinicopathological findings for the same tumors.RESULTS Both EMMPRIN and VEGF were occasionally expressed in pseudostratified columnar epithelium and frequently in lung carcinomas.Histologically,EMMPRIN and VEGF displayed higher levels in large (LCC) cell carcinomas than adenocarcinoma (AD),squamous (SQ) and small cell carcinomas (SCC) (P < 0.05).EMMPRIN was more highly expressed in SQ as compared with AD (P < 0.05),while the converse was true for VEGF (P < 0.05).Binding was generally more intense for EMMPRIN in samples from male compared to female patients (P < 0.05),whereas the latter tended to exhibit more VEGF expression (P < 0.05).Positive associations of VEGF expression with the TNM stage and amounts of EMMPRIN were noted in the lung carcinomas (P < 0.05).CONCLUSION EMMPRIN and VEGF possibly contribute to physiological repair of normal lung and histogenesis of lung carcinoma.Both proteins might be involved in the molecular basis for differences in the incidence of lung carcinoma between men and women.

  7. A novel baculovirus-derived promoter with high activity in the baculovirus expression system

    Directory of Open Access Journals (Sweden)

    María Martínez-Solís

    2016-06-01

    Full Text Available The baculovirus expression vector system (BEVS has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV. Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV vector system in different insect cell lines (Sf21, Se301, and Hi5 and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.

  8. High expression of FOXR2 in breast cancer correlates with poor prognosis.

    Science.gov (United States)

    Song, Haiping; He, Wenshan; Huang, Xiaoqing; Zhang, Huiqiong; Huang, Tao

    2016-05-01

    Forkhead box protein R2 (FOXR2) is associated with human central nervous system neoplasms. However, the expression level of FOXR2 in breast cancer specimens remains largely unknown. To identify whether FOXR2 can serve as a biomarker for the diagnosis and prognosis of breast cancer, real-time PCR and immunohistochemistry (IHC) staining were utilized to detect the expression of FOXR2. The messenger RNA (mRNA) and protein levels of FOXR2 in breast cancer samples were novelty higher compared to non-tumorous breast tissues. IHC results revealed FOXR2 expression was significantly correlated to classifications tumor size (p = 0.007) and Ki-67 (p = 0.019). The patients with high expression of FOXR2 had a significantly poor prognosis compared to those of low expression (p = 0.003), especially in the patients with tumor size ≥2 cm (p = 0.006) and lymph node metastasis status (p = 0.004). Furthermore, multivariate analysis indicated that FOXR2 expression was an independent prognostic factor for breast cancer patients (p = 0.035). This study first identifies that FOXR2 may be an important molecular marker for diagnosis and prognosis of breast cancer.

  9. Effects of rosiglitazone and high fat diet on lipase/esterase expression in adipose tissue.

    Science.gov (United States)

    Shen, Wen-Jun; Patel, Shailja; Yu, Zaixin; Jue, Dyron; Kraemer, Fredric B

    2007-02-01

    A number of intracellular lipase/esterase have been reported in adipose tissue either by functional assays of activity or through proteomic analysis. In the current work, we have studied the relative expression level of 12 members of the lipase/esterase family that are found in white adipose tissue. We found that the relative mRNA levels of ATGL and HSL are the most abundant, being 2-3 fold greater than TGH or ADPN; whereas other intracellular neutral lipase/esterases were expressed at substantially lower levels. High fat feeding did not alter the mRNA expression levels of most lipase/esterases, but did reduce CGI-58 and WBSCR21. Likewise, rosiglitazone treatment did not alter the mRNA expression levels of most lipase/esterases, but did increase ATGL, TGH, CGI-58 and WBSCR21, while reducing ADPN. WAT from HSL-/- mice showed no compensatory increase in any lipase/esterases, rather mRNA levels of most lipase/esterases were reduced. In contrast, BAT from HSL-/- mice showed an increase in ATGL expression, as well as a decrease in ES-1, APEH and WBSCR21. Analysis of the immunoreactive protein levels of some of the lipases confirmed the results seen with mRNA. In conclusion, these data highlight the complexity of the regulation of the expression of intracellular neutral lipase/esterases involved in lipolysis.

  10. THE GENE EXPRESSION PROFILE OF HIGHLY METASTATIC HUMAN OVARIAN CANCER CELL LINE BY GENE CHIP

    Institute of Scientific and Technical Information of China (English)

    吕桂泉; 许沈华; 牟瀚舟; 朱赤红; 羊正炎; 高永良; 楼洪坤; 刘祥麟; 杨文; 程勇

    2001-01-01

    To study the gene expression of high metastatic human ovarian carcinoma cell line (HO-8910PM) and to screen for novel metastasis- associated genes by cDNA microarray. Methods: The cDNA was retro-transcribed from equal quantity mRNA derived from tissues of highly metastatic ovarian carcinoma cell line and normal ovarian, and was labeled with Cy5 and Cy3 fluorescence as probes. The mixed probes were hybridized with BioDoor 4096 double dot human whole gene chip. The chip was scanned by scanArray 3000 laser scanner. The acquired image was analyzed by ImaGene 3.0 software. Results: By applying the cDNA microarray we found: A total of 323 genes whose expression level were 3 times higher or lower in HO-8910PM cell than normal ovarian epithelium cell were screened out, with 71 higher and 252 lower respectively. Among these 10 were new genes. 67 genes showed expression difference bigger than 6 times between HO-8910PM cell and normal ovarian epithelium cell, among these genes 12 were higher, 55 lower, and two new genes were found. Conclusion: cDNA microarray technique is effective in screening the differentially expressed genes between human ovarian cancer cell line (HO-8910PM) and normal ovarian epithelium cell. Using the cDNA microarray to analyze of human ovarian cancer cell line gene expression profile difference will help the gene diagnosis, treatment and protection.

  11. A highly sensitive and specific system for large-scale gene expression profiling

    Directory of Open Access Journals (Sweden)

    Wang Hui-Yun

    2008-01-01

    Full Text Available Abstract Background Rapid progress in the field of gene expression-based molecular network integration has generated strong demand on enhancing the sensitivity and data accuracy of experimental systems. To meet the need, a high-throughput gene profiling system of high specificity and sensitivity has been developed. Results By using specially designed primers, the new system amplifies sequences in neighboring exons separated by big introns so that mRNA sequences may be effectively discriminated from other highly related sequences including their genes, unprocessed transcripts, pseudogenes and pseudogene transcripts. Probes used for microarray detection consist of sequences in the two neighboring exons amplified by the primers. In conjunction with a newly developed high-throughput multiplex amplification system and highly simplified experimental procedures, the system can be used to analyze >1,000 mRNA species in a single assay. It may also be used for gene expression profiling of very few (n = 100 or single cells. Highly reproducible results were obtained from duplicate samples with the same number of cells, and from those with a small number (100 and a large number (10,000 of cells. The specificity of the system was demonstrated by comparing results from a breast cancer cell line, MCF-7, and an ovarian cancer cell line, NCI/ADR-RES, and by using genomic DNA as starting material. Conclusion Our approach may greatly facilitate the analysis of combinatorial expression of known genes in many important applications, especially when the amount of RNA is limited.

  12. High-level expression of housefly cecropin A in Escherichia coli using a fusion protein

    Institute of Scientific and Technical Information of China (English)

    Xueli Zheng; Wei Wang

    2010-01-01

    Objective:To investigate the effect of utilizing a molecular partner on high-level expression of Musca domestica (M. domestica) cecropin in Escherichia coli (E. coli) and to identify the expressed products. Methods:The genomic sequence of M. domestica cecropin A (MC) and M. domestica ubiquitin (UBI) were searched from Genbank and amplified by reverse transcriptase polymerase chain reaction (RT-PCR). Two expression plasmids, pET32a-MC and pET32a-UBI-MC, were constructed and transferred into E. coli and were then induced by Isopropylβ-D-1-Thiogalactopyranoside (IPTG). The expression of the fusion proteins Trx-MC and Trx-UBI-MC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fusion protein Trx-MC was verified by Western blot analysis. The bactericidal activity of the purified MC was quantitatively determined using E. coli BL21(DE3). Results:The result showed that the fusion proteins were successively expressed in E. coli BL21 cells. A band at the expected position of 24 kDa representing the Trx-MC target protein was positivelystained, and the band at 4 kDa representing the hydrolysis of mature MC protein was also observed at the expected position. The expression levels of Trx-UBI-MC were higher than that of Trx-MC in E. coli. MC exhibited antimicrobial activity. Conclusions:With high-level expression of housefly cecropin A in E. coli using a fusion protein, MC exhibited antimicrobial activity.

  13. A high resolution atlas of gene expression in the domestic sheep (Ovis aries).

    Science.gov (United States)

    Clark, Emily L; Bush, Stephen J; McCulloch, Mary E B; Farquhar, Iseabail L; Young, Rachel; Lefevre, Lucas; Pridans, Clare; Tsang, Hiu; Wu, Chunlei; Afrasiabi, Cyrus; Watson, Mick; Whitelaw, C Bruce; Freeman, Tom C; Summers, Kim M; Archibald, Alan L; Hume, David A

    2017-09-15

    Sheep are a key source of meat, milk and fibre for the global livestock sector, and an important biomedical model. Global analysis of gene expression across multiple tissues has aided genome annotation and supported functional annotation of mammalian genes. We present a large-scale RNA-Seq dataset representing all the major organ systems from adult sheep and from several juvenile, neonatal and prenatal developmental time points. The Ovis aries reference genome (Oar v3.1) includes 27,504 genes (20,921 protein coding), of which 25,350 (19,921 protein coding) had detectable expression in at least one tissue in the sheep gene expression atlas dataset. Network-based cluster analysis of this dataset grouped genes according to their expression pattern. The principle of 'guilt by association' was used to infer the function of uncharacterised genes from their co-expression with genes of known function. We describe the overall transcriptional signatures present in the sheep gene expression atlas and assign those signatures, where possible, to specific cell populations or pathways. The findings are related to innate immunity by focusing on clusters with an immune signature, and to the advantages of cross-breeding by examining the patterns of genes exhibiting the greatest expression differences between purebred and crossbred animals. This high-resolution gene expression atlas for sheep is, to our knowledge, the largest transcriptomic dataset from any livestock species to date. It provides a resource to improve the annotation of the current reference genome for sheep, presenting a model transcriptome for ruminants and insight into gene, cell and tissue function at multiple developmental stages.

  14. Mice that are fed a high-fat diet display increased hepcidin expression in adipose tissue.

    Science.gov (United States)

    Gotardo, Érica Martins Ferreira; dos Santos, Aline Noronha; Miyashiro, Renan Akira; Gambero, Sheley; Rocha, Thalita; Ribeiro, Marcelo Lima; Gambero, Alessandra

    2013-01-01

    Since the discovery that hepcidin is expressed in the adipose tissue of obese subjects, attention has been increasingly focused on alterations in iron homeostasis that are associated with adiposity. We examined the production of hepcidin, the expression of hepcidin-related genes and the iron content of the adipose tissue in obesity using Swiss mice fed a high-fat diet (HFD). The mice were maintained on a control diet or HFD for 12 or 24 wk, and body weight, adiposity and glucose homeostasis were evaluated. The expression of several genes (hepcidin, TfR1, TfR2, DMT1, FT-heavy, ferroportin, IRP-1, IRP-2 and HIF-1) and the protein expression of hepcidin and IL-6 were quantified. The iron level was assessed using a Prussian blue reaction in paraffin-embedded tissue. After 24 wk on the HFD, we observed increases in the levels of hepcidin in the serum and the visceral adipose tissue. The IL-6 levels also increased in the visceral adipose tissue. Adipocytes isolated from the visceral adipose tissues of lean and obese mice expressed hepcidin at comparable levels; however, isolated macrophages from the stromal vascular fraction expressed higher hepcidin levels. Adipose tissues from obese mice displayed increased tfR2 expression and the presence of iron. Our results indicate that IL-6 and iron may affect the signaling pathways governing hepcidin expression. Thus, the mice fed HFD for 24 wk represent a suitable model for the study of obesity-linked hepcidin alterations. In addition, hepcidin may play local roles in controlling iron availability and interfering with inflammation in adipose tissue.

  15. Association of high obesity with PAM50 breast cancer intrinsic subtypes and gene expression.

    Science.gov (United States)

    Kwan, Marilyn L; Kroenke, Candyce H; Sweeney, Carol; Bernard, Philip S; Weltzien, Erin K; Castillo, Adrienne; Factor, Rachel E; Maxfield, Kaylynn S; Stijleman, Inge J; Kushi, Lawrence H; Quesenberry, Charles P; Habel, Laurel A; Caan, Bette J

    2015-04-14

    Invasive breast cancers are now commonly classified using gene expression into biologically and clinically distinct tumor subtypes. However, the role of obesity in breast tumor gene expression and intrinsic subtype is unknown. Early-stage breast cancer (BC) patients (n = 1,676) were sampled from two prospective cohorts. The PAM50 qRT-PCR assay was used to: a) assess tumor gene expression levels for ESR1, PGR, ERBB2, and 10 proliferation genes and b) classify tumors into intrinsic subtype (Luminal A, Luminal B, Basal-like, HER2-enriched, Normal-like). Body mass index (BMI) around BC diagnosis (kg/m(2)) was categorized as: underweight (obese (30-34), and highly obese (≥35). In a cross-sectional analysis, we evaluated associations of BMI with gene expression using linear regression models, and associations of BMI with non-Luminal A intrinsic subtypes, compared with Luminal A subtype, using multinomial logistic regression. Statistical significance tests were two-sided. Highly obese women had tumors with higher expression of proliferation genes compared with normal weight women (adjusted mean difference = 0.44; 95% CI: 0.18, 0.71), yet mildly obese (adjusted mean difference = 0.16; 95% CI: -0.06, 0.38) and overweight (adjusted mean difference = 0.18; 95% CI: -0.01, 0.36) women did not. This association was stronger in postmenopausal women (p for interaction = 0.06). Being highly obese, however, was inversely associated with ESR1 expression (adjusted mean difference = -0.95; 95% CI: -1.47, -0.42) compared with being normal weight, whereas being mildly obese and overweight were not. In addition, women with Basal-like and Luminal B subtypes, relative to those with Luminal A subtype, were more likely to be highly obese, compared with normal-weight. ER expression may not increase correspondingly with increasing degree of obesity. Highly obese patients are more likely to have tumor subtypes associated with high proliferation and poorer prognosis.

  16. Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination

    Directory of Open Access Journals (Sweden)

    Lepelley Maud

    2012-03-01

    Full Text Available Abstract Background Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Results Two cysteine proteinase (CP and four cysteine proteinase inhibitor (CPI gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Conclusions Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is

  17. Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination.

    Science.gov (United States)

    Lepelley, Maud; Amor, Mohamed Ben; Martineau, Nelly; Cheminade, Gerald; Caillet, Victoria; McCarthy, James

    2012-03-01

    Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role in the programmed cell death

  18. High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

    Science.gov (United States)

    Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

    2016-03-01

    An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

  19. The Influence of Bovine Milk High or Low in Isoflavones on Hepatic Gene Expression in Mice

    Directory of Open Access Journals (Sweden)

    Mette T. Skaanild

    2010-01-01

    Full Text Available Isoflavones have generated much attention due to their potential positive effects in various diseases. Phytoestrogens especially equol can be found in bovine milk, as feed ration for dairy cows is comprised of plants containing phytoestrogens. The aim of this study was to analyze the changes in hepatic gene expression after dietary intake of milk high and low in isoflavones. In addition to pelleted feed female NMRI mice were offered water, water added either 17-estradiol, equol, Tween 80, and milk high and low in isoflavone content for a week. Gene expression was analyzed using an array qPCR kit. It was revealed that Tween 80 and 17-estradiol upregulated both phase I and phase II genes to the same extent whereas equol alone, high and low isoflavone milk did not alter the expression of phase I genes but decreased the expression of phase II genes. This study shows that dietary isoflavones can regulate the transcription of especially phase II liver enzymes which potentially could give rise to an increase in reactive oxygen metabolites that may contribute to the development of cancer.

  20. High VEGFC expression is associated with unique gene expression profiles and predicts adverse prognosis in pediatric and adult acute myeloid leukemia

    NARCIS (Netherlands)

    H.J.M. de Jonge (Hendrik); P.J.M. Valk (Peter); N.J.G.M. Veeger (Nic); A. ter Elst (Arja); M.L. den Boer (Monique); J. Cloos (Jacqueline); V. de Haas (Valerie); M.M. van den Heuvel-Eibrink (Marry); G.J. Kaspers (Gertjan); C.M. Zwaan (Christian Michel); W.A. Kamps (Willem); B. Löwenberg (Bob); E.S.J.M. de Bont (Eveline)

    2010-01-01

    textabstractHigh VEGFC mRNA expression of acute myeloid leukemia (AML) blasts is related to increased in vitro and in vivo drug resistance. Prognostic significance of VEGFC on long-term outcome and its associated gene expression profiles remain to be defined. We studied effect of VEGFC on treatment

  1. High VEGFC expression is associated with unique gene expression profiles and predicts adverse prognosis in pediatric and adult acute myeloid leukemia

    NARCIS (Netherlands)

    de Jonge, Hendrik J. M.; Valk, Peter J. M.; Veeger, Nic J. G. M.; ter Elst, Arja; den Boer, Monique L.; Cloos, Jacqueline; de Haas, Valerie; van den Heuvel-Eibrink, Marry M.; Kaspers, Gertjan J. L.; Zwaan, Christian M.; Kamps, Willem A.; Lowenberg, Bob; de Bont, Eveline S. J. M.

    2010-01-01

    High VEGFC mRNA expression of acute myeloid leukemia (AML) blasts is related to increased in vitro and in vivo drug resistance. Prognostic significance of VEGFC on long-term outcome and its associated gene expression profiles remain to be defined. We studied effect of VEGFC on treatment outcome and

  2. Suppression subtractive hybridization reveals differential gene expression in sunflower grown in high P.

    Science.gov (United States)

    Padmanabhan, Priya; Sahi, Shivendra V

    2011-06-01

    Sunflower (Helianthus annuus L.) is a commercially important oilseed crop. Previous studies proved that this crop is a promising plant species for phytoextraction of excess soil phosphorus (P) because of its superior P accumulating characteristics. Suppression subtractive hybridization (SSH) strategy was employed to isolate and characterize genes that are induced in response to high P in this crop. SSH library was prepared using cDNA generated from plants treated with high P as the 'tester'. Based on the results of dot blot analysis, 360 positive cDNA clones were selected from the SSH library for sequencing. A total of 89 non-redundant expressed sequence tags (ESTs) were identified as high P-responsive genes and they were classified into 6 functional groups. Several genes involved in metabolism showed markedly preferential expression in the library. For further confirmation, thirteen of the representative ESTs were selected from all categories for RT-PCR analysis and the results showed up-regulation of these genes in response to high P-treatment. The gene expression data derived from this study suggested that several of the up-regulated genes identified under high P-treatment might be involved in P-accumulation and tolerance in this plant.

  3. Expressed microRNA associated with high rate of egg production in chicken ovarian follicles.

    Science.gov (United States)

    Wu, N; Gaur, U; Zhu, Q; Chen, B; Xu, Z; Zhao, X; Yang, M; Li, D

    2017-04-01

    MicroRNA (miRNA) is a highly conserved class of small noncoding RNA about 19-24 nucleotides in length that function in a specific manner to post-transcriptionally regulate gene expression in organisms. Tissue miRNA expression studies have discovered a myriad of functions for miRNAs in various aspects, but a role for miRNAs in chicken ovarian tissue at 300 days of age has not hitherto been reported. In this study, we performed the first miRNA analysis of ovarian tissues in chickens with low and high rates of egg production using high-throughput sequencing. By comparing low rate of egg production chickens with high rate of egg production chickens, 17 significantly differentially expressed miRNAs were found (P chickens with high rates of egg production, suggesting that these miRNAs have an important role in ovary development and reproductive management of chicken. Furthermore, we uncovered that a significantly up-regulated miRNA-gga-miR-200a-3p-is ubiquitous in reproduction-regulation-related pathways. This miRNA may play a special central role in the reproductive management of chicken, and needs to be further studied for confirmation. © 2016 Stichting International Foundation for Animal Genetics.

  4. High interleukin-6 mRNA expression is a predictor of relapse in colon cancer

    DEFF Research Database (Denmark)

    Olsen, Jesper; Kirkeby, Lene T; Olsen, Jørgen;

    2015-01-01

    AIM: To investigate the expression of interleukin-6 (IL6) in colon cancer tissue, and to examine if the risk of relapse is influenced by IL6 expression. MATERIALS AND METHODS: Fresh-frozen biopsies from tumor and normal adjacent tissues were taken from patients with colon cancer during surgery...... for clinicopathological characteristics (Hazard Ratio=2.16, 95% CI=1.07-4.40; pcolon cancer tissue at the transcriptional level and is significantly associated with increased risk of relapse....... to normal adjacent tissue (pcancer stage. We found a significant association between high IL6 expression and risk of relapse (Hazard Ratio=2.23, 95% CI=1.10-4.53; p

  5. High interleukin-6 mRNA expression is a predictor of relapse in colon cancer

    DEFF Research Database (Denmark)

    Olsen, Jesper; Kirkeby, Lene T.; Olsen, Jørgen;

    2015-01-01

    Aim: To investigate the expression of interleukin-6 (IL6) in colon cancer tissue, and to examine if the risk of relapse is influenced by IL6 expression. Materials and Methods: Fresh-frozen biopsies from tumor and normal adjacent tissues were taken from patients with colon cancer during surgery...... for clinicopathological characteristics (Hazard Ratio=2.16, 95% CI=1.07-4.40; pcolon cancer tissue at the transcriptional level and is significantly associated with increased risk of relapse....... to normal adjacent tissue (pcancer stage. We found a significant association between high IL6 expression and risk of relapse (Hazard Ratio=2.23, 95% CI=1.10-4.53; p

  6. Highly expressed genes within hippocampal sector CA1: implications for the physiology of memory

    Directory of Open Access Journals (Sweden)

    Michael A. Meyer

    2014-06-01

    Full Text Available As the CA1 sector has been implicated to play a key role in memory formation, a dedicated search for highly expressed genes within this region was made from an on-line atlas of gene expression within the mouse brain (GENSAT. From a data base of 1013 genes, 16 were identified that had selective localization of gene expression within the CA1 region, and included Angpt2, ARHGEF6, CCK, Cntnap1, DRD3, EMP1, Epha2, Itm2b, Lrrtm2, Mdk, PNMT, Ppm1e, Ppp2r2d, RASGRP1, Slitrk5, and Sstr4. Of the 16 identified, the most selective and intense localization for both adult and post-natal day 7 was noted for ARHGEF6, which is known to be linked to non-syndromic mental retardation, and has also been localized to dendritic spines. Further research on the role played by ARHGEF6 in memory formation is strongly advocated.

  7. High expression of HLA-E in colorectal carcinoma is associated with a favorable prognosis

    Directory of Open Access Journals (Sweden)

    Monaco Elisa Lo

    2011-10-01

    Full Text Available Abstract Background Human Leukocyte Antigen (HLA-E is a non-classical class I HLA molecule that can be stabilized by ligands donated by other classical (HLA-A, -B, -C and non-classical (HLA-G family members. HLA-E engages a variety of immune receptors expressed by cytotoxic T lymphocytes (CTLs, Natural killer (NK cells and NK-CTLs. In view of the opposing outcomes (activation or inhibition of the different HLA-E receptors, the preferred role (if any of HLA-E expressed in vivo on tumor cells remains to be established. Methods Taking advantage of MEM-E/02, a recently characterized antibody to denatured HLA-E molecules, HLA-E expression was assessed by immunohistochemistry on an archival collection (formalin-fixed paraffin-embedded of 149 colorectal primary carcinoma lesions paired with their morphologically normal mucosae. Lymphoid infiltrates were assessed for the expression of the HLA-E-specific, inhibitory, non-rearranging receptor NKG2A. Results High HLA-E expression did not significantly correlate with the expression of classical HLA-B and HLA-C molecules, but it did correlate with high expression of its preferential ligand donor HLA-A. In addition, it correlated with lymphoid cell infiltrates expressing the inhibitory NKG2A receptor, and was an independent predictor of good prognosis, particularly in a subset of patients whose tumors express HLA-A levels resembling those of their paired normal counterparts (HLA-A. Thus, combination phenotypes (HLA-Elo-int/HLA-AE and HLA-Ehi/HLA-AE of classical and non-classical class I HLA molecules mark two graded levels of good prognosis. Conclusions These results suggest that HLA-E favors activating immune responses to colorectal carcinoma. They also provide evidence in humans that tumor cells entertain extensive negotiation with the immune system until a compromise between recognition and escape is reached. It is implied that this process occurs stepwise, as predicted by the widely accepted

  8. High expression of HMGA2 predicts poor survival in patients with clear cell renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Na N

    2016-11-01

    Full Text Available Ning Na,1,* Tujie Si,2,* Zhengyu Huang,1,* Bin Miao,1 Liangqing Hong,1 Heng Li,1 Jiang Qiu,2 Jianguang Qiu3 1Department of Kidney Transplantation, The Third Affiliated Hospital of Sun Yat-sen University, 2Department of Organ Transplant, The First Affiliated Hospital of Sun Yat-sen University, 3Department of Urology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: High-mobility group AT-hook 2 (HMGA2 is involved in a wide spectrum of biological processes and is upregulated in several tumors, but its role in renal carcinoma remains unclear. The aim of this study was to examine the expression of HMGA2 and its relationship to the overall survival (OS of patients with non-metastatic clear cell renal cell carcinoma (ccRCC following surgery. The expression of HMGA2 was evaluated retrospectively by immunohistochemistry (IHC in 162 patients with ccRCC who underwent nephrectomy in 2003 and 2004. An IHC analysis revealed that HMGA2 was expressed in the nuclei of tumor cells in 146 (90.1% patients with ccRCC. The level of HMGA2 was positively correlated with tumor size, lymph node metastasis, and Fuhrman Grade. A Kaplan–Meier analysis with log-rank test found that patients with high HMGA2 expression had a poor outcome and that patients with low HMGA2 expression had better survival. Cox regression analysis showed that HMGA2 expression could serve as an independent prognostic factor for ccRCC patients. The efficacy of the following prognostic models was improved when HMGA2 expression was added: tumor node metastasis stage, UCLA Integrated Scoring System, Mayo Clinic stage, size, grade, and necrosis score. In summary, this study showed that HMGA2 expression is an independent prognostic factor for OS in patients with ccRCC. HMGA2 was found to be a valuable biomarker for ccRCC progression. Keywords: renal carcinoma, high-mobility group protein A

  9. Concomitant high expression of BRAFV600E, P-cadherin and cadherin 6 is associated with High TNM stage and lymph node metastasis in conventional papillary thyroid carcinoma.

    Science.gov (United States)

    Zhao, Le; Jiang, Rong; Xu, Man; Zhu, Ping; Mo, Xiao-Mei; Wang, Ni; Chen, George G; Liu, Zhi-Min

    2016-05-01

    BRAFV600E mutation is the most common activating mutation associated with aggressive behaviours in human tumours including conventional papillary thyroid carcinoma (cPTC). P-cadherin and cadherin 6 have been shown to be mesenchymal-associated cadherins and promote cancer cell invasion and metastasis. The purpose of this study was to examine BRAFV600E, P-cadherin and cadherin 6 expressions in cPTC and to assess the association of their expression with clinicopathological indicators. BRAFV600E, P-cadherin and cadherin 6 protein expressions in 80 cPTCs, 61 nodular hyperplasia and 76 normal thyroid tissues were examined by immunohistochemistry. The correlation of their protein expression with clinicopathological indicators of cPTC was statistically analysed. Protein expression of BRAFV600E, P-cadherin and cadherin 6 was upregulated in cPTC. High protein expression of BRAFV600E, P-cadherin and cadherin 6 was significantly correlated with high TNM stage and lymph node metastasis (LNM) (P TNM stage and LNM when compared with BRAFV600E high expression combined with either P-cadherin or cadherin-6 high expression (P = 0·042, 0·017 for TNM stage and P = 0·003, 0·006 for LNM, respectively) and only BRAFV600E high expression (P TNM stage and LNM). Concomitant high expression of BRAFV600E, P-cadherin and cadherin 6 is strongly associated with high TNM stage and LNM in cPTC. © 2015 John Wiley & Sons Ltd.

  10. Monitoring yeast physiology during very high gravity wort fermentations by frequent analysis of gene expression.

    Science.gov (United States)

    Rautio, Jari J; Huuskonen, Anne; Vuokko, Heikki; Vidgren, Virve; Londesborough, John

    2007-09-01

    Brewer's yeast experiences constantly changing environmental conditions during wort fermentation. Cells can rapidly adapt to changing surroundings by transcriptional regulation. Changes in genomic expression can indicate the physiological condition of yeast in the brewing process. We monitored, using the transcript analysis with aid of affinity capture (TRAC) method, the expression of some 70 selected genes relevant to wort fermentation at high frequency through 9-10 day fermentations of very high gravity wort (25 degrees P) by an industrial lager strain. Rapid changes in expression occurred during the first hours of fermentations for several genes, e.g. genes involved in maltose metabolism, glycolysis and ergosterol synthesis were strongly upregulated 2-6 h after pitching. By the time yeast growth had stopped (72 h) and total sugars had dropped by about 50%, most selected genes had passed their highest expression levels and total mRNA was less than half the levels during growth. There was an unexpected upregulation of some genes of oxygen-requiring pathways during the final fermentation stages. For five genes, expression of both the Saccharomyces cerevisiae and S. bayanus components of the hybrid lager strain were determined. Expression profiles were either markedly different (ADH1, ERG3) or very similar (MALx1, ILV5, ATF1) between these two components. By frequent analysis of a chosen set of genes, TRAC provided a detailed and dynamic picture of the physiological state of the fermenting yeast. This approach offers a possible way to monitor and optimize the performance of yeast in a complex process environment.

  11. Emotional expression of high school students with visual impairments and their typically developing peers

    Directory of Open Access Journals (Sweden)

    Vučinić Vesna

    2015-01-01

    Full Text Available Emotions are adaptive reaction to events from the environment and they represent the central part of every person's life. Willing emotional expression influences people from the environment according to our expectations if they recognize our emotions. Studies on expressing emotions in persons with visual impairments indicate the existence of the same type of spontaneous emotional expressions as in typically developing population. Also, these studies point to difficulties in presenting willing emotional expressions. The aim of this research was to determine the difference in emotional expression between high school students with visual impairments and their typically developing peers. The sample consisted of 33 students with visual impairments and the same number of students with no developmental disabilities. Emotional states simulation scenario by Friedman et al. was used in this research. Emotional expression was assessed with regard to the level of success in simulating seven emotions (happiness, sadness, anger, disgust, surprise, fear, and neutral state. The participants' task was to simulate the given emotional states in three structured situations (uttering two sentences and a series of vowels. The simulation of emotions was recorded. On the basis of video recordings, three independent assessors measured the success in simulating emotions on a nine-point scale. By analyzing the obtained results, a statistically significant difference in emotional expression was determined between the participants with visual impairments and their peers with no developmental disabilities (F(1=3.692; p=0.05.Arithmetic mean differences are statistically significant for simulating disgust (p=0.002 and surprise (p=0.01. In the group of participants with visual impairments, gender was a significant factor in simulating the emotional state of happiness (p=0.024, while type of school was a significant factor in simulating sadness (p=0.027.

  12. Fucosyltransferase 8 expression in breast cancer patients: A high throughput tissue microarray analysis.

    Science.gov (United States)

    Yue, Liling; Han, Cuicui; Li, Zubin; Li, Xin; Liu, Deshui; Liu, Shulin; Yu, Haitao

    2016-05-01

    The aim of this study was to compare the expression of fucosyltransferase 8 (FUT8) in breast cancer tissue and to investigate the relationship between this marker with tumor progression and its applicability to differential diagnosis. An immunohistochemical study was performed for FUT8 using the tissue microarray technique. In addition, the mRNA and protein levels of FUT8 in the tissue were also tested by real-time PCR and Western blot. There was a significant difference in cytoplasmic expression of FUT8 between breast cancer tissue and matched normal tissue (ptissues ranging from negative, weak positive, positive and strong positive were 2.7%, 40.2%, 54% and 3.2%, respectively. High FUT8 protein expression correlated with lymphatic metastasis (p=0.008) and with stage status (p=0.039). We detected that reduced FUT8 expression correlated with disease-free survival (p=0.02) and overall survival (p=0.04) of breast cancer patients. Expression of FUT8 can stratify breast cancer tissue and may be considered a prognostic marker for breast cancer patients.

  13. Reduced expression of NGEP is associated with high-grade prostate cancers: a tissue microarray analysis.

    Science.gov (United States)

    Mohsenzadegan, Monireh; Madjd, Zahra; Asgari, Mojgan; Abolhasani, Maryam; Shekarabi, Mehdi; Taeb, Jaleh; Shariftabrizi, Ahmad

    2013-10-01

    New gene expressed in prostate (NGEP) is a newly diagnosed prostate-specific gene that is expressed only in normal prostate and prostate cancer cells. Discovery of tissue-specific markers may promote the development of novel targets for immunotherapy of prostate cancer. In the present study, the staining pattern and clinical significance of NGEP were evaluated in a series of prostate tissues composed of 123 prostate cancer, 19 high-grade prostatic intraepithelial neoplasia and 44 samples of benign prostate tissue included in tissue microarrays using immunohistochemistry. Our study demonstrated that NGEP localized mainly in the apical and lateral membranes and was also partially distributed in the cytoplasm of epithelial cells of normal prostate tissue. All of the examined prostate tissues expressed NGEP with a variety of intensities; the level of expression was significantly more in the benign prostate tissues compared to malignant prostate samples (P value tissues, and the intensity of expression is inversely proportional to the level of malignancy. NGEP could be an attractive target for immune-based therapy of prostate cancer patients as an alternative to the conventional therapies particularly in indolent patients.

  14. Persistence of high CD40 and CD40L expression after restorative proctocolectomy for ulcerative colitis

    Institute of Scientific and Technical Information of China (English)

    Lino Polese; Mauro Frego; Davide F D'Amico; Tmerio Angriman; Giuseppe De Franchis; Attilio Cecchetto; Giacomo C. Sturniolo; Renata D'Incà; Marco Scarpa; Cesare Ruffolo; Lorenzo Norberto

    2005-01-01

    AIM: To focus on the role of CD40 and CD40L in their pathogenesis.METHODS: We analyzed by immunohistochemistry the CD40 and CD40L expression in the pouch mucosa of 28 patients who had undergone RPC for UC, in the terminal ileum of 6 patients with UC and 11 healthy subjects. We also examined by flow cytometry the expression of CD40 by B lymphocytes and monocytes in the peripheral blood of 20 pouch patients, 15 UC patients and 11 healthy controls.RESULTS: Ileal pouch mucosa leukocytes presented a significantly higher expression of CD40 and CD40L as compared to controls. This alteration correlated with pouchitis, but was also present in the healthy pouch and in the terminal ileum of UC patients. CD40 expression of peripheral B lymphocytes was significantly higher in patients with UC and pouch, respect to controls. Increased CD40 levels in blood B cells of pouch patients correlatedwith the presence of spondyloarthropathy, but not with pouchitis, or inflammatory indices.CONCLUSION: High CD40 expression in the ileal pouch mucosa could be implied in the pathogenesis of pouchitis following proctocolectomy for UC, whereas its increased levels on peripheral blood B lymphocytes are associated with the presence of extraintestinal manifestations.

  15. High yield expression of catalytically active USP18 (UBP43 using a Trigger Factor fusion system

    Directory of Open Access Journals (Sweden)

    Basters Anja

    2012-08-01

    Full Text Available Abstract Background Covalent linkage of the ubiquitin-like protein ISG15 interferes with viral infection and USP18 is the major protease which specifically removes ISG15 from target proteins. Thus, boosting ISG15 modification by protease inhibition of USP18 might represent a new strategy to interfere with viral replication. However, so far no heterologous expression system was available to yield sufficient amounts of catalytically active protein for high-throughput based inhibitor screens. Results High-level heterologous expression of USP18 was achieved by applying a chaperone-based fusion system in E. coli. Pure protein was obtained in a single-step on IMAC via a His6-tag. The USP18 fusion protein exhibited enzymatic activity towards cell derived ISG15 conjugated substrates and efficiently hydrolyzed ISG15-AMC. Specificity towards ISG15 was shown by covalent adduct formation with ISG15 vinyl sulfone but not with ubiquitin vinyl sulfone. Conclusion The results presented here show that a chaperone fusion system can provide high yields of proteins that are difficult to express. The USP18 protein obtained here is suited to setup high-throughput small molecule inhibitor screens and forms the basis for detailed biochemical and structural characterization.

  16. Addressing Shewanella oneidensis "cytochromome": the first step towards high-throughput expression of cytochromes c.

    Science.gov (United States)

    Londer, Yuri Y; Giuliani, Sarah E; Peppler, Terese; Collart, Frank R

    2008-11-01

    Integrated studies that address proteins structure and function in the new era of systems biology and genomics often require the application of high-throughput approaches for parallel production of many different purified proteins from the same organism. Cytochromes c-electron transfer proteins carrying one or more hemes covalently bound to the polypeptide chain-are essential in most organisms. However, they are one of the most recalcitrant classes of proteins with respect to heterologous expression because post-translational incorporation of hemes is required for proper folding and stability. We have addressed this challenge by designing two families of vectors (total of 6 vectors) suitable for ligation-independent cloning and developing a pipeline for expression and solubility analysis of cytochromes c. This system has been validated by expression analysis of thirty genes from Shewanella oneidensis coding for cytochromes c or cytochromes c-type domains predicted to have 1-4 hemes. Out of 30 targets, 26 (87%) were obtained in soluble form in one or more vectors. This work establishes a methodology for high-throughput expression of this class of proteins and provides a clone resource for the microbiological and functional genomics research communities.

  17. The Expression Plasticity of Hypoxia Related Genes in High-Altitude and Plains Nanorana parkeri Populations

    Institute of Scientific and Technical Information of China (English)

    Qiong ZHANG; Xingzhi HAN; Robert H S KRAUS; Le YANG; Liqing FAN; Yinzi YE; Yi TAO

    2016-01-01

    For species that have a broad geographic distribution, adaptive variation may be attributable to gene expression plasticity. Nanorana parkeri is an anuran endemic to the southern Tibetan Plateau where it has an extensive altitudinal range (2850 to 5100 m asl). Low oxygen concentration is one of the main environmental characteristics of the Tibetan Plateau. Hypoxia-inducible factor α subunits (HIF-1α and HIF-2α, encoded by Endothelial PAS domain protein 1 (EPAS1)) and associated genes (e.g., vascular endothelial growth factor (VEGF) and Erythropoietin (EPO)) play crucial roles in maintaining oxygen homeostasis. In this study, we compared the expression of HIF-1A, VEGF, EPAS1 and EPO mRNA between two populations of N. parkeri: one population inhabiting the native high altitudes, and the second living in, and being acclimated to, the lower plains (70 m asl). The expression of HIF-1A, VEGF and EPAS1 mRNA in the high altitude population were significantly higher than in the acclimated population, whereas there was no significant difference for EPO between two groups. Our results indicated that gene expression plasticity may make significant contributions to local adaptation of species that have broad altitudinal distributions. In addition, we deepen our understanding of the adaptive potential of this species by evaluating the experiments in the scope of its evolutionary history.

  18. High level of MT-MMP expression is associated with invasiveness of cervical cancer cells.

    Science.gov (United States)

    Gilles, C; Polette, M; Piette, J; Munaut, C; Thompson, E W; Birembaut, P; Foidart, J M

    1996-01-17

    MMP-2 (gelatinase A) has been associated with the invasive potential of many cancer cells both in vitro and in vivo. It is now becoming clear that the activation of this enzyme might be a key step in tumor invasion. This activation process has been shown to be a membrane-associated pathway inducible by various agents such as collagen type I, concanavalin A or TGF-beta, but its physiological regulation is still largely unresolved. MT-MMP was recently discovered and described as a potential gelatinase-A activator. In the present study, we investigated the expression of MT-MMP (membrane-type metalloproteinase) in cervical cancer cells both in vitro and in vivo. Comparing several in vitro-transformed cervical cell lines, previously shown to display different invasive potentials, our results showed that the ability of cells to overexpress MT-MMP mRNA following ConA induction correlated with their ability to activate gelatinase A and with a highly invasive behavior. Moreover, using immunohistochemistry and in situ hybridization, we found a higher level of MT-MMP expression in invasive cervical carcinoma and lymph node metastases compared to its expression in non-invasive CIN III lesions. Our in vivo observations also clearly demonstrated a cooperation between stromal and tumor cells for the production of MT-MMP. Taken together, our results clearly correlated high level MT-MMP expression with invasiveness, and thus suggested that MT-MMP might play a crucial role in cervical tumor invasion.

  19. Effects of high temperature on photosynthesis and related gene expression in poplar

    Science.gov (United States)

    2014-01-01

    Background High temperature, whether transitory or constant, causes physiological, biochemical and molecular changes that adversely affect tree growth and productivity by reducing photosynthesis. To elucidate the photosynthetic adaption response and examine the recovery capacity of trees under heat stress, we measured gas exchange, chlorophyll fluorescence, electron transport, water use efficiency, and reactive oxygen-producing enzyme activities in heat-stressed plants. Results We found that photosynthesis could completely recover after less than six hours of high temperature treatment, which might be a turning point in the photosynthetic response to heat stress. Genome-wide gene expression analysis at six hours of heat stress identified 29,896 differentially expressed genes (15,670 up-regulated and 14,226 down-regulated), including multiple classes of transcription factors. These interact with each other and regulate the expression of photosynthesis-related genes in response to heat stress, controlling carbon fixation and changes in stomatal conductance. Heat stress of more than twelve hours caused reduced electron transport, damaged photosystems, activated the glycolate pathway and caused H2O2 production; as a result, photosynthetic capacity did not recover completely. Conclusions This study provides a systematic physiological and global gene expression profile of the poplar photosynthetic response to heat stress and identifies the main limitations and threshold of photosynthesis under heat stress. It will expand our understanding of plant thermostability and provides a robust dataset for future studies. PMID:24774695

  20. Expression of angiogenic regulators and skeletal muscle capillarity in selectively bred high aerobic capacity mice.

    Science.gov (United States)

    Audet, Gerald N; Meek, Thomas H; Garland, Theodore; Olfert, I Mark

    2011-11-01

    Selective breeding for high voluntary wheel running in untrained mice has resulted in a 'mini muscle' (MM) phenotype, which has increased skeletal muscle capillarity compared with muscles from non-selected control lines. Vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) are essential mediators of skeletal muscle angiogenesis; thus, we hypothesized that untrained MM mice with elevated muscle capillarity would have higher basal VEGF expression and lower basal TSP-1 expression, and potentially an exaggerated VEGF response to acute exercise. We examined skeletal muscle morphology and skeletal muscle protein expression of VEGF and TSP-1 in male mice from two (untrained) mouse lines selectively bred for high exercise capacity (MM and Non-MM), as well as one non-selected control mouse line (normal aerobic capacity). In the MM mice, gastrocnemius (GA) and plantaris (PLT) muscle capillarity (i.e. capillary-to-fibre ratio and capillary density) were greater compared with control mice (P capillarity in PLT was greater than in control mice (P capillarity among groups. In the GA, MM mice had 58% greater basal VEGF (P capillarity is associated with altered balance between positive and negative angiogenic regulators (i.e. VEGF versus TSP-1, respectively). Based on the greater capillarity and significant VEGF response to exercise in MM mice, these data suggest that VEGF expression may, at least in part, be genetically determined.

  1. CLONING AND HIGH EXPRESSION OF FULL LENGTH hTRF1 IN E. COLI

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To express human telomeric repeat binding factor (TRF1) at high level in E. coli. Method: Two primers were designed with KpnⅠand EcoRⅠ sites respectively, TRF1 cDNA fragments was amplified and cloned into plasmid pET29a, each step was confirmed by sequencing and restriction endonuclease map analysis. And the recombinant plasmid pET29a-TRF1 was then transformed into E. coli BL21 (DE3) PlysS. Fusion protein was purified by S-protein Kit and checked by SDS-PAGE and by western blot. Result: E. coli BL21 (DE3) PlysS expressing high level of 30 KD partial TRF1 was obtained, and TRF1 fusion protein was purified. The optimal induction time was at 2.5 h. Excessive expression system was established and 18.6% inductive protein was obtained. Conclusion: The expressed protein can be used for producing both polyclonal and monoclonal antibodies and for further study of the function and structure of TRF1 and its association with malignant tumor and leukemia.

  2. Expression Changes of Early Response Genes in Lung Due to High Volume Ventilation

    Institute of Scientific and Technical Information of China (English)

    WANG Yuelan; YAO Shanglong; XIONG Ping

    2005-01-01

    Summary: The expression changes of early response genes due to ventilation with high volume in adult rats in vivo were observed. Forty SD male rats were randomly divided into control and 30, 60, 90 and 120 min ventilation groups, respectively (n=8 in each group). The animals were ventilated with tidal volume of 42 ml/kg and a PEEP level of 0 cmH2O at a rate of 40 breaths per minute in room air with a ventilator was given to the small animals. The expression of Egr-1, C-jun and IL-1β mRNA and proteins was detected by RT-PCR and immunohistochemical technique, respectively. The pathological changes in lung tissues were examined by HE staining. The results indicated that the expression of Egr-1, C-jun and IL-1β mRNA was detectable at 30th min after overventilation, but there was no significant difference in comparison with that in control group until overventilation for 60 min. However, at 90 and 120 min there was a significent increase as compared with 30 min or control group (P<0.05). The expression of Egr-1, C-jun and IL-1β deteced by immunohistochemical assay also showed a similar tendency of the gradual increase. In the 120 min ventilation group, the expression intensity of Egr-1, C-jun and IL-1β proteins in lung cells was the strongest and the nuclear translocation was increased markedly in comparison with any other groups (P<0.05). HE staining suggested that the degree of lung injury was aggravated gradually with the ventialtion going on and had a similar tendency to the expression of these early response genes and proteins. The current data suggested that overventilation activated and upregulated the expression of early response genes and the expression of these genes may be taken as the early signal to predict the onset and degree of lung injury. These results may demonstrated partially that the expression of early response genes induced by the mechanical stretch is associated with biochamic lung injury.

  3. High glucose stimulates the expression of erythropoietin in rat glomerular epithelial cells

    OpenAIRE

    Lim, Seul Ki; Park, Soo Hyun

    2011-01-01

    It has been reported that the levels of erythropoietin are associated with diabetes mellitus. Glomerular epithelial cells, located in the renal cortex, play an important role in the regulation of kidney function and hyperglycemia-induced cell loss of glomerular epithelial cells is implicated in the onset of diabetic nephropathy. This study investigated the effect of high glucose on erythropoietin and erythropoietin receptor expression in rat glomerular epithelial cells. We found that 25 mM D-...

  4. High-throughput gene expression analysis in pigs as model for respiratory infections

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Brogaard, Louise; Schou, Kirstine Klitgaard

    Animal models are essential in understanding the mechanisms involved in human infectious disease and for the development of effective prevention and treatment strategies. It is increasingly realized that large animal models like the pig are exceptionally human like and serve as an excellent model...... highly controlled experimental infections and to study changes of symptoms, viral titer, and expression of microRNAs/mRNAs as the influenza infection progresses in time, generating information that would be difficult to obtain from human patients....

  5. Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Yacoby, I.; Tegler, L. T.; Pochekailov, S.; Zhang, S.; King, P. W.

    2012-04-01

    Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.

  6. Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae.

    Science.gov (United States)

    Takaya, Tomohiro; Koda, Risa; Adachi, Daisuke; Nakashima, Kazunori; Wada, Junpei; Bogaki, Takayuki; Ogino, Chiaki; Kondo, Akihiko

    2011-05-01

    In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5' untranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL#1) and double copies (pSENSU-FHL#2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL#1 and #2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL#2, methanolysis could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time.

  7. Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae

    Energy Technology Data Exchange (ETDEWEB)

    Takaya, Tomohiro; Koda, Risa; Adachi, Daisuke; Ogino, Chiaki; Kondo, Akihiko [Kobe Univ. (Japan). Dept. of Chemical Science and Engineering; Nakashima, Kazunori [Kobe Univ. (Japan). Organization of Advanced Science and Technology; Wada, Junpei; Bogaki, Takayuki [Ozeki Co., Nishinomiya-shi, Hyogo (Japan)

    2011-05-15

    In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5{sup '} untranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL1) and double copies (pSENSU-FHL2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL1 and 2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL2, methanolysis could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time. (orig.)

  8. TLMA Expression in Condyloma Acuminata from Patients with High and Low Risk HPV

    Institute of Scientific and Technical Information of China (English)

    涂亚庭; 陈善娟; 范超; 林能兴; 刘厚君; 刘志香

    2002-01-01

    Objective: To detect the activated expression oftelomerase in condyloma acuminata lesions in low-risk (6/11)and high-risk (16/18) human papilloma virus (HPV)infection and examine the role played by telomerase in theoccurrence, development and carcinomatous change ofcondyloma acuminata. Methods: Assaying the expression of telomerase and thetype of HPV in damaged skin of 42 CA patients and normalskin of 30 healthy control individuals through telomeraserepeat amplification protocol (TRAP) and polymerase chainreaction (PCR). Results: In all the normal skin controls, PCR for HPV wasnegative and only 16.7% of samples were positive for TLMAexpression; in CA lesions, HPV testing was positive in all (32cases were low-risk, 3 were high-risk, and 7 were of mixedtype) and all were positive for TLMA expression. Conclusion: TLMA may be activated by HPV infection,and in turn cause the hyperplasia of epidermal cells. It wasalso indicated that HPV, especially high risk types, canactivate TLMA. The activation of TLMA may play animportant role in abnormal hyperplasia and carcinomatouschanges in CA lesions.

  9. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Mandy Y M Lo

    Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  10. Kita driven expression of oncogenic HRAS leads to early onset and highly penetrant melanoma in zebrafish.

    Directory of Open Access Journals (Sweden)

    Cristina Santoriello

    Full Text Available BACKGROUND: Melanoma is the most aggressive and lethal form of skin cancer. Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed. METHODOLOGY AND PRINCIPAL FINDINGS: Using the combinatorial Gal4-UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter. Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth. By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish. The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line. Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors. In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period. CONCLUSIONS AND SIGNIFICANCE: This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors. Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens.

  11. Expression of a new chimeric protein with a highly repeated sequence in tobacco cells.

    Science.gov (United States)

    Saumonneau, Amélie; Rottier, Karine; Conrad, Udo; Popineau, Yves; Guéguen, Jacques; Francin-Allami, Mathilde

    2011-07-01

    In wheat, the high-molecular weight (HMW) glutenin subunits are known to contribute to gluten viscoelasticity, and show some similarities to elastomeric animal proteins as elastin. When combining the sequence of a glutenin with that of elastin is a way to create new chimeric functional proteins, which could be expressed in plants. The sequence of a glutenin subunit was modified by the insertion of several hydrophobic and elastic motifs derived from elastin (elastin-like peptide, ELP) into the hydrophilic repetitive domain of the glutenin subunit to create a triblock protein, the objective being to improve the mechanical (elastomeric) properties of this wheat storage protein. In this study, we investigated an expression model system to analyze the expression and trafficking of the wild-type HMW glutenin subunit (GS(W)) and an HMW glutenin subunit mutated by the insertion of elastin motifs (GS(M)-ELP). For this purpose, a series of constructs was made to express wild-type subunits and subunits mutated by insertion of elastin motifs in fusion with green fluorescent protein (GFP) in tobacco BY-2 cells. Our results showed for the first time the expression of HMW glutenin fused with GFP in tobacco protoplasts. We also expressed and localized the chimeric protein composed of plant glutenin and animal elastin-like peptides (ELP) in BY-2 protoplasts, and demonstrated its presence in protein body-like structures in the endoplasmic reticulum. This work, therefore, provides a basis for heterologous production of the glutenin-ELP triblock protein to characterize its mechanical properties.

  12. p21(WAF1/CIP1 RNA expression in highly HIV-1 exposed, uninfected individuals.

    Directory of Open Access Journals (Sweden)

    Joshua Herbeck

    Full Text Available Some individuals remain HIV-1 antibody and PCR negative after repeated exposures to the virus, and are referred to as HIV-exposed seronegatives (HESN. However, the causes of resistance to HIV-1 infection in cases other than those with a homozygous CCR5Δ32 deletion are unclear. We hypothesized that human p21WAF1/CIP1 (a cyclin-dependent kinase inhibitor could play a role in resistance to HIV-1 infection in HESN, as p21 expression has been associated with suppression of HIV-1 in elite controllers and reported to block HIV-1 integration in cell culture. We measured p21 RNA expression in PBMC from 40 HESN and 40 low exposure HIV-1 seroconverters (LESC prior to their infection using a real-time PCR assay. Comparing the 20 HESN with the highest exposure risk (median = 111 partners/2.5 years prior to the 20 LESC with the lowest exposure risk (median = 1 partner/2.5 years prior, p21 expression trended higher in HESN in only one of two experiments (P = 0.11 vs. P = 0.80. Additionally, comparison of p21 expression in the top 40 HESN (median = 73 partners/year and lowest 40 LESC (median = 2 partners/year showed no difference between the groups (P = 0.84. There was a weak linear trend between risk of infection after exposure and increasing p21 gene expression (R2 = 0.02, P = 0.12, but again only in one experiment. Hence, if p21 expression contributes to the resistance to viral infection in HESN, it likely plays a minor role evident only in those with extremely high levels of exposure to HIV-1.

  13. Recombinant myostatin reduces highly expressed microRNAs in differentiating C2C12 cells

    Directory of Open Access Journals (Sweden)

    Zachary A. Graham

    2017-03-01

    Full Text Available Myostatin is small glycopeptide that is produced and secreted by skeletal muscle. It is a potent negative regulator of muscle growth that has been associated with conditions of frailty. In C2C12 cells, myostatin limits cell differentiation. Myostatin acts through activin receptor IIB, activin receptor-like kinase (ALK and Smad transcription factors. microRNAs (miRNA are short, 22 base pair nucleotides that bind to the 3′ UTR of target mRNA to repress translation or reduce mRNA stability. In the present study, expression in differentiating C2C12 cells of the myomiRs miR-1 and 133a were down-regulated following treatment with 1 µg of recombinant myostatin at 1 d post-induction of differentiation while all myomiRs (miR-1, 133a/b and 206 were upregulated by SB431542, a potent ALK4/5/7 inhibitor which reduces Smad2 signaling, at 1 d and all, with the exception of miR-206, were upregulated by SB431542 at 3 d. The expression of the muscle-enriched miR-486 was greater following treatment with SB431542 but not altered by myostatin. Other highly expressed miRNAs in skeletal muscle, miR-23a/b and 145, were altered only at 1 d post-induction of differentiation. miR-27b responded differently to treatments at 1 d, where it was upregulated, as compared to 3 d, where it was downregulated. Neither myostatin nor SB431542 altered cell size or cell morphology. The data indicate that myostatin represses myomiR expression in differentiating C2C12 cells and that inhibition of Smad signaling with SB431542 can result in large changes in highly expressed miRNAs in differentiating myoblasts.

  14. Machine learning in computational biology to accelerate high-throughput protein expression

    DEFF Research Database (Denmark)

    Sastry, Anand; Monk, Jonathan M.; Tegel, Hanna

    2017-01-01

    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. Availability and implementation: 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...

  15. Comparative expression of wild-type and highly soluble mutant His103Leu of hydroxynitrile lyase from Manihot esculenta in prokaryotic and eukaryotic expression systems.

    Science.gov (United States)

    Dadashipour, Mohammad; Fukuta, Yasuhisa; Asano, Yasuhisa

    2011-05-01

    Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L. tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too.

  16. Cellular Stress Response Gene Expression During Upper and Lower Body High Intensity Exercises

    Science.gov (United States)

    Kochanowicz, Andrzej; Sawczyn, Stanisław; Niespodziński, Bartłomiej; Mieszkowski, Jan; Kochanowicz, Kazimierz

    2017-01-01

    Objectives The aim was to compare the effect of upper and lower body high-intensity exercise on chosen genes expression in athletes and non-athletes. Method Fourteen elite male artistic gymnasts (EAG) aged 20.6 ± 3.3 years and 14 physically active men (PAM) aged 19.9 ± 1.0 years performed lower and upper body 30 s Wingate Tests. Blood samples were collected before, 5 and 30 minutes after each effort to assess gene expression via PCR. Results Significantly higher mechanical parameters after lower body exercise was observed in both groups, for relative power (8.7 ± 1.2 W/kg in gymnasts, 7.2 ± 1.2 W/kg in controls, p = 0.01) and mean power (6.7 ± 0.7 W/kg in gymnasts, 5.4 ± 0.8 W/kg in controls, p = 0.01). No differences in lower versus upper body gene expression were detected for all tested genes as well as between gymnasts and physical active man. For IL-6 m-RNA time-dependent effect was observed. Conclusions Because of no significant differences in expression of genes associated with cellular stress response the similar adaptive effect to exercise may be obtained so by lower and upper body exercise. PMID:28141870

  17. PCI express bus design of large format array IRFPA high-speed acquisition system

    Science.gov (United States)

    Huang, Zewu; Zheng, Xing; Zeng, Xingxin; Liu, Ziji

    2012-10-01

    In this paper, a novel solution of PCI Express Bus was designed to improve the data transfer rate for large format array infrared imaging acquisition system. In this structure, an embedded PCI Express hard intellectual property (IP) block of Stratix IV GX FPGA was used, and the protocol stack module is totally compliant with PCI Express base specification Gen 2.0 which includes PHY-MAC, Data Link, and transaction layers. In order to communicate with CPU through computer PCIe root port, a pipeline structure was established with two SSRAMs to carry out the function of real-time data process. The DMA mode was adopted for the high-speed data transmission on the PCI Express Bus. Some other control logic parts such as detector drive signal generator - display controller and PCIe configuration module were also designed and introduced in this paper. According to the evaluation, the data transmission speed was up to 5.6Gbps, which means that this system could meet the qualifications of infrared imaging data acquisition. Compared with traditional infrared imaging data acquisition systems, this solution is more integrated and faster, so it is suitable for larger format and higher frame rate of infrared focal plane image acquisition in nowadays and future.

  18. High Expression of Endogenous Retroviral Envelope Gene in the Equine Fetal Part of the Placenta.

    Directory of Open Access Journals (Sweden)

    Valentina Stefanetti

    Full Text Available Endogenous retroviruses (ERVs are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora, the synepitheliochorial (Ruminantia, and the hemochorial placentation (human, mouse where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has "captured" a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated.

  19. High expression of GPR116 indicates poor survival outcome and promotes tumor progression in colorectal carcinoma.

    Science.gov (United States)

    Yang, Li; Lin, Xiao-Lu; Liang, Wei; Fu, Seng-Wang; Lin, Wen-Feng; Tian, Xiao-Qing; Gao, Yun-Jie; Chen, Hao-Yan; Dai, Jun; Ge, Zhi-Zheng

    2017-07-18

    Previous studies have found that G-protein-coupled receptor 116 (GPR116) is a regulator of breast cancer metastasis. However, the role of GPR116 in colorectal carcinoma (CRC) carcinogenesis and progression is unknown. In this study, We found GPR116 expression was significantly up-regulated in CRC specimens compared with corresponding non-cancerous tissues. Increased GPR116 expression in CRC was correlated with histological differentiation and distant metastasis. In addition, high expression of GPR116 was significantly associated with poor overall survival of CRC patients, which was also confirmed by GSE14333, GSE17536 and GSE33113 datasets from the Gene Expression Omnibus (GEO). Furthermore, we demonstrated that the ability of proliferation and invasion of CRC cell lines HCT116 and LOVO was markedly reduced after transfected with siRNA-GPR116. Meanwhile, GPR116 may drive EMT in CRC cells through AKT/EKR signaling pathway, resulting in metastasis. Thus, GPR116 may be a novel reliable prognostic indicator and a risk factor in CRC progression.

  20. Maternal High Fat Diet Affects Offspring's Vitamin K-Dependent Proteins Expression Levels.

    Directory of Open Access Journals (Sweden)

    Stuart Lanham

    Full Text Available Studies suggest bone growth & development and susceptibility to vascular disease in later life are influenced by maternal nutrition, during intrauterine and early postnatal life. There is evidence for a role of vitamin K-dependent proteins (VKDPs including Osteocalcin, Matrix-gla protein, Periostin, and Gas6, in bone and vascular development. This study extends the analysis of VKDPs previously conducted in 6 week old offspring, into offspring of 30 weeks of age, to assess the longer term effects of a maternal and postnatal high fat (HF diet on VKDP expression. Overall a HF maternal diet and offspring diet exacerbated the bone changes observed. Sex specific and tissue specific differences were observed in VKDP expression for both aorta and femoral tissues. In addition, significant correlations were observed between femoral OCN, Periostin Gas6, and Vkor expression levels and measures of femoral bone structure. Furthermore, MGP, OCN, Ggcx and Vkor expression levels correlated to mass and fat volume, in both sexes. In summary the current study has highlighted the importance of the long-term effects of maternal nutrition on offspring bone development and the correlation of VKDPs to bone structure.

  1. Analysis of Gene Expression in the K562-n High Tumorigenitic Human Leukemia Cell Line

    Institute of Scientific and Technical Information of China (English)

    Shuqing Lü; Xiaoping Xu; Fang Xia; JianMin Wang

    2005-01-01

    OBJECTIVE The human leukemia K562-n cell line displays much higher tumorigenic actively in nude mice compared with its parental K562 cell line. The molecular mechanism of the differences in tumorigenicity between K562-n and K562 in nude mice was examined.METHODS The differences in gene expression between K562 and K562-n cells were analyzed by using cDNA microarrays.RESULTS Among the12,800 genes examined, there was a significant difference in expression of 139 genes between K562-n and K562 cells.Eighty-five of these genes have been registered in the GeneBank and 54are unknown. The genes accessible from the GeneBank include:1)oncogenes and tumor-supressor genes; 2) genes related to transcription regulation, the cell cycle and apoptosis; 3) genes related to the cytoskeleton and cytokinetics; 4) genes related to metabolism and transport; 5) genes related to immune function. There were also some differently expressed genes with mixed functions.CONCLUSION There are many genes differentially expressed between K562-n and K562 cells .The high tumorigenicity of the human leukemia K562-n cell line in nude mice might be related to its specific geneexpression profile.

  2. [High expression of antimicrobial peptide Cecropin AD in Escherichia coli by fusion with EDDIE].

    Science.gov (United States)

    Zhang, Zhen; Ke, Tao; Zhou, Yuling; Ma, Xiangdong; Ma, Lixin

    2009-08-01

    In this study, we efficiently expressed the active antimicrobial peptide (CAD), which fused with the site-mutated coat protein (EDDIE) of the classical swine fever virus, in Escherichia coli. First, we obtained the e-cad fusion gene from the CAD gene and the EDDIE gene using overlapping PCR. Then to get the recombinant expression vector (pETED), the e-cad fusion gene was cloned into the pET30a vector by a site-directed homologous recombination technique. The EDDIE-CAD fusion protein expressed in E. coli as inclusion bodies, and its yield was more than 40% of total bacterial proteins. After renaturated in vitro and self-cleavage of the fusion protein, we obtained the antimicrobial peptide Cecropin AD. Antimicrobial experiments showed that the Cecropin AD efficiently inhibited the growth of G+ and G- bacteria, but it weakly inhibited the growth of Saccharomyces. This method provides an excellent way for high expression of antimicrobial peptides when fused with EDDIE.

  3. High Erk-1 activation and Gadd45a expression as prognostic markers in high risk pediatric haemolymphoproliferative diseases

    Directory of Open Access Journals (Sweden)

    Rondelli Roberto

    2009-03-01

    Full Text Available Abstract Studies on activated cell-signaling pathways responsible for neoplastic transformation are numerous in solid tumors and in adult leukemias. Despite of positive results in the evolution of pediatric hematopoietic neoplasias, there are some high-risk subtypes at worse prognosis. The aim of this study was to asses the expression and activation status of crucial proteins involved in cell-signaling pathways in order to identify molecular alterations responsible for the proliferation and/or escape from apoptosis of leukemic blasts. The quantitative and qualitative expression and activation of Erk-1, c-Jun, Caspase8, and Gadd45a was analyzed, by immunocytochemical (ICC and western blotting methods, in bone marrow blasts of 72 patients affected by acute myeloid leukemia (AML, T-cell acute lymphoblastic leukemia (ALL and stage IV non-Hodgkin Lymphoma (NHL. We found an upregulation of Erk-1, Caspase8, c-Jun, and Gadd45a proteins with a constitutive activation in 95.8%, 91.7%, 86.2%, 83.4% of analyzed specimens, respectively. It is worth noting that all AML patients showed an upregulation of all proteins studied and the high expression of GADD45a was associated to the lowest DFS median (p = 0.04. On univariate analysis, only Erk-1 phosphorylation status was found to be correlated with a significantly shorter 5-years DFS in all disease subgroups (p = 0.033 and the lowest DFS median in ALL/NHL subgroup (p = 0.04. Moreover, the simultaneous activation of multiple kinases, as we found for c-Jun and Erk-1 (r = 0.26; p = 0.025, might synergistically enhance survival and proliferation potential of leukemic cells. These results demonstrate an involvement of these proteins in survival of blast cells and, consequently, on relapse percentages of the different subgroups of patients.

  4. RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples

    OpenAIRE

    2004-01-01

    We have developed RNA expression microarrays (REMs), in which each spot on a glass support is composed of a population of cDNAs synthesized from a cell or tissue sample. We used simultaneous hybridization with test and reference (housekeeping) genes to calculate an expression ratio based on normalization with the endogenous reference gene. A test REM containing artificial mixtures of liver cDNA and dilutions of the bacterial LysA gene cDNA demonstrated the feasibility of detecting transcripts...

  5. Effect of high fat diet on pulmonary expression of parathyroid hormone-related protein and its downstream targets

    Directory of Open Access Journals (Sweden)

    Learta Oruqaj

    2016-10-01

    Significance: This study established that physiological regulation of leptin plasma levels by high fat diet affects the pulmonary PTHrP expression and of PTHrP downstream targets. Modification of pulmonary expression of PTH-1 receptors by high fat diet after myocardial infarction suggests that the identified interaction may participate in the obesity paradox.

  6. PathwayExplorer: web service for visualizing high-throughput expression data on biological pathways.

    Science.gov (United States)

    Mlecnik, Bernhard; Scheideler, Marcel; Hackl, Hubert; Hartler, Jürgen; Sanchez-Cabo, Fatima; Trajanoski, Zlatko

    2005-07-01

    While generation of high-throughput expression data is becoming routine, the fast, easy, and systematic presentation and analysis of these data in a biological context is still an obstacle. To address this need, we have developed PathwayExplorer, which maps expression profiles of genes or proteins simultaneously onto major, currently available regulatory, metabolic and cellular pathways from KEGG, BioCarta and GenMAPP. PathwayExplorer is a platform-independent web server application with an optional standalone Java application using a SOAP (simple object access protocol) interface. Mapped pathways are ranked for the easy selection of the pathway of interest, displaying all available genes of this pathway with their expression profiles in a selectable and intuitive color code. Pathway maps produced can be downloaded as PNG, JPG or as high-resolution vector graphics SVG. The web service is freely available at https://pathwayexplorer.genome.tugraz.at; the standalone client can be downloaded at http://genome.tugraz.at.

  7. Altered gene expression in highly purified enterocytes from patients with active coeliac disease

    Directory of Open Access Journals (Sweden)

    Jackson John

    2008-08-01

    Full Text Available Abstract Background Coeliac disease is a multifactorial inflammatory disorder of the intestine caused by ingestion of gluten in genetically susceptible individuals. Genes within the HLA-DQ locus are considered to contribute some 40% of the genetic influence on this disease. However, information on other disease causing genes is sparse. Since enterocytes are considered to play a central role in coeliac pathology, the aim of this study was to examine gene expression in a highly purified isolate of these cells taken from patients with active disease. Epithelial cells were isolated from duodenal biopsies taken from five coeliac patients with active disease and five non-coeliac control subjects. Contaminating T cells were removed by magnetic sorting. The gene expression profile of the cells was examined using microarray analysis. Validation of significantly altered genes was performed by real-time RT-PCR and immunohistochemistry. Results Enterocyte suspensions of high purity (98–99% were isolated from intestinal biopsies. Of the 3,800 genes investigated, 102 genes were found to have significantly altered expression between coeliac disease patients and controls (p Conclusion This study provides a profile of the molecular changes that occur in the intestinal epithelium of coeliac patients with active disease. Novel candidate genes were revealed which highlight the contribution of the epithelial cell to the pathogenesis of coeliac disease.

  8. Representing high throughput expression profiles via perturbation barcodes reveals compound targets.

    Science.gov (United States)

    Filzen, Tracey M; Kutchukian, Peter S; Hermes, Jeffrey D; Li, Jing; Tudor, Matthew

    2017-02-01

    High throughput mRNA expression profiling can be used to characterize the response of cell culture models to perturbations such as pharmacologic modulators and genetic perturbations. As profiling campaigns expand in scope, it is important to homogenize, summarize, and analyze the resulting data in a manner that captures significant biological signals in spite of various noise sources such as batch effects and stochastic variation. We used the L1000 platform for large-scale profiling of 978 representative genes across thousands of compound treatments. Here, a method is described that uses deep learning techniques to convert the expression changes of the landmark genes into a perturbation barcode that reveals important features of the underlying data, performing better than the raw data in revealing important biological insights. The barcode captures compound structure and target information, and predicts a compound's high throughput screening promiscuity, to a higher degree than the original data measurements, indicating that the approach uncovers underlying factors of the expression data that are otherwise entangled or masked by noise. Furthermore, we demonstrate that visualizations derived from the perturbation barcode can be used to more sensitively assign functions to unknown compounds through a guilt-by-association approach, which we use to predict and experimentally validate the activity of compounds on the MAPK pathway. The demonstrated application of deep metric learning to large-scale chemical genetics projects highlights the utility of this and related approaches to the extraction of insights and testable hypotheses from big, sometimes noisy data.

  9. Revealing complex function, process and pathway interactions with high-throughput expression and biological annotation data.

    Science.gov (United States)

    Singh, Nitesh Kumar; Ernst, Mathias; Liebscher, Volkmar; Fuellen, Georg; Taher, Leila

    2016-10-20

    The biological relationships both between and within the functions, processes and pathways that operate within complex biological systems are only poorly characterized, making the interpretation of large scale gene expression datasets extremely challenging. Here, we present an approach that integrates gene expression and biological annotation data to identify and describe the interactions between biological functions, processes and pathways that govern a phenotype of interest. The product is a global, interconnected network, not of genes but of functions, processes and pathways, that represents the biological relationships within the system. We validated our approach on two high-throughput expression datasets describing organismal and organ development. Our findings are well supported by the available literature, confirming that developmental processes and apoptosis play key roles in cell differentiation. Furthermore, our results suggest that processes related to pluripotency and lineage commitment, which are known to be critical for development, interact mainly indirectly, through genes implicated in more general biological processes. Moreover, we provide evidence that supports the relevance of cell spatial organization in the developing liver for proper liver function. Our strategy can be viewed as an abstraction that is useful to interpret high-throughput data and devise further experiments.

  10. Substrate-induced gene expression screening: a method for high-throughput screening of metagenome libraries.

    Science.gov (United States)

    Uchiyama, Taku; Miyazaki, Kentaro

    2010-01-01

    The SIGEX (substrate-induced gene expression) method is a novel approach for the screening of gene (genome) libraries. In addition to the commonly used function- and sequence-driven approaches to screening, SIGEX provides a third option; in SIGEX, positives are identified using a reporter gene, and the library is constructed using an "operon-trap" vector. This vector contains the reporter gene immediately downstream of the cloning site for the genomic insert so that the expression of the inserted gene(s) is coupled with that of the reporter gene. This system is especially suitable for screening catabolic genes that are induced in response to metabolically relevant compounds, such as substrates. If expression of the inserted gene(s) is activated in response to the addition of these compounds, then positive clones can be identified based on the reporter signal. The most effective selection is obtained by the use of a FACS (fluorescence-activated cell sorter) in conjunction with a FACS-compatible fluorescent reporter protein, such as GFP (green fluorescent protein). Activity-based screening of metagenomic libraries often suffers from low sensitivity and low throughput. In contrast, the high throughput, high sensitivity, and versatility of SIGEX make it a particularly suitable method for screening metagenomic libraries.

  11. High glucose enhance expression of matrix metalloproteinase—2 in smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    HAOFeng; YUJin-De

    2003-01-01

    AIM:To investigate the effects of high glucose on expression of matrix metalloproteinase-2(MMP-2) in rat aortic smooth muscle cells and the influence of matrix remodeling on atherogenesis in diabetic patients. METHODS: The smooth muscle cells were cultured from the thoracic aorta of Sprague-Dawley (SD) rat. MMP-2 mRNA was determined by reverse transcriptase-polymerase chain reaction(RT-PCR),MMP-2 protein was measured by Western blotting, and MMP-2 activity in conditioned medium was observed by zymography. RESULTS:In comparison with the control, there was no difference in the expression of MMP-2 when glucose concentration was 1g/L,whereas MMP-2 activity in smooth muscle cells was significantly increased by the glucose 5 g/L(P<0.01). CONCLUSION:High glucose enhanced the expression and activity of MMP-2 in smooth muscle cells, which may provide an explanation for the phenomenon that diabetes patients are prone to have atherosclerotic lesions.

  12. Representing high throughput expression profiles via perturbation barcodes reveals compound targets

    Science.gov (United States)

    Kutchukian, Peter S.; Li, Jing; Tudor, Matthew

    2017-01-01

    High throughput mRNA expression profiling can be used to characterize the response of cell culture models to perturbations such as pharmacologic modulators and genetic perturbations. As profiling campaigns expand in scope, it is important to homogenize, summarize, and analyze the resulting data in a manner that captures significant biological signals in spite of various noise sources such as batch effects and stochastic variation. We used the L1000 platform for large-scale profiling of 978 representative genes across thousands of compound treatments. Here, a method is described that uses deep learning techniques to convert the expression changes of the landmark genes into a perturbation barcode that reveals important features of the underlying data, performing better than the raw data in revealing important biological insights. The barcode captures compound structure and target information, and predicts a compound’s high throughput screening promiscuity, to a higher degree than the original data measurements, indicating that the approach uncovers underlying factors of the expression data that are otherwise entangled or masked by noise. Furthermore, we demonstrate that visualizations derived from the perturbation barcode can be used to more sensitively assign functions to unknown compounds through a guilt-by-association approach, which we use to predict and experimentally validate the activity of compounds on the MAPK pathway. The demonstrated application of deep metric learning to large-scale chemical genetics projects highlights the utility of this and related approaches to the extraction of insights and testable hypotheses from big, sometimes noisy data. PMID:28182661

  13. Characterization of Cardiac-Resident Progenitor Cells Expressing High Aldehyde Dehydrogenase Activity

    Directory of Open Access Journals (Sweden)

    Marc-Estienne Roehrich

    2013-01-01

    Full Text Available High aldehyde dehydrogenase (ALDH activity has been associated with stem and progenitor cells in various tissues. Human cord blood and bone marrow ALDH-bright (ALDHbr cells have displayed angiogenic activity in preclinical studies and have been shown to be safe in clinical trials in patients with ischemic cardiovascular disease. The presence of ALDHbr cells in the heart has not been evaluated so far. We have characterized ALDHbr cells isolated from mouse hearts. One percent of nonmyocytic cells from neonatal and adult hearts were ALDHbr. ALDHvery-br cells were more frequent in neonatal hearts than adult. ALDHbr cells were more frequent in atria than ventricles. Expression of ALDH1A1 isozyme transcripts was highest in ALDHvery-br cells, intermediate in ALDHbr cells, and lowest in ALDHdim cells. ALDH1A2 expression was highest in ALDHvery-br cells, intermediate in ALDHdim cells, and lowest in ALDHbr cells. ALDH1A3 and ALDH2 expression was detectable in ALDHvery-br and ALDHbr cells, unlike ALDHdim cells, albeit at lower levels compared with ALDH1A1 and ALDH1A2. Freshly isolated ALDHbr cells were enriched for cells expressing stem cell antigen-1, CD34, CD90, CD44, and CD106. ALDHbr cells, unlike ALDHdim cells, could be grown in culture for more than 40 passages. They expressed sarcomeric α-actinin and could be differentiated along multiple mesenchymal lineages. However, the proportion of ALDHbr cells declined with cell passage. In conclusion, the cardiac-derived ALDHbr population is enriched for progenitor cells that exhibit mesenchymal progenitor-like characteristics and can be expanded in culture. The regenerative potential of cardiac-derived ALDHbr cells remains to be evaluated.

  14. High expression of Sox10 correlates with tumor aggressiveness and poor prognosis in human nasopharyngeal carcinoma

    Directory of Open Access Journals (Sweden)

    Zhao Y

    2016-03-01

    Full Text Available Yu Zhao,1,* Zhi-gang Liu,1,* Jiao Tang,1 Ren-fang Zou,1 Xiao-yan Chen,2 Guan-min Jiang,3 Yan-fang Qiu,1 Hui Wang11Key Laboratory of Translational Radiation Oncology, Department of Radiation Oncology, 2Department of Pathology, 3Department of Clinical Laboratory, Hunan Cancer Hospital, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, People’s Republic of China*These authors contributed equally to this workPurpose: The aim of the study was to detect the expression of Sox10 in human nasopharyngeal carcinoma (NPC and investigate the relationship between its expression and the clinicopathological characteristics of NPC patients.Patients and methods: Tumor specimens (n=105 were retrospectively collected from patients with NPC diagnosed between 2004 and 2005 who presented at Hunan Cancer Hospital. Immunohistochemistry analyses were performed to characterize the expression of Sox10 in NPC. Kaplan–Meier survival and Cox regression analyses were employed to evaluate the prognosis of 105 NPC patients.Results: The results showed that Sox10 was markedly overexpressed in human NPC tissues. Analysis of clinicopathological parameters showed that high Sox10 expression was significantly correlated with the clinical stage (P=0.032, T classification (P=0.034, and lymph node metastasis (P=0.03. Cox regression analyses further showed that Sox10 expression was an independent prognostic factor for overall survival (P=0.005. This is the first time Sox10 has shown its importance in predicting NPC progressiveness and survival outcomes.Conclusion: Sox10 serves as a potential biomarker for NPC patients. It may hopefully become a novel therapeutic target for NPC patients.Keywords: Sox10, NPC, prognosis

  15. Proanthocyanidins Prevent High Glucose-Induced Eye Malformation by Restoring Pax6 Expression in Chick Embryo

    Directory of Open Access Journals (Sweden)

    Rui-Rong Tan

    2015-08-01

    Full Text Available Gestational diabetes mellitus (GDM is one of the leading causes of offspring malformations, in which eye malformation is an important disease. It has raised demand for therapy to improve fetal outcomes. In this study, we used chick embryo to establish a GDM model to study the protective effects of proanthocyanidins on eye development. Chick embryos were exposed to high glucose (0.2 mmol/egg on embryo development day (EDD 1. Proanthocyanidins (1 and 10 nmol/egg were injected into the air sac on EDD 0. Results showed that both dosages of proanthocyanidins could prevent the eye malformation and rescue the high glucose-induced oxidative stress significantly, which the similar effects were showed in edaravone. However, proanthocyanidins could not decrease the glucose concentration of embryo eye. Moreover, the key genes regulating eye development, Pax6, was down-regulated by high glucose. Proanthocyanidins could restore the suppressed expression of Pax6. These results indicated proanthocyanidins might be a promising natural agent to prevent high glucose-induced eye malformation by restoring Pax6 expression.

  16. Diet-dependent gene expression in honey bees: honey vs. sucrose or high fructose corn syrup.

    Science.gov (United States)

    Wheeler, Marsha M; Robinson, Gene E

    2014-07-17

    Severe declines in honey bee populations have made it imperative to understand key factors impacting honey bee health. Of major concern is nutrition, as malnutrition in honey bees is associated with immune system impairment and increased pesticide susceptibility. Beekeepers often feed high fructose corn syrup (HFCS) or sucrose after harvesting honey or during periods of nectar dearth. We report that, relative to honey, chronic feeding of either of these two alternative carbohydrate sources elicited hundreds of differences in gene expression in the fat body, a peripheral nutrient-sensing tissue analogous to vertebrate liver and adipose tissues. These expression differences included genes involved in protein metabolism and oxidation-reduction, including some involved in tyrosine and phenylalanine metabolism. Differences between HFCS and sucrose diets were much more subtle and included a few genes involved in carbohydrate and lipid metabolism. Our results suggest that bees receive nutritional components from honey that are not provided by alternative food sources widely used in apiculture.

  17. Expression profile of hypothalamic neuropeptides in chicken lines selected for high or low residual feed intake.

    Science.gov (United States)

    Sintubin, P; Greene, E; Collin, A; Bordas, A; Zerjal, T; Tesseraud, S; Buyse, J; Dridi, S

    2014-08-01

    The R(+) and R(-) chicken lines have been divergently selected for high (R(+)) or low (R(-)) residual feed intake. For the same body weight and egg production, the R(+) chickens consume 40% more food than their counterparts R(-) lines. In the present study we sought to determine the hypothalamic expression profile of feeding-related neuropeptides in these lines maintained under fed or food-deprived conditions. In the fed condition, the suppressor of cytokine signaling 3 (SOCS3) was 17-fold lower (Pfeeding-related genes that are differently expressed in the hypothalamus of R(+) and R(-) chickens and that might explain the difference in feed intake observed between the two lines. Published by Elsevier Ltd.

  18. High-level expression and secondary structure analysis of the bovine mature prion protein

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    By using the recombinant DNA technology, the gene of the bovine mature prion protein (bPrPCL) has been cloned into pET30a and the resulting plasmid has been expressed in E.coli BL21(DE3). After solubilizing in 8 mol/L urea, the expression product was purified by cation ion exchange chromatography. The purified product was refolded by dilution and the recovery was about 15%. Analysis of mass spectrum, circular dichroism (CD) spectrum and Fourier transform infrared (FTIR) spectrum demonstrate that the molecular weight of the bPrPCL is 23 630 u, the bPrPCL has a high α-helix content (36.1%) and low β-sheet content (11.9%).

  19. Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

    Science.gov (United States)

    Ribeiro, Thuanne Pires; Arraes, Fabricio Barbosa Monteiro; Lourenço-Tessutti, Isabela Tristan; Silva, Marilia Santos; Lisei-de-Sá, Maria Eugênia; Lucena, Wagner Alexandre; Macedo, Leonardo Lima Pepino; Lima, Janaina Nascimento; Santos Amorim, Regina Maria; Artico, Sinara; Alves-Ferreira, Márcio; Mattar Silva, Maria Cristina; Grossi-de-Sa, Maria Fatima

    2017-01-12

    Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2(-ΔΔCt) analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 μg g(-1) fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g(-1) fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.

  20. Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a in Plasma.

    Directory of Open Access Journals (Sweden)

    Masayuki Ozawa

    Full Text Available High lipoprotein(a [Lp(a] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a [apo(a], the unique component of Lp(a, is found only in primates and humans, the study of human Lp(a has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT techniques, we produced transgenic miniature pigs expressing human apo(a in the plasma. First, we placed the hemagglutinin (HA-tagged cDNA of human apo(a under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a; one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a. Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL of Lp(a in plasma, and the transgenic apo(a gene was transmitted to the offspring. Thus, we generated a human apo(a-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

  1. Ammonia metabolism capacity of HepG2 cells with high expression of human glutamine synthetase

    Institute of Scientific and Technical Information of China (English)

    Nan-Hong Tang; Xiao-Qian Wang; Xiu-Jin Li; Yan-Ling Chen

    2008-01-01

    BACKGROUND:Currently, one of the tough problems for the application of bioartiifcial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxiifcation. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS:hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and ampliifed. Then hGS mRNA was measured by semi-quantitative RT-PCR;hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH4+were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS:The expression of hGS mRNA in HepG2/pLNChGS cells (8.306±0.336) and HepG2/pLNAFhGS cells (21.358±1.716) was much stronger than in control cells (P CONCLUSION:The constructed hepatocytes (HepG2 cells) with speciifc high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.

  2. Thermal stress induces changes in gene expression and blood parameters in high and low feed efficiency meat quail.

    Science.gov (United States)

    Gasparino, Eliane; Voltolini, Débora Marques; Del Vesco, Ana Paula; Marcato, Simara Marcia; Zancanela, Vittor; de Oliveira Grieser, Daiane; de Souza Khatlab, Angélica; Guimarães, Simone Eliza Facioni; de Oliveira Neto, Adhemar Rodriges

    2015-05-01

    In this study, we analysed markers of stress, plasma creatinine and T3 content, and insulin-like growth factor I (IGF-I), growth hormone receptor (GHR), uncoupling protein (UCP), adenine nucleotide translocase (ANT) and cytochrome c oxidase subunit III (COX III) mRNA expression in the liver and muscle of high (0.22 g/g) and low (0.14 g/g) feed efficiency (FE) meat quail at three different air temperatures, comfortable, heat and cold stress, for 24 h. High FE quail presented higher plasma T3 and lower creatinine levels. IGF-I mRNA expression was higher in the livers of high FE quail than in the livers of low FE quail under both comfortable and cold stress conditions. In the muscle, regardless of the environment, high FE birds showed higher IGF-I mRNA expression. High FE birds also showed higher GHR mRNA expression under comfortable conditions. Regarding the environment, higher expression was observed in birds at comfortable conditions, and lower expression in birds under heat stress. UCP mRNA expression in the liver was lower in high FE birds and higher under heat stress compared with the other conditions. Low and high FE birds showed greater ANT mRNA expression in the muscle under cold stress. Greater mRNA COX III expressions were observed in the liver and muscle of quails under comfortable conditions. Our results suggest that temperature affects the expression of genes related to growth and mitochondrial energy production, and quail with different FEs respond differently to environmental stimuli. In comfortable conditions, high FE animals show higher IGF-I mRNA expression and plasma T3 and lower creatinine content.

  3. A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium.

    Science.gov (United States)

    Thorgersen, Michael P; Lancaster, W Andrew; Rajeev, Lara; Ge, Xiaoxuan; Vaccaro, Brian J; Poole, Farris L; Arkin, Adam P; Mukhopadhyay, Aindrila; Adams, Michael W W

    2017-02-15

    Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound.

  4. High-yield expression of recombinant SARS coronavirus nucleocapsid protein in methylotrophic yeast Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Ru-Shi Liu; Kun-Yu Yang; Jian Lin; Yi-Wei Lin; Zhi-Hong Zhang; Jun Zhang; Ning-Shao Xia

    2004-01-01

    AIM: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), theantigenicity of the protein is better than spike (S) protein.This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS.METHODS: Full length cDNA of SCoV nucleocapsid (SCoVN)protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into Pichia pastoris (P. pastoris) GS115 (HisMut+) by electroporation. His+Mut+recombinant strains were identified by PCR and cultivated on MM/MD plates. The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied. The expression level and activation were detected by SDS-PAGE and Western-blot respectively.RESULTS: All of the recombinants were His+Mut+ aftertransformation of P. pastoriswith linearized plasmids. The BMMY medium was optimal for recombinant ScoVN (rSCoVN)protein expression and growth of the recombinant strains.The final optimal concentration of methanol was 20 mL/L,the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved,and a maximum cell ,A at 600 nm of 62 was achieved in shake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum.The biological activity of rSCoVN expressed in P. pastoris was about 4-fold higher than that expressed in E.coliwhen the same rSCoVN protein

  5. SIVQ-aided laser capture microdissection: A tool for high-throughput expression profiling

    Science.gov (United States)

    Hipp, Jason; Cheng, Jerome; Hanson, Jeffrey C.; Yan, Wusheng; Taylor, Phil; Hu, Nan; Rodriguez-Canales, Jaime; Hipp, Jennifer; Tangrea, Michael A.; Emmert-Buck, Michael R.; Balis, Ulysses

    2011-01-01

    Introduction: Laser capture microdissection (LCM) facilitates procurement of defined cell populations for study in the context of histopathology. The morphologic assessment step in the LCM procedure is time consuming and tedious, thus restricting the utility of the technology for large applications. Results: Here, we describe the use of Spatially Invariant Vector Quantization (SIVQ) for histological analysis and LCM. Using SIVQ, we selected vectors as morphologic predicates that were representative of normal epithelial or cancer cells and then searched for phenotypically similar cells across entire tissue sections. The selected cells were subsequently auto-microdissected and the recovered RNA was analyzed by expression microarray. Gene expression profiles from SIVQ–LCM and standard LCM–derived samples demonstrated highly congruous signatures, confirming the equivalence of the differing microdissection methods. Conclusion: SIVQ–LCM improves the work-flow of microdissection in two significant ways. First, the process is transformative in that it shifts the pathologist's role from technical execution of the entire microdissection to a limited-contact supervisory role, enabling large-scale extraction of tissue by expediting subsequent semi-autonomous identification of target cell populations. Second, this work-flow model provides an opportunity to systematically identify highly constrained cell populations and morphologically consistent regions within tissue sections. Integrating SIVQ with LCM in a single environment provides advanced capabilities for efficient and high-throughput histological-based molecular studies. PMID:21572509

  6. Prostate cancer gene expression signature of patients with high body mass index

    Science.gov (United States)

    Sharad, Shashwat; Srivastava, Anjali; Ravulapalli, Suma; Parker, Patrick; Chen, Yongmei; Li, Hua; Petrovics, Gyorgy; Dobi, Albert

    2010-01-01

    The goal of this study was to evaluate prostate cancer gene expression signatures associated with elevated body mass index (BMI). Global gene expression profiles of prostate tumor cells and matching normal epithelial cells were compared between patients with features of normal- and high BMI at the time of radical prostatectomy. Knowledge-based analyses revealed an association of high BMI with altered levels of lipid metabolism and cholesterol homeostasis genes, such as stearoyl-CoA desaturase 1 (SCD1) and insulin-induced gene 1 (INSIG1), respectively, in prostate tumor cells. These genes were connected to known pathways of tumorigenesis revealed by the v-maf (musculoaponeurotic fibrosarcoma) oncogene homolog (MAF), notch receptor ligand, jagged 1 (JAG1), and the alanyl aminopeptidase (ANPEP/CD13) genes. This study highlighted that SCD1, a known target of statins, may play a mechanistic role in the recently noted beneficial effects of statin treatment in reducing biochemical recurrence of prostate cancer. An additional finding of our study is that some of the obesity related genes were upregulated in tumor-matched normal cells within the high BMI group, when compared to normal cells within the normal BMI cohort. PMID:21060327

  7. Linamarase Expression in Cassava Cultivars with Roots of Low- and High-Cyanide Content1

    Science.gov (United States)

    Santana, María Angélica; Vásquez, Valeria; Matehus, Juan; Aldao, Rafael Rangel

    2002-01-01

    This paper reports the expression and localization of linamarase in roots of two cassava (Manihot esculenta Crantz) cultivars of low and high cyanide. Two different patterns of linamarase activity were observed. In the low-cyanide type, young leaves displayed very high enzyme activity during the early plant growing stage (3 months), whereas in root peel, the activity increased progressively to reach a peak in 11-month-old plants. Conversely, in the high-cyanide cultivar (HCV), root peel linamarase activity decreased during the growth cycle, whereas in expanded leaves linamarase activity peaked in 11-month-old plants. The accumulation of linamarin showed a similar pattern in both cultivars, although a higher concentration was always found in the HCV. Linamarase was found mainly in laticifer cells of petioles and roots of both cultivars with no significant differences between them. At the subcellular level, there were sharp differences because linamarase was found mainly in the cell walls of the HCV, whereas in the low-cyanide cultivar, the enzyme was present in vacuoles and cell wall of laticifer cells. Reverse transcriptase-PCR on cassava tissues showed no expression of linamarase in cassava roots, thus, the transport of linamarase from shoots to roots through laticifers is proposed. PMID:12177481

  8. Prolonged application of high fluid shear to chondrocytes recapitulates gene expression profiles associated with osteoarthritis.

    Directory of Open Access Journals (Sweden)

    Fei Zhu

    Full Text Available BACKGROUND: Excessive mechanical loading of articular cartilage producing hydrostatic stress, tensile strain and fluid flow leads to irreversible cartilage erosion and osteoarthritic (OA disease. Since application of high fluid shear to chondrocytes recapitulates some of the earmarks of OA, we aimed to screen the gene expression profiles of shear-activated chondrocytes and assess potential similarities with OA chondrocytes. METHODOLOGY/PRINCIPAL FINDINGS: Using a cDNA microarray technology, we screened the differentially-regulated genes in human T/C-28a2 chondrocytes subjected to high fluid shear (20 dyn/cm(2 for 48 h and 72 h relative to static controls. Confirmation of the expression patterns of select genes was obtained by qRT-PCR. Using significance analysis of microarrays with a 5% false discovery rate, 71 and 60 non-redundant transcripts were identified to be ≥2-fold up-regulated and ≤0.6-fold down-regulated, respectively, in sheared chondrocytes. Published data sets indicate that 42 of these genes, which are related to extracellular matrix/degradation, cell proliferation/differentiation, inflammation and cell survival/death, are differentially-regulated in OA chondrocytes. In view of the pivotal role of cyclooxygenase-2 (COX-2 in the pathogenesis and/or progression of OA in vivo and regulation of shear-induced inflammation and apoptosis in vitro, we identified a collection of genes that are either up- or down-regulated by shear-induced COX-2. COX-2 and L-prostaglandin D synthase (L-PGDS induce reactive oxygen species production, and negatively regulate genes of the histone and cell cycle families, which may play a critical role in chondrocyte death. CONCLUSIONS/SIGNIFICANCE: Prolonged application of high fluid shear stress to chondrocytes recapitulates gene expression profiles associated with osteoarthritis. Our data suggest a potential link between exposure of chondrocytes/cartilage to abnormal mechanical loading and the pathogenesis

  9. High Expression of PHGDH Predicts Poor Prognosis in Non–Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jinhong Zhu

    2016-12-01

    Full Text Available Tumors have exceptionally high demands for energy and anabolism because of their rapid growth. The de novo serine synthesis pathway initiated by phosphoglycerate dehydrogenase (PHGDH has been recognized as a hallmark of metabolic adaption in carcinogenesis. The oncogenic role and prognostic value of PHGDH have been investigated in multiple cancer types, including breast cancer, melanoma, cervical cancer, and colon cancer. Due to the importance of PHGDH in cancer, we attempted to determine the clinical significance of PHGDH in 319 patients with non–small cell lung cancer (NSCLC. We evaluated the mRNA and protein expression levels of PHGDH gene, using quantitative reverse transcriptase polymerase chain reaction and tissue array–based immunohistochemistry, respectively. Significantly increased PHGDH expression in mRNA and protein levels was identified in tumor tissues versus matched adjacent nontumor tissues. More interestingly, immunohistochemical expression of PHGDH was significantly associated with lymph node metastasis (P = .021 and TNM stage (P = .016. Kaplan-Meier survival analysis indicated that NSCLC patients with low levels of PHGDH outperformed patients with high levels of PHGDH regarding 5-year overall survival. Significantly longer survival in the former suggested the prognostic implication of PHGDH in NSCLC. Multivariate survival analysis using Cox regression model demonstrated that high PHGDH levels and advanced TNM stage (III + IV were independent predictors of prognosis in NSCLC. Moreover, bioinformatics analysis confirmed the increase in PHGDH transcripts (data from The Cancer Genome Atlas and its prognostic value (Kaplan-Meier plotter in NSCLC. In conclusion, this study suggested the clinical implication of PHGDH in NSCLC. PHGDH may be a promising therapeutic target in NSCLC.

  10. AHNAK is highly expressed and plays a key role in cell migration and invasion in mesothelioma.

    Science.gov (United States)

    Sudo, Hitomi; Tsuji, Atsushi B; Sugyo, Aya; Abe, Masaaki; Hino, Okio; Saga, Tsuneo

    2014-02-01

    The worldwide incidence of the highly aggressive tumor mesothelioma is expected to increase. Mesothelioma is classified into three main histological subtypes: epithelioid, sarcomatoid and biphasic. Although the pathological diagnostic markers for epithelioid are established, to date no adequate marker for sarcomatoid mesothelioma has been found. Thus, a reliable diagnostic marker of sarcomatoid mesothelioma is necessary. In this study, to identify an unknown protein with 120 kDa expressed only in the mesothelioma cell line 211H, we conducted proteomic analysis and found five candidate proteins. One such protein, AHNAK, was highly expressed in all seven mesothelioma cell lines (211H, H28, H226, H2052, H2452, MESO1 and MESO4), but not in the mesothelial cell line MeT-5A by RT-PCR and immunofluorescence staining. Furthermore, we confirmed high AHNAK expression not only in xenografts but also in human mesothelioma specimens including sarcomatoid, epithelioid and biphasic mesothelioma using immunohistochemical staining. These findings suggest that AHNAK has the potential to be a new marker for detecting mesothelioma. Since AHNAK is involved in cell migration and invasion in other metastatic tumor cells, we conducted migration and invasion assays in mesothelioma cell lines. The number of migrating cells in six of seven mesothelioma cell lines and the number of invading cells in all seven cell lines were significantly increased compared with those in MeT-5A. Knockdown of AHNAK significantly reduced the cell migration and invasion ability in all seven mesothelioma cell lines. These results support further clinical evaluation of the association of AHNAK and metastasis in mesothelioma.

  11. Isolation and characterization of "GmScream" promoters that regulate highly expressing soybean (Glycine max Merr.) genes.

    Science.gov (United States)

    Zhang, Ning; McHale, Leah K; Finer, John J

    2015-12-01

    To increase our understanding of the regulatory components that control gene expression, it is important to identify, isolate and characterize new promoters. In this study, a group of highly expressed soybean (Glycine max Merr.) genes, which we have named "GmScream", were first identified from RNA-Seq data. The promoter regions were then identified, cloned and fused with the coding region of the green fluorescent protein (gfp) gene, for introduction and analysis in different tissues using 3 tools for validation. Approximately half of the GmScream promoters identified showed levels of GFP expression comparable to or higher than the Cauliflower Mosaic Virus 35S (35S) promoter. Using transient expression in lima bean cotyledonary tissues, the strongest GmScream promoters gave over 6-fold higher expression than the 35S promoter while several other GmScream promoters showed 2- to 3-fold higher expression. The two highest expressing promoters, GmScreamM4 and GmScreamM8, regulated two different elongation factor 1A genes in soybean. In stably transformed soybean tissues, GFP driven by the GmScreamM4 or GmScreamM8 promoter exhibited constitutive high expression in most tissues with preferentially higher expression in proliferative embryogenic tissues, procambium, vascular tissues, root tips and young embryos. Using deletion analysis of the promoter, two proximal regions of the GmScreamM8 promoter were identified as contributing significantly to high levels of gene expression.

  12. iNOS-derived peroxynitrite mediates high glucose-induced inflammatory gene expression in vascular smooth muscle cells through promoting KLF5 expression and nitration.

    Science.gov (United States)

    Zhang, Man-Li; Zheng, Bin; Tong, Fei; Yang, Zhan; Wang, Zhi-Bo; Yang, Bao-Ming; Sun, Yan; Zhang, Xin-Hua; Zhao, Yi-Lin; Wen, Jin-Kun

    2017-07-12

    Inducible NO synthase (iNOS) expression and peroxynitrite formation are significantly increased in diabetic vascular tissues. Transcription factor KLF5 activates iNOS gene transcription and is involved in vascular inflammatory injury and remodeling. However, mutual regulation between KLF5, iNOS and peroxynitrite in diabetic vascular inflammation, as well as the underlying mechanisms, remain largely unknown. In this study, we found a marked increase in KLF5 and iNOS expression in vascular smooth muscle cells (VSMC) of diabetic patients. High glucose-induced expression of KLF5 and iNOS was also observed in cultured mouse VSMCs. Further investigation showed that high glucose induced KLF5 nitration by iNOS-mediated peroxynitrite generation, and nitrated KLF5 increased its interaction with NF-κB p50 and thus cooperatively activated the expression of inflammatory cytokines TNF-α and IL-1β. Furthermore, we showed that the VSMC-specific knockout of KLF5 dramatically reduced inflammatory cytokine expression in the vascular tissues of diabetic mice. Moreover, 17β-estradiol (E2) inhibited high glucose-mediated effects in VSMCs, and in the response to E2, estrogen receptor (ER) α competed with KLF5 for binding to NF-κB p50, which in turn leads to the suppression of inflammatory gene expression in VSMCs. Together, the present findings were the first to show that KLF5 expression and nitration by iNOS-mediated peroxynitrite are necessary for the induction of TNF-α and IL-1β expression in VSMCs of diabetic vascular tissues. Copyright © 2017. Published by Elsevier B.V.

  13. Highly selective and rapid arsenic removal by metabolically engineered Escherichia coli cells expressing Fucus vesiculosus metallothionein.

    Science.gov (United States)

    Singh, Shailendra; Mulchandani, Ashok; Chen, Wilfred

    2008-05-01

    An arsenic-chelating metallothionein (fMT) from the arsenic-tolerant marine alga Fucus vesiculosus was expressed in Escherichia coli, resulting in 30- and 26-fold-higher As(III) and As(V) binding, respectively. Coexpression of the As(III)-specific transporter GlpF with fMT further improved arsenic accumulation and offered high selectivity toward As. Resting E. coli cells coexpressing fMT and GlpF completely removed trace amounts (35 ppb) of As(III) within 20 min, providing a promising technology for compliance with the As limit of 10 ppb newly recommended by the U.S. EPA.

  14. Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (Fc epsilon RI)

    OpenAIRE

    1992-01-01

    It has been suggested that epidermal Langerhans cells (LC) bearing immunoglobulin E (IgE) may be involved in the genesis of atopic disease. The identity of the IgE receptor(s) on LC remained unclear, although it represents a crucial point in understanding cellular events linked to the binding of allergens to LC via IgE. In this report, we demonstrate that epidermal LC express the high affinity receptor for the Fc fragment of IgE (Fc epsilon RI) which has, so far, only been described on mast c...

  15. Highly Selective and Rapid Arsenic Removal by Metabolically Engineered Escherichia coli Cells Expressing Fucus vesiculosus Metallothionein▿

    Science.gov (United States)

    Singh, Shailendra; Mulchandani, Ashok; Chen, Wilfred

    2008-01-01

    An arsenic-chelating metallothionein (fMT) from the arsenic-tolerant marine alga Fucus vesiculosus was expressed in Escherichia coli, resulting in 30- and 26-fold-higher As(III) and As(V) binding, respectively. Coexpression of the As(III)-specific transporter GlpF with fMT further improved arsenic accumulation and offered high selectivity toward As. Resting E. coli cells coexpressing fMT and GlpF completely removed trace amounts (35 ppb) of As(III) within 20 min, providing a promising technology for compliance with the As limit of 10 ppb newly recommended by the U.S. EPA. PMID:18326684

  16. High-expression of recombinant human concensus interferon-α by Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    HAO Yuyou; SHI Qiqi; HE Yun; ZHUANG Yingping; WANG Yonghong; ZHANG Siliang; CHU Ju; LIU Zhimin

    2007-01-01

    The present work focused on the high expression of recombinant human consensus interferon-α (cIFN) by Pichia pastoris.The cycle of glycerol feeding,the strategy of methanol feeding and the optimum pH for protein induction were studied.The optimized strategies were a 4-h glycerol-feeding period,induction pH being kept at 5.0 and methanol concentration being kept under 5 g/L.The maximum dry cell weight,cIFN production and bioactivity obtained were 168,1.24 g/L and 5.4 × 107 U/mL,respectively.

  17. A New Expression for the Full Energy Peak Efficiency of a High Pure Germanium Detector

    CERN Document Server

    Medhat, M E; Awaad, Z

    2001-01-01

    An empirical expression for the full energy photo-peak efficiency in terms of gamma-ray energy (E) and the vertical distance from the detector surface (d) (i.e. efficiency = function (d,E)) has been obtained for a high pure germanium detector (HPGe) using different standard sources. Comparison of the calculated efficiencies and the experimentally measured values for the energy range from 59.5-1332.2 keV and a source-to-detector distance of 5-30 cm showed that the theoretical values agree with the experiment.

  18. Association between high expression macrophage migration inhibitory factor (MIF) alleles and West Nile virus encephalitis.

    Science.gov (United States)

    Das, Rituparna; Loughran, Kerry; Murchison, Charles; Qian, Feng; Leng, Lin; Song, Yan; Montgomery, Ruth R; Loeb, Mark; Bucala, Richard

    2016-02-01

    Infection with mosquito-borne West Nile virus (WNV) is usually asymptomatic but can lead to severe WNV encephalitis. The innate cytokine, macrophage migration inhibitory factor (MIF), is elevated in patients with WNV encephalitis and promotes viral neuroinvasion and mortality in animal models. In a case-control study, we examined functional polymorphisms in the MIF locus in a cohort of 454 North American patients with neuroinvasive WNV disease and found patients homozygous for high-expression MIF alleles to be >20-fold (p=0.008) more likely to have WNV encephalitis. These data indicate that MIF is an important determinant of severity of WNV neuropathogenesis and may be a therapeutic target.

  19. Modulation of PPAR Expression and Activity in Response to Polyphenolic Compounds in High Fat Diets

    Science.gov (United States)

    Domínguez-Avila, J. Abraham; González-Aguilar, Gustavo A.; Alvarez-Parrilla, Emilio; de la Rosa, Laura A.

    2016-01-01

    Peroxisome proliferator-activated receptors (PPAR) are transcription factors that modulate energy metabolism in liver, adipose tissue and muscle. High fat diets (HFD) can negatively impact PPAR expression or activity, favoring obesity, dyslipidemia, insulin resistance and other conditions. However, polyphenols (PP) found in vegetable foodstuffs are capable of positively modulating this pathway. We therefore focused this review on the possible effects that PP can have on PPAR when administered together with HFD. We found that PP from diverse sources, such as coffee, olives, rice, berries and others, are capable of inducing the expression of genes involved in a decrease of adipose mass, liver and serum lipids and lipid biosynthesis in animal and cell models of HFD. Since cells or gut bacteria can transform PP into different metabolites, it is possible that a synergistic or antagonistic effect ultimately occurs. PP molecules from vegetable sources are an interesting option to maintain or return to a state of energy homeostasis, possibly due to an adequate PPAR expression and activity. PMID:27367676

  20. Identification of genes preferentially expressed by highly virulent piscine Streptococcus agalactiae upon interaction with macrophages.

    Directory of Open Access Journals (Sweden)

    Chang-Ming Guo

    Full Text Available Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood-brain barrier (BBB. The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB.

  1. Low and high expressing alleles of the LMNA gene: implications for laminopathy disease development.

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    Sofía Rodríguez

    Full Text Available Today, there are at least a dozen different genetic disorders caused by mutations within the LMNA gene, and collectively, they are named laminopathies. Interestingly, the same mutation can cause phenotypes with different severities or even different disorders and might, in some cases, be asymptomatic. We hypothesized that one possible contributing mechanism for this phenotypic variability could be the existence of high and low expressing alleles in the LMNA locus. To investigate this hypothesis, we developed an allele-specific absolute quantification method for lamin A and lamin C transcripts using the polymorphic rs4641(C/TLMNA coding SNP. The contribution of each allele to the total transcript level was investigated in nine informative human primary dermal fibroblast cultures from Hutchinson-Gilford progeria syndrome (HGPS and unaffected controls. Our results show differential expression of the two alleles. The C allele is more frequently expressed and accounts for ∼70% of the lamin A and lamin C transcripts. Analysis of samples from six patients with Hutchinson-Gilford progeria syndrome showed that the c.1824C>T, p.G608G mutation is located in both the C and the T allele, which might account for the variability in phenotype seen among HGPS patients. Our method should be useful for further studies of human samples with mutations in the LMNA gene and to increase the understanding of the link between genotype and phenotype in laminopathies.

  2. Zebrafish Cx35: cloning and characterization of a gap junction gene highly expressed in the retina.

    Science.gov (United States)

    McLachlan, Elizabeth; White, Thomas W; Ugonabo, Chioma; Olson, Carl; Nagy, James I; Valdimarsson, Gunnar

    2003-09-15

    The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304-amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT-PCR, at 2 days post-fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos.

  3. Perception of emotional facial expressions in individuals with high Autism-spectrum Quotient (AQ

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    Ervin Poljac

    2012-10-01

    Full Text Available Autism is characterized by difficulties in social interaction, communication, restrictive and repetitive behaviours and specific impairments in emotional processing. The present study employed The Autism Spectrum Quotient (Baron-Cohen et al. 2006 to quantify autistic traits in a group of 260 healthy individuals and to investigate whether this measure is related to the perception of facial emotional expressions. The emotional processing of twelve participants that scored significantly higher than the average on the AQ was compared to twelve participants with significantly lower AQ scores. Perception of emotional expressions was estimated by The Facial Recognition Task (Montagne et al. 2007. There were significant differences between the two groups with regard to accuracy and sensitivity of the perception of emotional facial expressions. Specifically, the group with high AQ score was less accurate and needed higher emotional content to recognize emotions of anger, disgust, happiness and sadness. This result implies a selective impairment that might be helpful in understanding the psychopathology of autism spectrum disorders.

  4. High-Resolution Genomic and Expression Profiling Reveals 105 Putative Amplification Target Genes in Pancreatic Cancer

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    Eija H. Mahlamaki

    2004-09-01

    Full Text Available Comparative genomic hybridization (CGH studies have provided a wealth of information on common copy number aberrations in pancreatic cancer, but the genes affected by these aberrations are largely unknown. To identify putative amplification target genes in pancreatic cancer, we performed a parallel copy number and expression survey in 13 pancreatic cancer cell lines using a 12,232-clone cDNA microarray, providing an average resolution of 300 kb throughout the human genome. CGH on cDNA microarray allowed highly accurate mapping of copy number increases and resulted in identification of 24 independent amplicons, ranging in size from 130 kb to 11 Mb. Statistical evaluation of gene copy number and expression data across all 13 cell lines revealed a set of 105 genes whose elevated expression levels were directly attributable to increased copy number. These included genes previously reported to be amplified in cancer as well as several novel targets for copy number alterations, such as p21-activated kinase 4 (PAK4, which was previously shown to be involved in cell migration, cell adhesion, and anchorage-independent growth. In conclusion, our results implicate a set of 105 genes that is likely to be actively involved in the development and progression of pancreatic cancer.

  5. High expression of N-acetylglucosaminyltransferase IVa promotes invasion of choriocarcinoma.

    Science.gov (United States)

    Niimi, K; Yamamoto, E; Fujiwara, S; Shinjo, K; Kotani, T; Umezu, T; Kajiyama, H; Shibata, K; Ino, K; Kikkawa, F

    2012-12-04

    Gestational trophoblastic diseases (GTDs) are related to trophoblasts, and human chorionic gonadotropin (hCG) is secreted by GTDs as well as normal placentas. However, the asparagine-linked sugar chains on hCG contain abnormal biantennary structures in invasive mole and choriocarcinoma, but not normal pregnancy or hydatidiform mole. N-acetylglucosaminyltransferase-IV (GnT-IV) catalyses β1,4-N-acetylglucosamine branching on asparagine-linked oligosaccharides, which are consistent with the abnormal sugar chain structures on hCG. We investigated GnT-IVa expression in GTDs and placentas by immunohistochemistry, western blot, and RT-PCR. We assessed the effects of GnT-IVa knockdown in choriocarcinoma cells in vitro and in vivo. The GnT-IVa was highly expressed in trophoblasts of invasive mole and choriocarcinoma, and moderately in extravillous trophoblasts during the first trimester, but not in hydatidiform mole or other normal trophoblasts. The GnT-IVa knockdown in choriocarcinoma cells significantly reduced migration and invasive capacities, and suppressed cellular adhesion to extracellular matrix proteins. The extent of β1,4-N-acetylglucosamine branching on β1 integrin was greatly reduced by GnT-IVa knockdown, although the expression of β1 integrin was not changed. In vivo studies further demonstrated that GnT-IVa knockdown suppressed tumour engraftment and growth. These findings suggest that GnT-IVa is involved in regulating invasion of choriocarcinoma through modifications of the oligosaccharide chains of β1 integrin.

  6. Modulation of PPAR Expression and Activity in Response to Polyphenolic Compounds in High Fat Diets

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    J. Abraham Domínguez-Avila

    2016-06-01

    Full Text Available Peroxisome proliferator-activated receptors (PPAR are transcription factors that modulate energy metabolism in liver, adipose tissue and muscle. High fat diets (HFD can negatively impact PPAR expression or activity, favoring obesity, dyslipidemia, insulin resistance and other conditions. However, polyphenols (PP found in vegetable foodstuffs are capable of positively modulating this pathway. We therefore focused this review on the possible effects that PP can have on PPAR when administered together with HFD. We found that PP from diverse sources, such as coffee, olives, rice, berries and others, are capable of inducing the expression of genes involved in a decrease of adipose mass, liver and serum lipids and lipid biosynthesis in animal and cell models of HFD. Since cells or gut bacteria can transform PP into different metabolites, it is possible that a synergistic or antagonistic effect ultimately occurs. PP molecules from vegetable sources are an interesting option to maintain or return to a state of energy homeostasis, possibly due to an adequate PPAR expression and activity.

  7. High expression of Lin28 is associated with tumour aggressiveness and poor prognosis of patients in oesophagus cancer.

    Science.gov (United States)

    Hamano, R; Miyata, H; Yamasaki, M; Sugimura, K; Tanaka, K; Kurokawa, Y; Nakajima, K; Takiguchi, S; Fujiwara, Y; Mori, M; Doki, Y

    2012-04-10

    Lin28 is a negative regulator of the tumour suppressor microRNA, let-7, suggesting its role in tumourigenesis. However, the clinical significance of Lin28 expression in oesophageal cancer has not been elucidated. Lin28 and Lin28B expression was examined by immunohistochemistry in 161 tissues from patients with oesophageal cancer who had undergone curative surgery. The relationship between the expressions of Lin28 and Lin28B and various clinicopathological factors was examined. In vitro assays were conducted to determine the role of Lin28 in aggressiveness of oesophageal cancers using oesophageal cancer cell line. Lin28 and Lin28B were overexpressed in oesophageal cancer cells compared with non-cancerous epithelial cells, especially in the invasive front. High expression of Lin28 and Lin28B correlated significantly with lymph node metastasis and poor prognosis. High expression of Lin28B expression correlated significantly with low expression of let-7. Multivariate analysis also identified Lin28B expression as an independent prognostic factor. In vitro assays showed that the proliferative and invasive activities were significantly reduced in Lin28B-knockdown cells, compared with control cells. High expression of Lin28 is associated with poor prognosis and high tumour aggressiveness in oesophageal cancer and these effects are mediated through increased proliferation and invasiveness of oesophageal cancer cells.

  8. Expression of Selenoprotein Genes Is Affected by Obesity of Pigs Fed a High-Fat Diet.

    Science.gov (United States)

    Zhao, Hua; Li, Ke; Tang, Jia-Yong; Zhou, Ji-Chang; Wang, Kang-Ning; Xia, Xin-Jie; Lei, Xin Gen

    2015-07-01

    Relations of the 25 mammalian selenoprotein genes with obesity and the associated inflammation remain unclear. This study explored impacts of high-fat diet-induced obesity on inflammation and expressions of selenoprotein and obesity-related genes in 10 tissues of pigs. Plasma and 10 tissues were collected from pigs (n = 10) fed a corn-soy-based control diet or that diet containing 3-7% lard from weanling to finishing (180 d). Plasma concentrations (n = 8) of cytokines and thyroid hormones and tissue mRNA abundance (n = 4) of 25 selenoprotein genes and 16 obesity-related genes were compared between the pigs fed the control and high-fat diets. Stepwise regression was applied to analyze correlations among all these measures, including the previously reported body physical and plasma biochemical variables. The high-fat diet elevated (P obesity-related genes in 3 patterns. Specifically, the high-fat diet up-regulated 12 selenoprotein genes in 6 tissues, down-regulated 13 selenoprotein genes in 7 tissues, and exerted no effect on 5 genes in any tissue. Body weights and plasma triglyceride concentrations of pigs showed the strongest regressions to tissue mRNA abundances of selenoprotein and obesity-related genes. Among the selenoprotein genes, selenoprotein V and I were ranked as the strongest independent variables for the regression of phenotypic and plasma measures. Meanwhile, agouti signaling protein, adiponectin, and resistin genes represented the strongest independent variables of the obesity-related genes for the regression of tissue selenoprotein mRNA. The high-fat diet induced inflammation in pigs and affected their gene expression of selenoproteins associated with thioredoxin and oxidoreductase systems, local tissue thyroid hormone activity, endoplasmic reticulum protein degradation, and phosphorylation of lipids. This porcine model may be used to study interactive mechanisms between excess fat intake and selenoprotein function. © 2015 American Society for

  9. A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

    Energy Technology Data Exchange (ETDEWEB)

    Thorgersen, Michael P. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Lancaster, W. Andrew [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Rajeev, Lara [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Ge, Xiaoxuan [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Vaccaro, Brian J. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Poole, Farris L. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; Arkin, Adam P. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Mukhopadhyay, Aindrila [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Adams, Michael W. W. [Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology

    2016-12-02

    Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Here in this paper, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. Finally, while other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by

  10. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth

    DEFF Research Database (Denmark)

    Jakociune, Dziuginta; Herrero-Fresno, Ana; Jelsbak, Lotte

    2016-01-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality......, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino...

  11. Nuclear Kaiso expression is associated with high grade and triple-negative invasive breast cancer.

    Directory of Open Access Journals (Sweden)

    Jeroen F Vermeulen

    Full Text Available Kaiso is a BTB/POZ transcription factor that is ubiquitously expressed in multiple cell types and functions as a transcriptional repressor and activator. Little is known about Kaiso expression and localization in breast cancer. Here, we have related pathological features and molecular subtypes to Kaiso expression in 477 cases of human invasive breast cancer. Nuclear Kaiso was predominantly found in invasive ductal carcinoma (IDC (p = 0.007, while cytoplasmic Kaiso expression was linked to invasive lobular carcinoma (ILC (p = 0.006. Although cytoplasmic Kaiso did not correlate to clinicopathological features, we found a significant correlation between nuclear Kaiso, high histological grade (p = 0.023, ERα negativity (p = 0.001, and the HER2-driven and basal/triple-negative breast cancers (p = 0.018. Interestingly, nuclear Kaiso was also abundant in BRCA1-associated breast cancer (p<0.001 and invasive breast cancer overexpressing EGFR (p = 0.019. We observed a correlation between nuclear Kaiso and membrane-localized E-cadherin and p120-catenin (p120 (p<0.01. In contrast, cytoplasmic p120 strongly correlated with loss of E-cadherin and low nuclear Kaiso (p = 0.005. We could confirm these findings in human ILC cells and cell lines derived from conditional mouse models of ILC. Moreover, we present functional data that substantiate a mechanism whereby E-cadherin controls p120-mediated relief of Kaiso-dependent gene repression. In conclusion, our data indicate that nuclear Kaiso is common in clinically aggressive ductal breast cancer, while cytoplasmic Kaiso and a p120-mediated relief of Kaiso-dependent transcriptional repression characterize ILC.

  12. Impact of high predation risk on genome-wide hippocampal gene expression in snowshoe hares.

    Science.gov (United States)

    Lavergne, Sophia G; McGowan, Patrick O; Krebs, Charles J; Boonstra, Rudy

    2014-11-01

    The population dynamics of snowshoe hares (Lepus americanus) are fundamental to the ecosystem dynamics of Canada's boreal forest. During the 8- to 11-year population cycle, hare densities can fluctuate up to 40-fold. Predators in this system (lynx, coyotes, great-horned owls) affect population numbers not only through direct mortality but also through sublethal effects. The chronic stress hypothesis posits that high predation risk during the decline severely stresses hares, leading to greater stress responses, heightened ability to mobilize cortisol and energy, and a poorer body condition. These effects may result in, or be mediated by, differential gene expression. We used an oligonucleotide microarray designed for a closely-related species, the European rabbit (Oryctolagus cuniculus), to characterize differences in genome-wide hippocampal RNA transcript abundance in wild hares from the Yukon during peak and decline phases of a single cycle. A total of 106 genes were differentially regulated between phases. Array results were validated with quantitative real-time PCR, and mammalian protein sequence similarity was used to infer gene function. In comparison to hares from the peak, decline phase hares showed increased expression of genes involved in metabolic processes and hormone response, and decreased expression of immune response and blood cell formation genes. We found evidence for predation risk effects on the expression of genes whose putative functions correspond with physiological impacts known to be induced by predation risk in snowshoe hares. This study shows, for the first time, a link between changes in demography and alterations in neural RNA transcript abundance in a natural population.

  13. High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

    Directory of Open Access Journals (Sweden)

    Craveiro R.B.

    2006-01-01

    Full Text Available Carboxypeptidase M (CPM is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1. This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa, suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

  14. Feeding strategies enhance high cell density cultivation and protein expression in milliliter scale bioreactors.

    Science.gov (United States)

    Faust, Georg; Janzen, Nils H; Bendig, Christoph; Römer, Lin; Kaufmann, Klaus; Weuster-Botz, Dirk

    2014-10-01

    Miniature bioreactors under parallel fed-batch operations are not only useful screening tools for bioprocess development but also provide a suitable basis for eventual scale-up. In this study, three feeding strategies were investigated: besides the established intermittent feeding by a liquid handler, an optimized microfluidic device and a new enzymatic release system were applied for parallel fed-batch cultivation of Escherichia coli HMS174(DE3) and BL21(DE3) strains in stirred-tank bioreactors on a 10 mL scale. Lower fluctuation in dissolved oxygen (DO) and higher optical densities were measured in fed-batch processes applying the microfluidic device or the enzymatic glucose/fructose release system (conversion of intermittently added sucrose by an invertase), but no difference in dry cell weights (DCW) were observed. With all three feeding strategies high cell densities were realized on a milliliter scale with final optical density measured at 600 nm (OD600 ) of 114-133 and final DCW concentrations of 69-70 g L(-1) . The effect of feeding strategies on the expression of two heterologous proteins was investigated. Whereas no impact was observed on the expression of the spider silk protein eADF4(C16), the fluorescence of enhanced green fluorescence protein (eGFP) was reproducibly lower, if an intermittent glucose feed was applied. Thus, the impact of feeding strategy on expression is strongly dependent on the E. coli strain and/or expressed protein. As a completely continuous feed supply is difficult to realize in miniature bioreactors, the enzymatic release approach from this study can be easily applied in all microfluidic system to reduce fluctuations of glucose supply and DO concentrations.

  15. High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

    Directory of Open Access Journals (Sweden)

    R.B. Craveiro

    2006-02-01

    Full Text Available Carboxypeptidase M (CPM is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1. This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa, suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

  16. High Expression of NKG2A/CD94 and Low Expression of Granzyme B Are Associated with Reduced Cord Blood NK Cell Activity

    Institute of Scientific and Technical Information of China (English)

    Yanyan Wang; Han Xu; Xiaodong Zheng; Haiming Wei; Rui Sun; Zhigang Tian

    2007-01-01

    Human umbilical cord blood (CB) has recently been used as a source of stem cells in transplantation. NK cells derived from CB are the key effector cells involved in graft-versus-host disease (GVHD) and graft-versus-leukemia(GVL). It was reported that the activity of CB NK cells was lower than that of adult peripheral blood (PB) NK cells.In this study, we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in CB and PB NK cells. The expressions of activating NK receptors, CD16, NKG2D and NKp46, did not show significant difference between CB and PB NK cells. But the expression of inhibitory receptor NKG2A/CD94 was significantly higher on CB NK cells. As to the effector function molecules, granzyme B was expressed significantly lower in CB NK cells, but the expressions of intracellular perforin, IFN-γ, TNF-α and cell surface FasL and TRAIL did not show difference between CB and PB NK cells. The results indicated that the high expression of NKG2A/CD94 and low expression of granzyme B may be related with the reduced activity of CB NK cells.

  17. Pathway-based factor analysis of gene expression data produces highly heritable phenotypes that associate with age

    OpenAIRE

    Brown, Andrew Anand; Ding, Zhihao; Viñuela, Ana; Glass, Dan; Parts, Leopold; Spector, Tim; Winn, John; Durbin, Richard

    2015-01-01

    Statistical factor analysis methods have previously been used to remove noise components from high-dimensional data prior to genetic association mapping and, in a guided fashion, to summarize biologically relevant sources of variation. Here, we show how the derived factors summarizing pathway expression can be used to analyze the relationships between expression, heritability, and aging. We used skin gene expression data from 647 twins from the MuTHER Consortium and applied factor analysis to...

  18. Peripheral blood mononuclear cell gene expression in healthy adults rapidly transported to high altitude

    Directory of Open Access Journals (Sweden)

    Herman NM

    2014-12-01

    Full Text Available Nicole M Herman,1 Diane E Grill,2 Paul J Anderson,1 Andrew D Miller,1 Jacob B Johnson,1 Kathy A O’Malley,1 Maile L Ceridon Richert,1 Bruce D Johnson1 1Department of Cardiovascular Diseases, 2Department of Biostatistics, Mayo Clinic Rochester, MN, USA Abstract: Although mechanisms of high altitude illness have been studied extensively, the processes behind the development of these conditions are still unclear. Few genome-wide studies on rapid exposure to high altitude have been performed. Each year, scientists and support workers are transferred by plane from McMurdo Station in Antarctica (sea level to the Amundsen-Scott South Pole Station at 2,835 meters. This uniform and rapid transfer to altitude provides a unique opportunity to study the effects of hypobaric hypoxia on gene expression that may help illustrate the body's adaptations to these conditions. We hypothesized that an extensive number of genes would change with rapid exposure to altitude and further expected that these genes would correspond to inflammatory pathways proposed as a mechanism in development of acute mountain sickness. Peripheral venous blood samples were drawn from 98 healthy subjects at sea level and again on day two at altitude. Microarray analysis was performed on these samples. In total, 1,118 probe sets with significant P-values and fold changes (90% upregulated were identified and entered into MetaCore™ software. Several pathways, including oxidative phosphorylation, cytoskeleton remodeling, and platelet aggregation, were significantly represented by the data set and all were upregulated. Many genes changed expression, and the vast majority of these increased. Increased metabolism in peripheral blood mononuclear cells suggests increased inflammatory activity. Keywords: peripheral blood mononuclear cells, microarray, gene expression, acute mountain sickness

  19. Hierarchical IL-5 expression defines a subpopulation of highly differentiated human Th2 cells.

    Science.gov (United States)

    Upadhyaya, Bhaskar; Yin, Yuzhi; Hill, Brenna J; Douek, Daniel C; Prussin, Calman

    2011-09-15

    Each of the three Th2 cytokine genes, IL-4, IL-5, and IL-13, has different functions. We hypothesized that Th2 heterogeneity could yield Th2 subpopulations with different cytokine expression and effector functions. Using multiple approaches, we demonstrate that human Th2 cells are composed of two major subpopulations: a minority IL-5(+) (IL-5(+), IL-4(+), IL-13(+)) and majority IL-5(-) Th2 (IL-5(-), IL-4(+), IL-13(+)) population. IL-5(+) Th2 cells comprised only 20% of all Th2 cells. Serial rounds of in vitro differentiation initially yielded IL-5(-) Th2, but required multiple rounds of differentiation to generate IL-5(+) Th2 cells. IL-5(+) Th2 cells expressed less CD27 and greater programmed cell death-1 than IL-5(-) Th2 cells, consistent with their being more highly differentiated, Ag-exposed memory cells. IL-5(+) Th2 cells expressed greater IL-4, IL-13, and GATA-3 relative to IL-5(-) Th2 cells. GATA-3 and H3K4me(3) binding to the IL5 promoter (IL5p) was greater in IL-5(+) relative to IL-5(-) Th2 cells, whereas there was no difference in their binding to the IL4p and IL13p. Conversely, H3K27me(3) binding to the IL5p was greater in IL-5(-) Th2 cells. These findings demonstrate Th2 lineage heterogeneity, in which the IL5 gene is regulated in a hierarchical manner relative to other Th2 genes. IL-5(+) Th2 cells are phenotypically distinct and have epigenetic changes consistent with greater IL5p accessibility. Recurrent antigenic exposure preferentially drives the differentiation of IL-5(+) Th2 cells. These results demonstrate that IL-5(+) and IL-5(-) Th2 cells, respectively, represent more and less highly differentiated Th2 cell subpopulations. Such Th2 subpopulations may differentially contribute to Th2-driven pathology.

  20. High expression of CD26 accurately identifies human bacteria-reactive MR1-restricted MAIT cells.

    Science.gov (United States)

    Sharma, Prabhat K; Wong, Emily B; Napier, Ruth J; Bishai, William R; Ndung'u, Thumbi; Kasprowicz, Victoria O; Lewinsohn, Deborah A; Lewinsohn, David M; Gold, Marielle C

    2015-07-01

    Mucosa-associated invariant T (MAIT) cells express the semi-invariant T-cell receptor TRAV1-2 and detect a range of bacteria and fungi through the MHC-like molecule MR1. However, knowledge of the function and phenotype of bacteria-reactive MR1-restricted TRAV1-2(+) MAIT cells from human blood is limited. We broadly characterized the function of MR1-restricted MAIT cells in response to bacteria-infected targets and defined a phenotypic panel to identify these cells in the circulation. We demonstrated that bacteria-reactive MR1-restricted T cells shared effector functions of cytolytic effector CD8(+) T cells. By analysing an extensive panel of phenotypic markers, we determined that CD26 and CD161 were most strongly associated with these T cells. Using FACS to sort phenotypically defined CD8(+) subsets we demonstrated that high expression of CD26 on CD8(+)  TRAV1-2(+) cells identified with high specificity and sensitivity, bacteria-reactive MR1-restricted T cells from human blood. CD161(hi) was also specific for but lacked sensitivity in identifying all bacteria-reactive MR1-restricted T cells, some of which were CD161(dim) . Using cell surface expression of CD8, TRAV1-2, and CD26(hi) in the absence of stimulation we confirm that bacteria-reactive T cells are lacking in the blood of individuals with active tuberculosis and are restored in the blood of individuals undergoing treatment for tuberculosis.

  1. The European Expression Of Interest For High Purity U-233 Materials

    Energy Technology Data Exchange (ETDEWEB)

    Giaquinto, Joseph M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Younkin, James R. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2017-04-01

    The purpose of this letter report is to document the response for an Expression of Interest (EOI) sent to the European Safeguards and research and development (R&D) scientific communities for the distribution of small amounts of high purity 233U materials for use in safeguards, nonproliferation, and basic R&D in the nuclear disciplines. The intent for the EOI was to gauge the level of international interest for these materials from government and research institutions with programmatic missions in the nuclear security or nuclear R&D arena. The information contained herein is intended to provide information to assist key decision makers in DOE as to the ultimate disposition path for the high purity materials currently being recovered at Oak Ridge National Laboratory (ORNL) and only those items for which there is no United States (U.S.) sponsor identified.

  2. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.

    Science.gov (United States)

    Ma, Yi; Yu, Jieying; Lin, Jinglian; Wu, Shaomin; Li, Shan; Wang, Jufang

    2016-01-01

    Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  3. High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene.

    Science.gov (United States)

    Banguela, Alexander; Arrieta, Juan G; Rodríguez, Raisa; Trujillo, Luis E; Menéndez, Carmen; Hernández, Lázaro

    2011-06-10

    Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long β(2,6)-linked fructosyl chains with β(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10(4) fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded β(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30mgg(-1) of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan.

  4. Highly and moderately aggressive mouse ovarian cancer cell lines exhibit differential gene expression

    Science.gov (United States)

    Zhang, Wensheng; Kale, Shubha P.; McFerrin, Harris; Davenport, Ian; Wang, Guangdi; Skripnikova, Elena; Li, Xiao-Lin; Bowen, Nathan J.; McDaniels, Leticia B; Meng, Yuan-Xiang; Polk, Paula; Liu, Yong-Yu; Zhang, Qian-Jin

    2017-01-01

    Patients with advanced epithelial ovarian cancer often experience disease recurrence after standard therapies, a critical factor in determining their five-year survival rate. Recent reports indicated that long-term or short-term survival is associated with varied gene expression of cancer cells. Thus, identification of novel prognostic biomarkers should be considered. Since the mouse genome is similar to the human genome, we explored potential prognostic biomarkers using two groups of mouse ovarian cancer cell lines (group 1: IG-10, IG-10pw, and IG-10pw/agar; group 2: IG-10 clones 2, 3, and 11) which display highly and moderately aggressive phenotypes in vivo. Mice injected with these cell lines have different survival time and rates, capacities of tumor, and ascites formations, reflecting different prognostic potentials. Using an Affymetrix Mouse Genome 430 2.0 Array, a total of 181 genes were differentially expressed (Pdeath. None of the genes from a set of the 72 genes overexpressed in the moderately aggressive cell lines had a function annotation in the David database. Our results suggested that the overexpressed MYC and 109 gene set represented highly aggressive ovarian cancer potential biomarkers while overexpressed AR and 72 gene set represented moderately aggressive ovarian cancer potential biomarkers. Based on our knowledge, the current study is first time to report the potential biomarkers relevant to different aggressive ovarian cancer. These potential biomarkers provide important information for investigating human ovarian cancer prognosis. PMID:26935058

  5. Gold nanoparticles combined with highly expressed amber suppressor tRNA: a future antibacterial agent

    Directory of Open Access Journals (Sweden)

    Xiaoda Song

    2010-10-01

    Full Text Available "nAmber suppressor tRNA is a mutant allele coding for a tRNA, whose anticodon is altered in such a way that the suppressor tRNA inserts an amino acid at an amber codon in translation which leads to suppressing (preventing termination. And some Amber suppressor tRNA strains were found. We propose that gold nanoparticles combined with highly expressed amber suppressor tRNA which can be uptake by cells and recognized by AARS (aminoacyl tRNA synthetase will lead to the formation of C-terminally extended proteins. These proteins probably will not work properly, leading bacteria's death. Because of the difference of tRNA between prokaryotic and eukaryotic cells, even between different bacteria species, this amber suppressor tRNA is orthogonal for other species and cannot be recognized by AARS, therefore has no toxicity to other species. May it be an excellent antibacterial agent in the future? In this article we provide a screening method for the highly expressed amber suppressor tRNA using randomly bases mutation, radioactive selection, activity test in vivo, and finally linkage of the amber suppressor tRNA to gold nanoparticles.

  6. Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.

    Science.gov (United States)

    Spidel, Jared L; Vaessen, Benjamin; Chan, Yin Yin; Grasso, Luigi; Kline, J Bradford

    2016-12-01

    Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  7. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yi Ma

    2016-01-01

    Full Text Available Human epidermal growth factor (hEGF is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  8. High fibroblast growth factor 19 (FGF19) expression predicts worse prognosis in invasive ductal carcinoma of breast.

    Science.gov (United States)

    Buhmeida, Abdelbaset; Dallol, Ashraf; Merdad, Adnan; Al-Maghrabi, Jaudah; Gari, Mamdooh A; Abu-Elmagd, Muhammad M; Chaudhary, Adeel G; Abuzenadah, Adel M; Nedjadi, Taoufik; Ermiah, Eramah; Al-Thubaity, Fatima; Al-Qahtani, Mohammed H

    2014-03-01

    Metabolic diseases like diabetes and obesity are major risk factors for breast cancer. Aberrant expression of metabolic effectors such as fibroblast growth factor 19 (FGF19) could be therefore associated with the disease. The expression of FGF19 was examined in 193 archival breast tumor samples by immunohistochemistry and evaluated semi-quantitatively by determining the staining index and correlating it with clinicopathological parameters using Fisher's exact test. The correlation between FGF19 expression and 5-year disease-specific survival rate was determined using the univariate Kaplan-Meier analysis. The prognostic value of FGF19 expression was evaluated using the multivariate Cox regression analysis. Of the 193 tumors analyzed, 40% were classified with low FGF19 expression, whereas 60% were categorized as tumors with high FGF19 expression. There was a highly significant correlation between high FGF19 expression and patients' age (p = 0.008) as well as 5-year disease-specific survival (p = 0.001). However, FGF19 expression did not show any significant correlations with other clinicopathological parameters, including hormonal status, tumor grade, tumor size, or lymph node status. Univariate Kaplan-Meier log rank analysis showed that patients with high FGF19 expression exhibited a significantly shorter disease-specific 5-year survival (p = 0.007). This effect was exacerbated by lymph node metastasis (p = 0.001), negative estrogen receptor (ER) status (p = 0.002), or old age (p = 0.013). Multivariate analysis showed that high FGF19 expression could be an independent prognostic marker of disease-specific survival in breast cancer patients (p = 0.030). Quantification of FGF19 expression appears to provide valuable prognostic information in breast cancer, particularly in older patients with lymph node metastasis and negative ER status.

  9. High Expression of Insulin-like Growth Factor H (IGF-Ⅱ) Using Bac-to-Bac Expression System

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective In order to obtain mature insulin-like growth factor- Ⅱ ( IGF- Ⅱ ), we used Bac-to-Bac baculovirus expression system. Methods Firstly the IGF- Ⅱ cDNA was cloned into a donor plasmid pFastBac1 and the recombinant pFastBac1 was then introduced into competent cells DH 10Bac. Recombinant bacmids were constructed by transposing a mini-Tn7 element from a donor plasmid pFastBac1 to the mini-attTn7 attachment site on the bacmid where the Tn7 transposition functions were provided in trans by a helper plasmid, and then used to transfect Sf9 insect cells to get recombinant baculovirus. The recombinant baculovirus was used to infect insect cells. Results Agarose gel analysis showed that recombinant donor plasmid pFastBac1 was constructed successfully; Agarose gel analysis of PCR products confirmed recombinant bacmid ; SDS-PAGE and Western Blotting showed that a 7KD protein band appeared. Conclusion The mature IGF- Ⅱ with immunogenecity has been expressed and produced by using Bac-to-Bac expression system.

  10. High-Salt Intake Suppressed MicroRNA-133a Expression in Dahl SS Rat Myocardium

    Directory of Open Access Journals (Sweden)

    Tong-Shuai Guo

    2014-06-01

    Full Text Available Salt-sensitive individuals show earlier and more serious cardiac damage than nonsalt-sensitive ones. Some studies have suggested that microRNA-133a could reduce cardiac hypertrophy and myocardial fibrosis. The current study aims to investigate the different functions of high-salt intake on salt-sensitive (SS rats and Sprague-Dawley (SD rats and the involvement of microRNA-133a in these roles. After high-salt intervention, the left ventricular mass (LVW and left ventricular mass index (LVMI of the salt-sensitive high salt (SHS group were obviously higher than those of the salt-sensitive low salt (SLS group. However, the difference between the Sprague-Dawley high salt (DHS group and the Sprague-Dawley low salt (DLS group was not significant. Compared with SLS group, collagen I and connective tissue growth factor (CTGF in the heart of SHS group were significantly higher, whereas no statistical difference was observed between the DHS group and the DLS group. Compared with low-salt diet, microRNA-133a in the heart of both strains were significantly decreased, but that in the SHS group decreased more significantly. These results suggest that high salt intervention could down-regulate the expression of myocardial microRNA-133a, which may be one of the mechanisms involved in myocardial fibrosis in salt-sensitive hypertension.

  11. Tumor Classification Using High-Order Gene Expression Profiles Based on Multilinear ICA

    Directory of Open Access Journals (Sweden)

    Ming-gang Du

    2009-01-01

    Full Text Available Motivation. Independent Components Analysis (ICA maximizes the statistical independence of the representational components of a training gene expression profiles (GEP ensemble, but it cannot distinguish relations between the different factors, or different modes, and it is not available to high-order GEP Data Mining. In order to generalize ICA, we introduce Multilinear-ICA and apply it to tumor classification using high order GEP. Firstly, we introduce the basis conceptions and operations of tensor and recommend Support Vector Machine (SVM classifier and Multilinear-ICA. Secondly, the higher score genes of original high order GEP are selected by using t-statistics and tabulate tensors. Thirdly, the tensors are performed by Multilinear-ICA. Finally, the SVM is used to classify the tumor subtypes. Results. To show the validity of the proposed method, we apply it to tumor classification using high order GEP. Though we only use three datasets, the experimental results show that the method is effective and feasible. Through this survey, we hope to gain some insight into the problem of high order GEP tumor classification, in aid of further developing more effective tumor classification algorithms.

  12. Emotional Facial and Vocal Expressions during Story Retelling by Children and Adolescents with High-Functioning Autism

    Science.gov (United States)

    Grossman, Ruth B.; Edelson, Lisa R.; Tager-Flusberg, Helen

    2013-01-01

    Purpose: People with high-functioning autism (HFA) have qualitative differences in facial expression and prosody production, which are rarely systematically quantified. The authors' goals were to qualitatively and quantitatively analyze prosody and facial expression productions in children and adolescents with HFA. Method: Participants were 22…

  13. The High Resolution Stereo Camera (HRSC) Experiment On The European Mars Express Mission

    Science.gov (United States)

    Neukum, G.; Hoffmann, H.; HRSC Science Experiment Team

    Imaging Mars is one of the main goals of the European Mars Express mission and will be performed by the German High Resolution Stereo Camera (HRSC). The HRSC ex- periment is a pushbroom scanning instrument with 9 CCD line detectors mounted in parallel on the focal plane. Its unique feature is the ability to obtain nearly simul- taneously imaging data of a specific site at high resolution, with along-track triple stereo, with four colours, and at five different phase angles, thus avoiding any time- dependent variations of the observation conditions. An additional Super-Resolution Channel (HRSC-SRC), a framing device, will yield nested-in images in the meter- range thus serving as the sharpening eye for detailed photogeologic studies. The spa- tial resolution from the nominal periapsis altitude of 250 km will be 10 m/pixel and 2.3 m/pixel for the HRSC-SRC. The manufacture of the flight hardware has been ac- complished. Before delivery of the flight model to ESA in January 2002, tests of the instrument were performed demonstrating its imaging capabilities and performances. During the nominal operational lifetime of the mission of 1 Martian year, it will be possible to cover at least 50% of the Martian surface at a spatial resolution of better than 15 m/pixel. More than 70% of the Martian surface can be observed at a spatial resolution of better than 30 m/pixel, while more than 1% of the surface will be im- aged at about 2.5 m/pixel. HRSC on Mars Express will be able to close the existing gap between medium to low-resolution coverage on the one hand and the very high resolution images of Mars Global Surveyor as well as the in-situ observations and measurements by landers on the other hand. The HRSC on Mars Express will make a major contribution to the study of Martian geosciences with special emphasis on the evolution of the Martian surface in general, the evolution of volcanism, and the role of water throughout the Martian history. The instrument will obtain images

  14. Myostatin expression, lymphocyte population, and potential cytokine production correlate with predisposition to high-fat diet induced obesity in mice.

    Directory of Open Access Journals (Sweden)

    Jeri-Anne Lyons

    Full Text Available A strong relationship exists between increased inflammatory cytokines and muscle insulin resistance in obesity. This study focused on identifying a relationship between metabolic propensity and myostatin expression in muscle and spleen cells in response to high-fat diet intake. Using a comparative approach, we analyzed the effects of high-fat diet intake on myostatin and follistatin expression, spleen cell composition, and potential cytokine expression in high-fat diet induced obesity (HFDIO resistant (SWR/J and susceptible (C57BL/6 mice models. Results demonstrated overall increased myostatin expression in muscle following high-fat diet intake in HFDIO-susceptible mice, while myostatin expression levels decreased initially in muscle from high-fat diet fed resistant mice. In HFDIO-resistant mice, myostatin expression decreased in spleen, while myostatin increased in spleen tissue from HFDIO-susceptible mice. Proinflammatory cytokine (IL-17, IL-1β, and IFNγ potential increased in splenocytes from HFDIO-susceptible mice. In comparison, C57BL/6 mice fed a high-fat diet exhibited higher frequencies of CD4(+/CD44(hi and CD8(+/CD44(hi cells in the spleen compared to control fed mice. Together, these results suggest that susceptibility to high-fat diet induced obesity could be influenced by local myostatin activity in a tissue-specific manner and that splenocytes exhibit differential cytokine production in a strain-dependent manner. This study sets the stage for future investigations into the interactions between growth, inflammation, and metabolism.

  15. In situ Expression of Functional Genes Reveals Nitrogen Cycling at High Temperatures in Terrestrial Hydrothermal Systems

    Science.gov (United States)

    Loiacono, S. T.; Meyer-Dombard, D. R.

    2011-12-01

    An essential element for life, nitrogen occurs in all living organisms and is critical for the synthesis of amino acids, proteins, nucleic acids, and other forms of biomass. Thus, nitrogen cycling likely plays a vital role in microbial metabolic processes as well as nutrient availability. For microorganisms in "extreme" environments, this means developing adaptations that allow them to survive in harsh conditions and still perform the metabolisms essential to sustain life. Recent studies have screened biofilms and thermal sediments of Yellowstone National Park (YNP) thermal features for the presence of nifH genes, which code for a key enzyme in the nitrogen fixation process [1-4]. Furthermore, analysis of nitrogen isotopes in biofilms across a temperature and chemical gradient revealed that nitrogen fixation likely varies across the chemosynthetic/photosynthetic ecotone [5]. Although research has evaluated and confirmed the presence of nifH genes in various thermophilic microbial communities, the existence of a gene in the DNA of an organism does not verify its use. Instead, other methods, such as culturing, isotope tracer assays, and gene expression studies are required to provide direct evidence of biological nitrogen fixation. Culturing and isotope tracer approaches have successfully revealed high-temperature biological nitrogen fixation in both marine hydrothermal vent microbial communities [6] and in acidic, terrestrial hydrothermal sediment [3]. Transcriptomics-based techniques (using mRNA extracted from samples to confirm in situ expression of targeted genes) have been much more limited in number, and only a few studies have, to date, investigated in situ expression of the nifH gene in thermophilic microbial communities [2, 7]. This study explores the presence and expression of nifH genes in several features of the Lower Geyser Basin (LGB) of YNP. Nucleic acids from chemosynthetic and photosynthetic microbial communities were extracted and then amplified

  16. High expression level of bone degrading proteins as a possible inducer of osteolytic features in pigmented villonodular synovitis.

    Science.gov (United States)

    Geldyyev, Aman; Koleganova, Nadezda; Piecha, Grzegorz; Sueltmann, Holger; Finis, Katharina; Ruschaupt, Markus; Poustka, Annemarie; Gross, Marie-Luise; Berger, Irina

    2007-10-08

    Protein expression of osteopontin (OPN), osteoprotegerin (OPG), bone sialoprotein (BSP), osteocalcin (OC), RANKL and PTHrP was determined by use of immunohistochemical analysis on tissue arrays (48 cases of PVNS, 20 cases of active (a-RA), non-active rheumatoid arthritis (na-RA), and osteoarthritis (OA)). Additionally, gene expression was analysed using complimentary DNA (cDNA) microarrays. All PVNS cases showed a higher level of both protein and gene expression of RANKL, OPN and BSP in comparison with OA cases. Expression of OPG was not significantly different in PVNS compared to OA. The RANKL/OPG expression ratio was significantly higher in PVNS than in OA. High expressions level of proteins involved in bone degradation in PVNS may promote an intra-osseous propagation of the lesion. This evidence suggests that PVNS might respond to treatment using specific inhibitors of RANKL, OPN and BSP.

  17. MicroRNA expression analysis in the liver of high fat diet-induced obese mice

    Directory of Open Access Journals (Sweden)

    Won-Mo Yang

    2016-12-01

    Full Text Available A previous study indicated a causal link between certain miRNAs induced by obesity and the development of hepatic insulin resistance and type 2 diabetes. Here we provide accompanying data collected using Affymetrix GeneChip miRNAs microarrays to identify the changes in miRNAs expression in the liver of mice fed a high fat diet (HFD. Differentially expressed microRNA analyses in the liver of the HFD-fed mice revealed a range of upregulated (>1.5-fold or downregulated (<0.5-fold miRNAs. Among those upregulated miRNAs, in silico target analysis, such as TargetScan, PicTar, and miRWalk, identified miRNAs with the putative binding sites on the 3’UTRs of INSR and/or IRS-1. Interpretation of the data and further extensive insights into the implication of miRNAs, particularly miR-15b, in hepatic insulin resistance can be found in "Obesity-induced miR-15b is linked causally to the development of insulin resistance through the repression of the insulin receptor in hepatocytes." (W.M. Yang, H.J. Jeong, S.W. Park, W. Lee, 2015[1].

  18. High expression of REGγ is associated with metastasis and poor prognosis of patients with breast cancer.

    Science.gov (United States)

    Chai, Fan; Liang, Yan; Bi, Jiong; Chen, Li; Zhang, Fan; Cui, Youhong; Bian, Xiuwu; Jiang, Jun

    2014-01-01

    REGgamma (REGγ) has been recently found in several types of human cancer, however, its clinical significance in metastasis and prognosis of breast cancer remains unknown. In this study, immunohistochemical staining and western blot analysis were performed to evaluate REGγ expression in both mouse and human breast cancer specimens. We found that in MMTV-PyMT mice, 14 out of 20 (70%) mouse mammary carcinomas were REGγ positive, which was significantly higher than control (0/20, 0%, P breast cancer tissues with the paired peritumoural normal breast tissues and 140 breast benign disease tissue samples showed that REGγ was undetectable in normal breast tissues and nonmetastatic axillary lymph nodes (ALNs), whereas 111 out of 136 (81.6%) breast cancer tissue samples were REGγ positive, which was significantly higher than breast benign disease tissues (9/140, 6.4%, P breast cancer (OR = 4.369, P = 0.008). Our results suggest that the high expression of REGγ might predict metastasis and poor prognosis in breast cancer.

  19. Spanning high-dimensional expression space using ribosome-binding site combinatorics.

    Science.gov (United States)

    Zelcbuch, Lior; Antonovsky, Niv; Bar-Even, Arren; Levin-Karp, Ayelet; Barenholz, Uri; Dayagi, Michal; Liebermeister, Wolfram; Flamholz, Avi; Noor, Elad; Amram, Shira; Brandis, Alexander; Bareia, Tasneem; Yofe, Ido; Jubran, Halim; Milo, Ron

    2013-05-01

    Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.

  20. The myostatin gene of Mytilus chilensis evidences a high level of polymorphism and ubiquitous transcript expression.

    Science.gov (United States)

    Núñez-Acuña, Gustavo; Gallardo-Escárate, Cristian

    2014-02-15

    Myostatin (MSTN) is a protein of the Transforming Growth Factor-β (TGF-β) superfamily and plays a crucial role in muscular development for higher vertebrates. However, its biological function in marine invertebrates remains undiscovered. This study characterizes the full-length sequence of the Mytilus chilensis myostatin gene (Mc-MSTN). Furthermore, tissue transcription patterns and putative single nucleotide polymorphisms (SNPs) were also identified. The Mc-MSTN cDNA sequence showed 3528 base pairs (bp), consisting of 161 bp of 5' UTR, 2,110 bp of 3' UTR, and an open reading frame of 1,257 bp encoding for 418 amino acids and with an RXXR proteolytic site and nine cysteine-conserved residues. Gene transcription analysis revealed that the Mc-MSTN has ubiquitous expression among several tissues, with higher expression in the gonads and mantle than in the digestive gland, gills, and hemolymph. Furthermore, high levels of polymorphisms were detected (28 SNPs in 3'-UTR and 9 SNPs in the coding region). Two SNPs were non-synonymous and involved amino acid changes between Glu/Asp and Thr/Ile. Until now, the MSTN gene has been mainly related to muscle growth in marine bivalves. However, the present study suggests a putative biological function not entirely associated to muscle tissue and contributes molecular evidence to the current debate about the function of the MSTN gene in marine invertebrates.

  1. Genomic Integration of High-Risk HPV Alters Gene Expression in Oropharyngeal Squamous Cell Carcinoma.

    Science.gov (United States)

    Walline, Heather M; Komarck, Christine M; McHugh, Jonathan B; Bellile, Emily L; Brenner, J Chad; Prince, Mark E; McKean, Erin L; Chepeha, Douglas B; Wolf, Gregory T; Worden, Francis P; Bradford, Carol R; Carey, Thomas E

    2016-10-01

    High-risk HPV (hrHPV) is the leading etiologic factor in oropharyngeal cancer. HPV-positive oropharyngeal tumors generally respond well to therapy, with complete recovery in approximately 80% of patients. However, it remains unclear why some patients are nonresponsive to treatment, with 20% of patients recurring within 5 years. In this study, viral factors were examined for possible clues to differences in tumor behavior. Oropharynx tumors that responded well to therapy were compared with those that persisted and recurred. Viral oncogene alternate transcripts were assessed, and cellular sites of viral integration were mapped and sequenced. Effects of integration on gene expression were assessed by transcript analysis at the integration sites. All of the tumors demonstrated active viral oncogenesis, indicated by expression of HPV E6 and E7 oncogenes and alternate E6 splicing. In the responsive tumors, HPV integration occurred exclusively in intergenic chromosome regions, except for one tumor with viral integration into TP63. Each recurrent tumor exhibited complex HPV integration patterns into cancer-associated genes, including TNFRSF13B, SCN2A, SH2B1, UBE2V2, SMOC1, NFIA, and SEMA6D Disrupted cellular transcripts were identified in the region of integration in four of the seven affected genes.

  2. Expression and purification of bioactive high-purity human midkine in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Zhong-hui ZHANG; Li-juan DU; Di XIANG; Shun-ying ZHU; Ming-yuan WU; Hui-li LU; Yan YU; Wei HAN

    2009-01-01

    Midkine is a heparin-binding growth factor,which plays important roles in the regulation of cell growth and differentiation.The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important growth factor.In the present work,rhMK was expressed in Escherichia coli (E.coli) BL21 (DE3).The expression of midkine was efficiently induced by isopropyl-β-D-thiogalactopyranoside (IPTG).After sonication,midkine was recovered in an insoluble form,and was dissolved in guaoidine hydrochloride buffer.Renaturation of the denatured protein was carried out in the defined protein refolding buffer,and the refolded protein was purified using S-Sepharose ion-exchange chromatography.The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC).The purified rhMK enhanced the proliferation of NIH3T3 cells.

  3. Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells

    Directory of Open Access Journals (Sweden)

    Andersen Søren S.L.

    2004-01-01

    Full Text Available In synaptic vesicles, the estimated concentration of the excitatory amino acid glutamate is 100-150 mM. It was recently discovered that VGLUT1, previously characterized as an inorganic phosphate transporter (BNPI with 9-11 predicted transmembrane spanning domains, is capable of transporting glutamate. The expression and His-tag based purification of recombinant VGLUT1 from PC12 cells and High Five insect cells is described. Significantly better virus and protein expression was obtained using High Five rather than Sf9 insect cells. PC12 cell expressed VGLUT1 is functional but not the Baculovirus expressed protein. The lack of functionality of the Baculovirus expressed VGLUT1 is discussed. The data indicate that VGLUT1 readily oligomerizes/dimerizes. The data are discussed in the context of developing this system further in order to reconstitute vesicular glutamate uptake in vitro using lipid-detergent vesicles.

  4. Stage III-IV Uterine Prolapse Risk Factors: Sacrouterine Ligaments High Estrogen Receptor Alpha and Collagen III Expression and Low Elastin Expression

    Directory of Open Access Journals (Sweden)

    I Wayan Megadhana

    2016-08-01

    Full Text Available Background: Uterine prolapse is common, non-life-threatening, but has a negative impact on women psychosocial and economic life. Damage to levator ani muscle is the early onset of uterine prolapse, while the damage of sacrouterine ligaments aggravates the stage. The strength of sacrouterine ligament depends on tissue cellularity, the formation of collagen I/III ratio, and the decreased expression of elastin. The lower the ratio of collagen I/III, the higher the risk of stage III-IV uterine prolapse. The ratio of collagen I/III formation is allegedly influencing through the expression of estrogen receptor alpha, by increasing collagen III synthesis and decreasing the degradation. Objective: We aimed to investigate whether high estrogen receptor alpha and collagen III expression, and the low elastin expression in the sacrouterine ligaments were stage III-IV uterine prolapse risk factors. Method: In March to August 2014, a non-matching case control study was conducted in 3 hospitals in Denpasar, and the materials were processed in the Faculty of Veterinary Medicine Laboratory of Udayana University. The case was uterine prolapse stage III-IV, the control was the non-uterine prolapse. We collected 1.5 cm residual sacrouterine ligaments from the edge of the cervix fixed with 10% buffered formalin from patients who underwent a total hysterectomy. They were examined immunohistochemically to identify estrogen receptor alpha expression, collagen III, and elastin. Results: Our sample was 44, divided equally between the case and control group. Compared to the control, in the case group, the proportion was significantly higher for the high estrogen receptor alpha expression (OR=5.71, 95%CI 1.56-20.93, p=0.007, high collagen III (OR=6.50, 95% CI 1.64- 25.76, p=0.005, and low elastin (OR=5.40, 95%CI 1.37-21.26, p=0.012. Conclusion: the high expression of estrogen receptor alpha and collagen III and low expressions of elastin in sacrouterine ligaments served as

  5. In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

    Directory of Open Access Journals (Sweden)

    Bekim Sadikovic

    Full Text Available Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.

  6. Gene expression profiling in human peripheral blood mononuclear cells using high-density filter-based cDNA microarrays.

    Science.gov (United States)

    Walker, J; Rigley, K

    2000-05-26

    Microarray technology has provided the ability to analyse the expression profiles for thousands of genes in parallel. The need for highly specialised equipment to use certain types of microarrays has restricted the application of this technology to a small number of dedicated laboratories. High-density filter-based cDNA microarrays provide a low-cost option for performing high-throughput gene expression analysis. We have used a model system in which filter-based cDNA microarrays representing over 4000 known human genes were used to monitor the kinetics of gene expression in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemagluttinin (PHA). Using software-based cluster analysis, we identified 104 genes that altered in expression levels in response to PHA stimulation of PBMCs and showed that there was a considerable overlap between genes with similar temporal expression profiles and similar functional roles. Comparison of microarray quantitation with quantitative PCR showed almost identical expression profiles for a number of genes. Coupled with the fact that our findings are in agreement with a large number of independent observations, we conclude that the use of filter-based cDNA microarrays is a valid and accurate method for high-throughput gene expression profiling.

  7. Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death.

    Science.gov (United States)

    Norholm, Morten H H; Nour-Eldin, Hussam H; Brodersen, Peter; Mundy, John; Halkier, Barbara A

    2006-04-17

    We report the biochemical characterization in Xenopus oocytes of the Arabidopsis thaliana membrane protein, STP13, as a high affinity, hexose-specific H(+)-symporter. Studies with kinase activators suggest that it is negatively regulated by phosphorylation. STP13 promoter GFP reporter lines show GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP13 in PCD is supported by microarray data from e.g. plants undergoing senescence and a strong correlation between STP13 transcripts and the PCD phenotype in different accelerated cell death (acd11) mutants.

  8. Evaluation and Application of Two High-Iron Transgenic Rice Lines Expressing a Pea Ferritin Gene

    Institute of Scientific and Technical Information of China (English)

    YE Hong-xai; LI Mei; Guo Ze-jian; Shu Qing-yao; xu Xiao-hui; BAO Jin-song; SHEN Sheng-quan

    2008-01-01

    A totaI of 105 transgenic rice lines independently transformed with a pea ferritin gene (Fer)were previously obtained.After seven generations of selfing and β-glucuronidase(GUS)assisted selection,82 transgenic lines with stable agronomic traits were got.Among the 82 transgenic lines,two high-iron transgenic rice lines Fer34 and Fer65,with the iron contents in the milled rice being 4.82 and 3.46 times of that of the wild type Xiushui 11,respectively were identified.In the two transgenic lines,the exogenous Fer gene was highly expressed,and inherited as a single locus.The transgene had no negative effect on the agronomic traits of rice plant,other mineral nutritional components,appearance quailty and eating quailty of the milled rice,indicating that these two lines were elite high-iron breeding lines.Furthermore,the practical application and further studies facilitating utilization of the two elite breeding lines were discussed.

  9. High abundance and expression of transposases in bacteria from the Baltic Sea.

    Science.gov (United States)

    Vigil-Stenman, Theoden; Ininbergs, Karolina; Bergman, Birgitta; Ekman, Martin

    2017-07-21

    Transposases are mobile genetic elements suggested to have an important role in bacterial genome plasticity and host adaptation but their transcriptional activity in natural bacterial communities is largely unexplored. Here we analyzed metagenomes and -transcriptomes of size fractionated (0.1-0.8, 0.8-3.0 and 3.0-200 μm) bacterial communities from the brackish Baltic Sea, and adjacent marine waters. The Baltic Sea transposase levels, up to 1.7% of bacterial genes and 2% of bacterial transcripts, were considerably higher than in marine waters and similar to levels reported for extreme environments. Large variations in expression were found between transposase families and groups of bacteria, with a two-fold higher transcription in Cyanobacteria than in any other phylum. The community-level results were corroborated at the genus level by Synechococcus transposases reaching up to 5.2% of genes and 6.9% of transcripts, which is in contrast to marine Synechococcus that largely lack these genes. Levels peaked in Synechococcus from the largest size fraction, suggesting high frequencies of lateral gene transfer and high genome plasticity in colony-forming picocyanobacteria. Together, the results support an elevated rate of transposition-based genome change and adaptation in bacterial populations of the Baltic Sea, and possibly also of other highly dynamic estuarine waters.The ISME Journal advance online publication, 21 July 2017; doi:10.1038/ismej.2017.114.

  10. High-throughput gene expression profiling of memory differentiation in primary human T cells

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    Russell Kate

    2008-08-01

    Full Text Available Abstract Background The differentiation of naive T and B cells into memory lymphocytes is essential for immunity to pathogens. Therapeutic manipulation of this cellular differentiation program could improve vaccine efficacy and the in vitro expansion of memory cells. However, chemical screens to identify compounds that induce memory differentiation have been limited by 1 the lack of reporter-gene or functional assays that can distinguish naive and memory-phenotype T cells at high throughput and 2 a suitable cell-line representative of naive T cells. Results Here, we describe a method for gene-expression based screening that allows primary naive and memory-phenotype lymphocytes to be discriminated based on complex genes signatures corresponding to these differentiation states. We used ligation-mediated amplification and a fluorescent, bead-based detection system to quantify simultaneously 55 transcripts representing naive and memory-phenotype signatures in purified populations of human T cells. The use of a multi-gene panel allowed better resolution than any constituent single gene. The method was precise, correlated well with Affymetrix microarray data, and could be easily scaled up for high-throughput. Conclusion This method provides a generic solution for high-throughput differentiation screens in primary human T cells where no single-gene or functional assay is available. This screening platform will allow the identification of small molecules, genes or soluble factors that direct memory differentiation in naive human lymphocytes.

  11. Cryptochrome genes are highly expressed in the ovary of the African clawed frog, Xenopus tropicalis.

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    Yoko Kubo

    Full Text Available Cryptochromes (CRYs are flavoproteins sharing high homology with photolyases. Some of them have function(s including transcription regulation in the circadian clock oscillation, blue-light photoreception for resetting the clock phase, and light-dependent magnetoreception. Vertebrates retain multiple sets of CRY or CRY-related genes, but their functions are yet unclear especially in the lower vertebrates. Although CRYs and the other circadian clock components have been extensively studied in the higher vertebrates such as mice, only a few model species have been studied in the lower vertebrates. In this study, we identified two CRYs, XtCRY1 and XtCRY2 in Xenopus tropicalis, an excellent experimental model species. Examination of tissue specificity of their mRNA expression by real-time PCR analysis revealed that both the XtCRYs showed extremely high mRNA expression levels in the ovary. The mRNA levels in the ovary were about 28-fold (XtCry1 and 48-fold (XtCry2 higher than levels in the next abundant tissues, the retina and kidney, respectively. For the functional analysis of the XtCRYs, we cloned circadian positive regulator XtCLOCK and XtBMAL1, and found circadian enhancer E-box in the upstream of XtPer1 gene. XtCLOCK and XtBMAL1 exhibited strong transactivation from the XtPer1 E-box element, and both the XtCRYs inhibited the XtCLOCK:XtBMAL1-mediated transactivation, thereby suggesting this element to drive the circadian transcription. These results revealed a conserved main feedback loop in the X. tropicalis circadian clockwork and imply a possible physiological importance of CRYs in the ovarian functions such as synthesis of steroid hormones and/or control of estrus cycles via the transcription regulation.

  12. miR-155 Over-expression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-prone DSB Repair

    Science.gov (United States)

    Czochor, Jennifer R.; Sulkowski, Parker; Glazer, Peter M.

    2016-01-01

    miR-155 is an oncogenic microRNA (miR) that is often over-expressed in cancer and is associated with poor prognosis. miR-155 can target several DNA repair factors including RAD51, MLH1, and MSH6, and its over-expression results in an increased mutation frequency in vitro, although the mechanism has yet to be fully understood. Here, we demonstrate that over-expression of miR-155 drives an increased mutation frequency both in vitro and in vivo, promoting genomic instability by affecting multiple DNA repair pathways. miR-155 over-expression causes a decrease in homologous recombination, but yields a concurrent increase in the error-prone non-homologous end-joining (NHEJ) pathway. Despite repressing established targets MLH1 and MSH6, the identified mutation pattern upon miR-155 over-expression does not resemble that of a mismatch repair-deficient background. Further investigation revealed that all four subunits of polymerase delta, a high-fidelity DNA replication and repair polymerase, are down-regulated at the mRNA level in the context of miR-155 over-expression. FOXO3a, a transcription factor and known target of miR-155, has one or more putative binding site(s) in the promoter of all four polymerase delta subunits. Finally, suppression of FOXO3a by miR-155 or by siRNA knockdown is sufficient to repress the expression of the catalytic subunit of polymerase delta, POLD1, at the protein level, indicating that FOXO3a contributes to the regulation of polymerase delta levels. PMID:26850462

  13. Scutellarein inhibits hypoxia- and moderately-high glucose-induced proliferation and VEGF expression in human retinal endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Rong GAO; Bang-hao ZHU; Shi-bo TANG; Jiang-feng WANG; Jun REN

    2008-01-01

    Aim: This study was designed to examine the effect of scutellarein on high glu-cose- and hypoxia-stimulated proliferation of human retinal endothelial cells (HREC). Methods: HREC were cultured under normal glucose (NG), moderate, and high glucose (NG supplemented with 10 or 25 mmol/L D-glucose) and/or hypoxic (cobalt chloride treated) conditions. Cell proliferation was evaluated by a cell counting kit. The expression of vascular endothelial growth factor (VEGF) was assessed by Western blot analysis. Results: The proliferation of HREC was significantly elevated in response to moderately-high glucose and hypoxic conditions. The combination of high glucose and hypoxia did not have any additive effects on cell proliferation. Consistent with the proliferation data, the expression of VEGF was also upregulated under both moderately-high glucose and hypoxic conditions. The treatment with scutellarein (1 × 10-11-1 × 10-5 mol/L) significantly inhibited high glucose- or hypoxia-induced cell proliferation and VEGF expression. Conclusion: Both hypoxia and moderately-high glucose were potent stimuli for cell proliferation and VEGF expression in HREC without any significant additive effects. Scutellarein is capable of inhibiting the proliferation of HREC, which is possibly related to its ability to suppress the VEGF expression.

  14. [High-efficiency expression of a receptor-binding domain of SARS-CoV spike protein in tobacco chloroplasts].

    Science.gov (United States)

    Zhong, Xue; Qi, Guangxun; Yang, Jing; Xing, Guojie; Liu, Jianfeng; Yang, Xiangdong

    2014-06-01

    Chloroplast-based expression system is promising for the hyper-expression of plant-derived recombinant therapeutic proteins and vaccines. To verify the feasibility of obtaining high-level expression of the SARS subunit vaccine and to provide a suitable plant-derived vaccine production platform against the severe acute respiratory syndrome coronavirus (SARS-CoV), a 193-amino acid fragment of SARS CoV spike protein receptor-binding domain (RBD), fused with the peptide vector cholera toxin B subunit (CTB), was expressed in tobacco chloroplasts. Codon-optimized CTB-RBD sequence was integrated into the chloroplast genome and homoplasmy was obtained, as confirmed by PCR and Southern blot analysis. Western blot showed expression of the recombinant fusion protein mostly in soluble monomeric form. Quantification of the recombinant fusion protein CTB-RBD was conducted by ELISA analysis from the transplastomic leaves at different developmental stages, attachment positions and time points in a day and the different expression levels of the CTB-RBD were observed with the highest expression of 10.2% total soluble protein obtained from mature transplastomic leaves. Taken together, our results demonstrate the feasibility of highly expressing SARS subunit vaccine RBD, indicating its potential in subsequent development of a plant-derived recombinant subunit vaccine and reagents production for antibody detection in SARS serological tests.

  15. Relationship between the high-risk HPV infection and the expression of oncogenes, anti-oncogenes in cervical dysplasia

    Institute of Scientific and Technical Information of China (English)

    Li-Ping Shi; Xiu-Jie Sheng

    2017-01-01

    Objective:To study the relationship between the infection of high-risk HPV in cervical precancerous lesion and the expression of oncogene, anti-oncogene.Methods:218 cases ofcervical intraepithelial neoplasia patients in our hospital during May 2014–May 2016 were chosed and divided into high-risk HPV group (n=107), low-risk HPV group (n=111) according to cervical tissue HPV test; another 100 cases of patients received cervical biopsy and confirmed as benign lesions were enrolled in the control group. RT-PCR method was used to detect the mRNA expression of proto-oncogene and anti-oncogene in three groups, Western-blot method was used to detect the protein expression of Sox-2 and Wnt/β-catenin signal pathway.Results: mRNA expression of oncogene DEK, Bmi-1, c-fos, K-ras, Prdx4 in high-risk HPV group were higher than low-risk HPV group and control group (P<0.05); mRNA expression of anti-oncogene P27, P16, DAPK, PTEN, eIF4E3 in high-risk HPV group were lower than low-risk HPV group and control group (P<0.05); expression of Sox-2 and Wnt/β-catenin signaling pathway protein Sox-2,β-catenin, wnt-1, wnt-3a in high-risk HPV group were higher than low-risk HPV group and control group (P<0.05).Conclusions:High-risk HPV infection can increase the expression of oncogenes and reduce the expression of anti-oncogenes in cervical dysplasia tissues on Sox-2- and Wnt/β-catenin signaling pathway manners.

  16. Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions.

    Science.gov (United States)

    Butaye, Katleen M J; Goderis, Inge J W M; Wouters, Piet F J; Pues, Jonathan M-T G; Delauré, Stijn L; Broekaert, Willem F; Depicker, Ann; Cammue, Bruno P A; De Bolle, Miguel F C

    2004-08-01

    Basic and applied research involving transgenic plants often requires consistent high-level expression of transgenes. However, high inter-transformant variability of transgene expression caused by various phenomena, including gene silencing, is frequently observed. Here, we show that stable, high-level transgene expression is obtained using Arabidopsis thaliana post-transcriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation (T1) A. thaliana plants transformed with a beta-glucuronidase (GUS) gene (uidA) driven by the 35S cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise, when sgs2 mutants were transformed with a cyclin-dependent kinase inhibitor 6 gene under control of p35S, all transformants showed a clear phenotype typified by serrated leaves, whereas such phenotype was only observed in about one of five wild type transformants. p35S-driven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked contrast to the variable expression patterns observed in wild type T2 populations. We further show that T-DNA constructs flanked by matrix attachment regions of the chicken lysozyme gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12-fold in sgs3 plants, reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild type background. MAR-based plant transformation vectors used in a PTGS mutant background might be of high value for efficient high-throughput screening of transgene-based phenotypes as well as for obtaining extremely high transgene expression in plants.

  17. High-grade neuroendocrine carcinomas of the lung highly express enhancer of zeste homolog 2, but carcinoids do not.

    Science.gov (United States)

    Findeis-Hosey, Jennifer J; Huang, Jiaoti; Li, Faqian; Yang, Qi; McMahon, Loralee A; Xu, Haodong

    2011-06-01

    Enhancer of zeste homolog 2, the catalytic subunit of polycomb repressive complex 2, is a histone methyltransferase and plays an important role in cell proliferation and cell cycle regulation. It has been shown to be overexpressed in a number of malignant neoplasms. This study aimed to determine the expression pattern of enhancer of zeste homolog 2 in neuroendocrine tumors of the lung and the potential of enhancer of zeste homolog 2 to serve as a biomarker to segregate carcinoids from high-grade neuroendocrine carcinomas. Fifty-four cases, including 25 typical carcinoids, 7 atypical carcinoids, 9 large-cell neuroendocrine carcinomas, and 13 small-cell lung carcinomas, were immunohistochemically studied using a monoclonal antibody against enhancer of zeste homolog 2. All 13 small-cell lung carcinomas demonstrated moderate to strong nuclear staining with 12 exhibiting more than 90% of tumor cells staining. All 9 large-cell neuroendocrine carcinomas were moderately to strongly positive for enhancer of zeste homolog 2, with 6 cases having staining in more than 80% of tumor cells. In contrast, all 25 typical carcinoids and 6 atypical carcinoids showed only rare scattered enhancer of zeste homolog 2-positive tumor cells, with 1 case of atypical carcinoid exhibiting moderate staining in 40% of tumor cells. A subsequent validation study of the 14 specimens of lung or mediastinal lymph node biopsy and fine-needle aspiration, including 6 small-cell lung carcinomas, 2 large-cell neuroendocrine carcinomas, 5 typical carcinoids, and 1 atypical carcinoid, was performed. Enhancer of zeste homolog 2 was diffusely and strongly positive in all small-cell lung carcinomas and large-cell neuroendocrine carcinomas, even with severe crush artifact, whereas it was only positive in rare tumor cells in carcinoids. These findings support the formulation that enhancer of zeste homolog 2 may play an important role in the regulation of biologic behavior of high-grade neuroendocrine carcinomas

  18. Complex Reconstitution and Characterization by Combining Co-expression Techniques in Escherichia coli with High-Throughput.

    Science.gov (United States)

    Vincentelli, Renaud; Romier, Christophe

    2016-01-01

    Single protein expression technologies have strongly benefited from the Structural Genomics initiatives that have introduced parallelization at the laboratory level. Specifically, the developments made in the wake of these initiatives have revitalized the use of Escherichia coli as major host for heterologous protein expression. In parallel to these improvements for single expression, technologies for complex reconstitution by co-expression in E. coli have been developed. Assessments of these co-expression technologies have highlighted the need for combinatorial experiments requiring automated protocols. These requirements can be fulfilled by adapting the high-throughput approaches that have been developed for single expression to the co-expression technologies. Yet, challenges are laying ahead that further need to be addressed and that are only starting to be taken into account in the case of single expression. These notably include the biophysical characterization of the samples at the small-scale level. Specifically, these approaches aim at discriminating the samples at an early stage of their production based on various biophysical criteria leading to cost-effectiveness and time-saving. This chapter addresses these various issues to provide the reader with a broad and comprehensive overview of complex reconstitution and characterization by co-expression in E. coli.

  19. Effects of dietary high fructose corn syrup on regulation of energy intake and leptin gene expression in rats

    Directory of Open Access Journals (Sweden)

    Guadalupe López-Rodríguez

    2015-12-01

    Full Text Available OBJECTIVE: To evaluate in Wistar rats the effect of chronic use of high fructose corn syrup on serum lipids, body weight, energy intake regulation, and expression of associated genes. METHODS: For 11 weeks, male rats were fed a standard diet with either water (control or 15% high fructose corn syrup solution, or fed a high-fat diet. The rats' food intake and body weight were measured weekly. Expression of leptin and fatty acid synthase genes was quantified in their brain and adipose tissue upon sacrifice at age 119 days using real-time polymerase chain reaction. RESULTS: The intake of 15% high fructose corn syrup did not affect the rats' weight, only the rats on the high-fat diet gained significant weight. The rats in both diets had lower levels of leptin expression and high levels of fatty acid synthase in the brain, which were associated with high serum triglycerides. CONCLUSION: Fifteen percent high fructose corn syrup intake and the high-fat diet reduced leptin gene expression in the brain of Wistar rats, with differential effects on weight gain.

  20. The High Resolution Stereo Camera (HRSC) Experiment onboard the European Mars Express (MEX) Mission

    Science.gov (United States)

    Neukum, G.; HRSC Team

    2003-04-01

    A major goal of the European Mars Express mission is to image the Martian surface at high spatial resolution, in stereo and in color. This task will be met by the High Resolution Stereo Camera (HRSC), a multiple-line pushbroom scanner. 9 CCD lines are mounted in parallel and simultaneously acquire images at high spatial resolution, in triple-stereo, in four colors and at five viewing angles. During the nominal mission, the HRSC will cover at least 50% of the Martian surface at 10-15 m/pixel, 70% at better than 30 m/pixel and 100% at better than 100 m/pixel resolution. The instrument is equipped with an additional super-resolution channel reaching a spatial resolution of up to 2 m/pixel. This channel is boresighted with the HRSC stereo scanner and will obtain nested-in images or image strips. Up to a few % of the Martian surface can be covered by the super-resolution channel during the mission. This channel will be of particular importance for highest-resolution coverage of landing sites such as planned for the Mars Express Beagle 2 site and the two Mars Surveyor 2003 rover sites. Scientifically, the HRSC experiment concentrates on the geological and climatological evolution of Mars with special emphasis on the role of water throughout the Martian history. An international team of 40 Co-Investigators from 28 scientific institutions and 10 countries will run the experiment and analyze the data over the two-year nominal mission with a possible extension over an additional two years. The data will be processed in such a way that they will be usable by the scientific community at large six months after receipt. The experiment hardware and software development is finished and the instrument is being assembled and tested at the ESA-MEX spacecraft. The launch of the mission is scheduled from Baikonur in late May 2003. First data from the cruise phase to Mars will be received in the June-July period of 2003.

  1. NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ruizhao, E-mail: liruizhao1979@126.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Zhang, Li, E-mail: Zhanglichangde@163.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Southern Medical University, Guangzhou, Guangdong (China); Shi, Wei, E-mail: shiwei.gd@139.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Zhang, Bin, E-mail: zhangbinyes@yahoo.com.cn [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Liang, Xinling, E-mail: xinlingliang@yahoo.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Liu, Shuangxin, E-mail: mplsxi@yahoo.com.cn [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Wang, Wenjian, E-mail: wwjph@yahoo.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China)

    2013-04-15

    Background: Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. Methods: Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca{sup 2+} was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. Results: HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500 nM) or 11R-VIVIT (100 nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca{sup 2+}]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. Conclusion: Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway

  2. Different pattern of Galleria mellonella jhbp gene expression in high five and Sf9 cells.

    Science.gov (United States)

    Andruszewska, Grażyna; Ożyhar, Andrzej; Kochman, Marian; Schmidt, Marcin

    2013-03-01

    Juvenile hormone binding protein (JHBP) is the key element of the system that transmits hormone signals to target tissues. Recently, we found that the core promoter of the jhbp gene is strongly under the control of the TATA box and the transcription start site. In this report, we have shown that the jhbp promoter contains distal regulatory elements whose functionality clearly depends on the particular cell environment and that the scope of research from one cell line is insufficient to generalize the conclusions of the analysis. Cf1/Usp (where Usp is ultraspiracle protein previously known as Cf1, chorion factor 1) elements suppressed transcription of the reporter gene in the High Five cell line but not in the Sf9 cell line. However, upstream from all three Cf1/Usp elements there is a DNA sequence, containing the Zeste element, which activates jhbp in both systems. We found that juvenile hormone strongly inhibited the activity of the jhbp promoter in the Sf9 cell line, whereas it did not have an effect in the High Five cell line. A second key hormone that controls insect development--20-hydroxyecdysone, was also found to suppress the transcription of jhbp. This is the first report describing how these two hormones affect jhbp gene expression in different cell lines.

  3. Cytochrome P450 2C24: expression, tissue distribution, high-throughput assay, and pharmacological inhibition

    Directory of Open Access Journals (Sweden)

    Jun Yang

    2012-04-01

    Full Text Available Cytochrome P450 (CYP-mediated epoxidation of arachidonic acid (AA contributes to important biological functions, including the pain-relieving responses produced by analgesic drugs. However, the relevant epoxygenase(s remain unidentified. Presently, we describe the tissue distribution, high-throughput assay, and pharmacological characteristics of the rat epoxygenase CYP2C24. Following cloning from male rat liver, recombinant baculovirus containing the C-terminal His-tagged cDNA was constructed and used to express the protein in Spodoptera frugiperda (Sf9 cells. Enzymatic activity was detected with membranes, NADPH regenerating system and CYP reductase, and optimized for high throughput screening by use of the Vivid Blue© BOMCC fluorescence substrate. Quantitative real-time PCR identified CYP2C24 m-RNA in liver, kidney, heart, lung, gonad and brain. Screening of CYP2C24 activity against a panel of inhibitors showed a very strong correlation with activity against the human homologue CYP2C19. In agreement with recent findings on CYP2C19, the epoxygenase blockers PPOH and MS-PPOH inhibited CYP2C24 only weakly, confirming that these drugs are not universal epoxygenase inhibitors. Finally, comparisons of the CYP2C24 inhibitor profile with anti-analgesic activity suggests that this isoform does not contribute to brain analgesic drug action. The present methods and pharmacological data will aid in study of the biological significance of this CYP isoform.

  4. Nasal associated lymphoid tissue of the Syrian golden hamster expresses high levels of PrPC.

    Directory of Open Access Journals (Sweden)

    Melissa D Clouse

    Full Text Available The key event in the pathogenesis of the transmissible spongiform encephalopathies is a template-dependent misfolding event where an infectious isoform of the prion protein (PrPSc comes into contact with native prion protein (PrPC and changes its conformation to PrPSc. In many extraneurally inoculated models of prion disease this PrPC misfolding event occurs in lymphoid tissues prior to neuroinvasion. The primary objective of this study was to compare levels of total PrPC in hamster lymphoid tissues involved in the early pathogenesis of prion disease. Lymphoid tissues were collected from golden Syrian hamsters and Western blot analysis was performed to quantify PrPC levels. PrPC immunohistochemistry (IHC of paraffin embedded tissue sections was performed to identify PrPC distribution in tissues of the lymphoreticular system. Nasal associated lymphoid tissue contained the highest amount of total PrPC followed by Peyer's patches, mesenteric and submandibular lymph nodes, and spleen. The relative levels of PrPC expression in IHC processed tissue correlated strongly with the Western blot data, with high levels of PrPC corresponding with a higher percentage of PrPC positive B cell follicles. High levels of PrPC in lymphoid tissues closely associated with the nasal cavity could contribute to the relative increased efficiency of the nasal route of entry of prions, compared to other routes of infection.

  5. Identification, expression, and characterization of the highly conserved D-xylose isomerase in animals

    Institute of Scientific and Technical Information of China (English)

    Ming Ding; Yigang Teng; Qiuyu Yin; Wei Chen; Fukun Zhao

    2009-01-01

    D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway? With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase (EC 5.3.1.5). The xylose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg2+. At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331μmol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.

  6. Nasal associated lymphoid tissue of the Syrian golden hamster expresses high levels of PrPC.

    Science.gov (United States)

    Clouse, Melissa D; Shikiya, Ronald A; Bartz, Jason C; Kincaid, Anthony E

    2015-01-01

    The key event in the pathogenesis of the transmissible spongiform encephalopathies is a template-dependent misfolding event where an infectious isoform of the prion protein (PrPSc) comes into contact with native prion protein (PrPC) and changes its conformation to PrPSc. In many extraneurally inoculated models of prion disease this PrPC misfolding event occurs in lymphoid tissues prior to neuroinvasion. The primary objective of this study was to compare levels of total PrPC in hamster lymphoid tissues involved in the early pathogenesis of prion disease. Lymphoid tissues were collected from golden Syrian hamsters and Western blot analysis was performed to quantify PrPC levels. PrPC immunohistochemistry (IHC) of paraffin embedded tissue sections was performed to identify PrPC distribution in tissues of the lymphoreticular system. Nasal associated lymphoid tissue contained the highest amount of total PrPC followed by Peyer's patches, mesenteric and submandibular lymph nodes, and spleen. The relative levels of PrPC expression in IHC processed tissue correlated strongly with the Western blot data, with high levels of PrPC corresponding with a higher percentage of PrPC positive B cell follicles. High levels of PrPC in lymphoid tissues closely associated with the nasal cavity could contribute to the relative increased efficiency of the nasal route of entry of prions, compared to other routes of infection.

  7. Loss of p27Kip1 expression in high grade human prostate adenocarcinoma

    Directory of Open Access Journals (Sweden)

    MOHD ROHAIZAD MD. RODUAN

    2014-08-01

    Full Text Available p27Kip1 has been hypothesized to play a major role in carcinogenesis. Most of the published data reported that loss of p27Kip1 expression was strongly associated with development and progression of tumour. The purpose of this study was to analyze p27Kip1 expression in normal, benign and malignant prostate cancer tissues and their association with the clinicopathological parameters. The expression of p27Kip1 was evaluated by an immunohistochemistry method. p27Kip1 expression was significantly higher in normal and benign prostate tissues (P<0.01. In contrast, some malignant tissues had no p27Kip1 expression and most had weak p27Kip1 expression. p27Kip1 expression was found to be decreased significantly with increasing Gleason scores (P=0.003. Most of prostate adenocarcinomas (PCa with Gleason 8 and 9 showed loss of p27Kip1 expression. The expression was also positively correlated with prostate specific antigen level and age in PCa group (P=0.003 and 0.043 respectively, whereas no association was found between the p27Kip1 expression with tumour amount and age in benign prostatic hyperplasia group. This study suggests that loss of p27Kip1expression is essential during development and progression of prostate cancer.

  8. Cyclophosphamide alters the gene expression profile in patients treated with high doses prior to stem cell transplantation.

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    Ibrahim El-Serafi

    Full Text Available BACKGROUND: Hematopoietic stem cell transplantation is a curative treatment for several haematological malignancies. However, treatment related morbidity and mortality still is a limiting factor. Cyclophosphamide is widely used in condition regimens either in combination with other chemotherapy or with total body irradiation. METHODS: We present the gene expression profile during cyclophosphamide treatment in 11 patients conditioned with cyclophosphamide for 2 days followed by total body irradiation prior to hematopoietic stem cell transplantation. 299 genes were identified as specific for cyclophosphamide treatment and were arranged into 4 clusters highly down-regulated genes, highly up-regulated genes, early up-regulated but later normalized genes and moderately up-regulated genes. RESULTS: Cyclophosphamide treatment down-regulated expression of several genes mapped to immune/autoimmune activation and graft rejection including CD3, CD28, CTLA4, MHC II, PRF1, GZMB and IL-2R, and up-regulated immune-related receptor genes, e.g. IL1R2, IL18R1, and FLT3. Moreover, a high and significant expression of ANGPTL1 and c-JUN genes was observed independent of cyclophosphamide treatment. CONCLUSION: This is the first investigation to provide significant information about alterations in gene expression following cyclophosphamide treatment that may increase our understanding of the cyclophosphamide mechanism of action and hence, in part, avoid its toxicity. Furthermore, ANGPTL1 remained highly expressed throughout the treatment and, in contrast to several other alkylating agents, cyclophosphamide did not influence c-JUN expression.

  9. High-Level γ-Glutamyl-Hydrolase (GGH) Expression is Linked to Poor Prognosis in ERG Negative Prostate Cancer

    Science.gov (United States)

    Melling, Nathaniel; Rashed, Masoud; Schroeder, Cornelia; Hube-Magg, Claudia; Kluth, Martina; Lang, Dagmar; Simon, Ronald; Möller-Koop, Christina; Steurer, Stefan; Sauter, Guido; Jacobsen, Frank; Büscheck, Franziska; Wittmer, Corinna; Clauditz, Till; Krech, Till; Tsourlakis, Maria Christina; Minner, Sarah; Huland, Hartwig; Graefen, Markus; Budäus, Lars; Thederan, Imke; Salomon, Georg; Schlomm, Thorsten; Wilczak, Waldemar

    2017-01-01

    γ-glutamyl-hydrolase (GGH) is a ubiquitously-expressed enzyme that regulates intracellular folate metabolism for cell proliferation, DNA synthesis, and repair. Employing GGH immunohistochemistry on a tissue microarray with 12,427 prostate cancers, we found that GGH expression was negative to low in normal prostate epithelium, whereas 88.3% of our 10,562 interpretable cancers showed GGH expression. GGH staining was considered as low intensity in 49.6% and as high intensity in 38.6% of cancers. High GGH expression was linked to the TMPRSS2:ERG-fusion positive subset of cancers (p < 0.0001), advanced pathological tumor stage, and high Gleason grade (p < 0.0001 each). Further analysis revealed that these associations were merely driven by the subset of ERG-negative cancers, High GGH expression was weakly linked to early biochemical recurrence in ERG negative cancers (p < 0.0001) and independent from established histo-pathological parameters. Moreover, GGH expression was linked to features of genetic instability, including presence of recurrent deletions at 3p, 5q, 6q, and 10q (PTEN, p ≤ 0.01 each), as well as to accelerated cell proliferation as measured by Ki67 immunohistochemistry (p < 0.0001). In conclusion, the results of our study identify GGH as an ERG subtype specific molecular marker with modest prognostic relevance, which may have clinical relevance if analyzed in combination with other molecular markers. PMID:28146062

  10. Expression of calretinin in high-grade hormone receptor-negative invasive breast carcinomas: correlation with histological and molecular subtypes.

    Science.gov (United States)

    Micello, Donata; Bossi, Alberto; Marando, Alessandro; Dainese, Emanuele; Sessa, Fausto; Capella, Carlo

    2017-07-01

    Calretinin expression has been reported in neoplasms arising in various organs, including the breast. We investigated the relationship of calretinin expression with different histological and molecular subtypes of invasive breast carcinomas (IBCs) and its prognostic significance in high-grade female hormone receptor-negative IBCs. A total of 196 cases of IBCs of different histological subtypes were analyzed for immunohistochemical expression of calretinin, human epidermal growth factor receptor 2 (HER2), basal-like (BL), apocrine, and proliferative markers and grouped in different molecular subtypes. We found significant morphological differences in the group of formally classified invasive ductal carcinoma of no special type (IDC-NST), which we further subdivided into two types (type I IDC-NST and type II IDC-NST) according to their morphology. Calretinin expression was found in 55.1% of the IBCs and was strongly associated with carcinoma with medullary features (P = 0.014) and type II IDC-NST (P correlated (P negative subtypes and even less in MA/HER2+ ones. Calretinin expression was significantly associated with high (≥50) Ki-67 (P = 0.02), but not with parameters like age, tumor size, lymph node status, overall survival (OS), and disease-free survival. Calretinin expression is most common in high-grade IBCs with histological medullary features, type II IDC-NST and BL phenotype, and is associated with high neoplastic proliferative index.

  11. High RBM3 expression is associated with an improved survival and oxaliplatin response in patients with metastatic colorectal cancer.

    Science.gov (United States)

    Siesing, Christina; Sorbye, Halfdan; Dragomir, Anca; Pfeiffer, Per; Qvortrup, Camilla; Pontén, Fredrik; Jirström, Karin; Glimelius, Bengt; Eberhard, Jakob

    2017-01-01

    High expression of the RNA-binding motif protein 3 (RBM3) has been shown to correlate, with prolonged survival in several malignant diseases and with the benefit of platinum-based chemotherapy in ovarian cancer. The aim of this study was to evaluate RBM3 in metastatic colorectal cancer (mCRC) as a prognostic factor for overall survival and in relation to benefit of first-line chemotherapy. Immunohistochemical staining was conducted and evaluated in tumours from 455 mCRC patients. Kaplan-Meier analysis and Cox regression proportional hazards models were used to access the impact of RBM3 expression on overall survival (OS) and progression-free survival (PFS). High RBM3 expression, both nuclear and cytoplasmic, was an independent prognostic factor for prolonged OS (hazard ratio [HR] 0.67, 95% confidence interval [CI] 0.50-0.90 and HR 0.66, 95% CI 0.48-0.91, respectively). PFS was significantly longer in patients with high RBM3 expression who had received first-line oxaliplatin based treatment, compared to those who had received irinotecan based treatment, both regarding nuclear and cytoplasmic expression (p-value 0.020 and 0.022 respectively). High RBM3 expression is an independent predictor of prolonged survival in mCRC patients, in particular in patients treated with first-line oxaliplatin based chemotherapy.

  12. Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation

    Directory of Open Access Journals (Sweden)

    Wood David W

    2010-10-01

    Full Text Available Abstract Background Elastin-like polypeptides (ELPs are useful tools that can be used to non-chromatographically purify proteins. When paired with self-cleaving inteins, they can be used as economical self-cleaving purification tags. However, ELPs and ELP-tagged target proteins have been traditionally expressed using highly enriched media in shake flask cultures, which are generally not amenable to scale-up. Results In this work, we describe the high cell-density expression of self-cleaving ELP-tagged targets in a supplemented minimal medium at a 2.5 liter fermentation scale, with increased yields and purity compared to traditional shake flask cultures. This demonstration of ELP expression in supplemented minimal media is juxtaposed to previous expression of ELP tags in extract-based rich media. We also describe several sets of fed-batch conditions and their impact on ELP expression and growth medium cost. Conclusions By using fed batch E. coli fermentation at high cell density, ELP-intein-tagged proteins can be expressed and purified at high yield with low cost. Further, the impact of media components and fermentation design can significantly impact the overall process cost, particularly at large scale. This work thus demonstrates an important advances in the scale up of self-cleaving ELP tag-mediated processes.

  13. High expression of miR-21 in tumor stroma correlates with increased cancer cell proliferation in human breast cancer

    DEFF Research Database (Denmark)

    Rask, Lene; Balslev, Eva; Jørgensen, Stine

    2011-01-01

    Low-risk and high-risk breast cancer patients are stratified primarily according to their lymph node (LN) status and grading. However, some low-risk patients relapse, and some high-risk patients have a favorable clinical outcome, implying a need for better prognostic and predictive tests. Micro...... RNAs are often aberrantly expressed in cancer and microRNA-21 is upregulated in a variety of cancers, including breast cancer. High miR-21 levels have been associated with poor prognosis. To determine the cellular localization of miR-21 and to compare its expression levels with histopathological...... features, we performed in situ hybridization and semi-quantitative assessment of the miR-21 signal on 12 LN negative grade I (assumed low risk), and 12 LN positive grade II (high risk) breast cancers. miR-21 was predominantly seen in cancer associated fibroblast-like cells, with no difference in expression...

  14. Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A.

    Science.gov (United States)

    Johnston, J M; Denning, G; Doering, C B; Spencer, H T

    2013-06-01

    We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain-deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino-acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: (1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), (2) HIV versus SIV viral vector systems and (3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha, cytomegalovirus (CMV) or phosphoglycerate kinase promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.

  15. Bacterial translational regulations: high diversity between all mRNAs and major role in gene expression

    Directory of Open Access Journals (Sweden)

    Picard Flora

    2012-10-01

    Full Text Available Abstract Background In bacteria, the weak correlations at the genome scale between mRNA and protein levels suggest that not all mRNAs are translated with the same efficiency. To experimentally explore mRNA translational level regulation at the systemic level, the detailed translational status (translatome of all mRNAs was measured in the model bacterium Lactococcus lactis in exponential phase growth. Results Results demonstrated that only part of the entire population of each mRNA species was engaged in translation. For transcripts involved in translation, the polysome size reached a maximum of 18 ribosomes. The fraction of mRNA engaged in translation (ribosome occupancy and ribosome density were not constant for all genes. This high degree of variability was analyzed by bioinformatics and statistical modeling in order to identify general rules of translational regulation. For most of the genes, the ribosome density was lower than the maximum value revealing major control of translation by initiation. Gene function was a major translational regulatory determinant. Both ribosome occupancy and ribosome density were particularly high for transcriptional regulators, demonstrating the positive role of translational regulation in the coordination of transcriptional networks. mRNA stability was a negative regulatory factor of ribosome occupancy and ribosome density, suggesting antagonistic regulation of translation and mRNA stability. Furthermore, ribosome occupancy was identified as a key component of intracellular protein levels underlining the importance of translational regulation. Conclusions We have determined, for the first time in a bacterium, the detailed translational status for all mRNAs present in the cell. We have demonstrated experimentally the high diversity of translational states allowing individual gene differentiation and the importance of translation-level regulation in the complex process linking gene expression to protein

  16. Drosophila clueless is highly expressed in larval neuroblasts, affects mitochondrial localization and suppresses mitochondrial oxidative damage.

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    Aditya Sen

    Full Text Available Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu's specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu's role in mitochondrial function is not critical during larval development, but is important for pupae and adults.

  17. Expression of glutamine:fructose-6-phosphate amidotransferase in human tissues: evidence for high variability and distinct regulation in diabetes.

    Science.gov (United States)

    Nerlich, A G; Sauer, U; Kolm-Litty, V; Wagner, E; Koch, M; Schleicher, E D

    1998-02-01

    Recent in vitro and in vivo studies suggested that the increased flux of glucose through the hexosamine biosynthetic pathway may contribute to glucose-induced insulin resistance and to the induction of the synthesis of growth factors. Because glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first and rate-limiting step in the formation of hexosamine products, this enzyme is the key regulator in this pathway and is therefore possibly also involved in the alterations occurring in preclinical or manifest diabetic patients. To study the expression of GFAT in human tissues, we produced and characterized a peptic antiserum specifically recognizing GFAT protein and a riboprobe for the detection of GFAT mRNA. Immunohistochemical and nonradioactive in situ hybridization analysis revealed high levels of expression of GFAT protein and mRNA in adipocytes and skeletal muscle. Furthermore, a marked GFAT expression was found in vascular smooth muscle cells with unexpectedly high variability and lower levels in other cells, e.g., peripheral nerve sheath cells or endocrine-active cells, including the pancreatic islet cell. GFAT protein expression was below detection level in endothelium, osteocytes, lymphocytes, granulocytes, and in most quiescent fibroblasts. In renal tissue, GFAT was expressed in tubular epithelial cells, while glomerular cells remained essentially unstained. Renal sections obtained from patients with diabetic nephropathy showed significant GFAT expression in some glomerular epithelial and mesangial cells, indicating that GFAT expression may be induced by manifest diabetes. Our data indicate that GFAT is expressed in most tissues involved in the development of diabetic late complications. Furthermore, the results suggest that GFAT gene expression is highly regulated.

  18. Elevated microRNA-126 is associated with high vascular endothelial growth factor receptor 2 expression levels and high microvessel density in colorectal cancer

    DEFF Research Database (Denmark)

    Hansen, Torben Frøstrup; Andersen, Claus Lindbjerg; Nielsen, Boye Schnack

    2011-01-01

    analysed by ELISA. Angiogenesis, visualised by the endothelial cell marker CD105 combined with caldesmon, was assessed by immunohistochemistry and the microvessel density (MVD) technique. In situ hybridisation was performed for miRNA-126. Tumours were classified as low or high miRNA‑126-expressing using...... the median as the cut-off. The median gene expression levels of VEGFR-2 were significantly lower in the tumours expressing low levels of miRNA-126, 0.30 (95% CI, 0.24‑0.36), compared to those expressing high levels of miRNA-126, 0.48 (95% CI, 0.28-0.60), p=0.02. A positive association was observed with VEGFR......-2 protein concentrations, p=0.06. The median MVD was significantly lower in the tumours expressing low levels of miRNA-126, 5.8 (95% CI, 5.33‑6.67), compared to those expressing high levels, 8.0 (95% CI, 6.33‑9.00), p

  19. High bone sialoprotein (BSP expression correlates with increased tumor grade and predicts a poorer prognosis of high-grade glioma patients.

    Directory of Open Access Journals (Sweden)

    Tao Xu

    Full Text Available OBJECTIVES: To investigate the expression and prognostic value of bone sialoprotein (BSP in glioma patients. METHODS: We determined the expression of BSP using real-time RT-PCR and immunohistochemistry in tissue microarrays containing 15 normal brain and 270 glioma samples. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate analyses were performed by the stepwise forward Cox regression model. RESULTS: Both BSP mRNA and protein levels were significantly elevated in high-grade glioma tissues compared with those of normal brain and low-grade glioma tissues, and BSP expression positively correlated with tumor grade (P<0.001. Univariate and multivariate analysis showed high BSP expression was an independent prognostic factor for a shorter progression-free survival (PFS and overall survival (OS in both grade III and grade IV glioma patients [hazard ratio (HR = 2.549 and 3.154 for grade III glioma, and HR = 1.637 and 1.574 for grade IV glioma, respectively]. Patients with low BSP expression had a significantly longer median OS and PFS than those with high BSP expression. Small extent of resection and lineage of astrocyte served as independent risk factors of both shorter PFS and OS in grade III glioma patients; GBM patients without O(6-methylguanine (O(6-meG DNA methyltransferase (MGMT methylation and Karnofsky performance score (KPS less than 70 points were related to poor prognosis. Lack of radiotherapy related to shorter OS but not affect PFS in both grade III and grade IV glioma patients. CONCLUSION: High BSP expression occurs in a significant subset of high-grade glioma patients and predicts a poorer outcome. The study identifies a potentially useful molecular marker for the categorization and targeted therapy of gliomas.

  20. Expression of interferon gamma by a highly virulent strain of Newcastle disease virus decreases its pathogenicity in chickens.

    Science.gov (United States)

    Susta, Leonardo; Cornax, Ingrid; Diel, Diego G; Garcia, Stivalis Cardenas; Miller, Patti J; Liu, Xiufan; Hu, Shunlin; Brown, Corrie C; Afonso, Claudio L

    2013-01-01

    The role of interferon gamma (IFN-γ) expression during Newcastle disease virus (NDV) infection in chickens is unknown. Infection of chickens with highly virulent NDV results in rapid death, which is preceded by increased expression of IFN-γ in target tissues. IFN-γ is a cytokine that has pleiotropic biological effects including intrinsic antiviral activity and immunomodulatory effects that may increase morbidity and mortality during infections. To better understand how IFN-γ contributes to NDV pathogenesis, the coding sequence of the chicken IFN-γ gene was inserted in the genome of the virulent NDV strain ZJ1 (rZJ1-IFNγ), and the effects of high levels of IFN-γ expression during infection were determined in vivo and in vitro. IFN-γ expression did not significantly affect NDV replication in fibroblast or in macrophage cell lines. However, it affected the pathogenesis of rZJ1-IFNγ in vivo. Relative to the virus expressing the green fluorescent protein (rZJ1-GFP) or lacking the IFN-γ insert (rZJ1-rev), expression of IFN-γ by rZJ1-IFNγ produced a marked decrease of pathogenicity in 4-week-old chickens, as evidenced by lack of mortality, decreased disease severity, virus shedding, and antigen distribution. These results suggest that early expression of IFN-γ had a significant protective role against the effects of highly virulent NDV infection in chickens, and further suggests that the level and timing of expression of this cytokine may be critical for the disease outcome. This is the first description of an in vivo attenuation of a highly virulent NDV by avian cytokines, and shows the feasibility to use NDV for cytokine delivery in chicken organs. This approach may facilitate the study of the role of other avian cytokines on the pathogenesis of NDV.

  1. Reduced Variance of Gene Expression at Numerous Loci in a Population of Chickens Selected for High Feather Pecking

    DEFF Research Database (Denmark)

    Hughes, A L; Buitenhuis, A J

    2010-01-01

    Changes in gene expression in response to selection were studied by comparing microarray expression profiles among a population of domestic chickens selected for high feather pecking (FP) with a control population and a population selected for low FP. No transcripts showed significant differences...... and gentle FP were distinct, suggesting that very distinct underlying neural mechanisms underlie these 2 behaviors, with SFP showing more signs of an association with synaptic plasticity and with an immunosuppressive stress response...

  2. Differential expression of HDACs and KATs in high and low regeneration capacity neurons during spinal cord regeneration.

    Science.gov (United States)

    Chen, Jie; Laramore, Cindy; Shifman, Michael I

    2016-06-01

    After spinal cord injury (SCI) in mammals, injured axons fail to regenerate. By contrast, lampreys recover from complete spinal transection and axons regenerate selectively in their correct paths. Yet the large, identified reticulospinal neurons in the lamprey brain vary greatly in their regenerative abilities - some have high regeneration capacity (probability of regeneration >50%) and others have low regeneration capacity (regenerating and non-regenerating neurons located in the same brain region and projecting to the same axon tracts suggests that differences in their regenerating abilities depend upon factors intrinsic to the neurons. Previous work has suggested that axon regeneration, especially in PNS, could depend on epigenetic mechanisms of histone modifications, such as the acetylation of histone tails. Our data indicated that expression of the enzymes responsible for regulating the acetylation of histone (KATs and HDACs) - KAT2A, KAT5 and P300 and HDAC3 did not change after SCI in either high regeneration capacity or low regeneration capacity neurons. In the present report, we show a novel and unexpected relationship between neuron regeneration abilities and expression of HDAC1. While HDAC1 expression was downregulated in both high and low regeneration capacity neurons 2 and 4weeks after SCI, it was upregulated at 7weeks at almost all RS neurons. However, at 10weeks post-transection only high regeneration capacity neurons displayed elevated HDAC1 mRNA expression and HDAC1 expression was again downregulated in low regeneration capacity neurons. Moreover, we show that HDAC1 is preferentially expressed in regenerated neurons, but not in non-regenerating neurons. Together, these results suggest that SCI causes significant changes in HDAC1 expression and that HDAC1 expression in regenerating neurons may modulates a survival or regeneration programs. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Global gene expression analysis of Saccharomyces cerevisiae grown under redox potential-controlled very-high-gravity conditions.

    Science.gov (United States)

    Liu, Chen-Guang; Lin, Yen-Han; Bai, Feng-Wu

    2013-11-01

    Redox potential (ORP) plays a pivotal role in yeast viability and ethanol production during very-high-gravity (VHG) ethanol fermentation. In order to identify the correlation between redox potential profiles and gene expression patterns, global gene expression of Saccharomyces cerevisiae was investigated. Results indicated that significant changes in gene expression occurred at the periods of 0 - 6 h and 30 - 36 h, respectively. Changes noted in the period of 0 - 6 h were mainly related to carbohydrate metabolism. In contrast, gene expression variation at 30 - 36 h could be attributed primarily to stress response. Although CDC19 was down-regulated, expression of PYK2, PDC6 and ADH2 correlated inversely with ORP. Meanwhile, expression of GPD1 decreased due to the depletion of dissolved oxygen in the fermentation broth, but expression of GPD2 correlated with ORP. Transcription of genes encoding heat shock proteins was characterized by uphill, downhill, valley and plateau expression profiles, accordingly to specific function in stress response. These results highlight the role of ORP in modulating yeast physiology and metabolism under VHG conditions.

  4. Dissociation between PGC-1alpha and GLUT-4 expression in skeletal muscle of rats fed a high-fat diet.

    Science.gov (United States)

    Higashida, Kazuhiko; Higuchi, Mitsuru; Terada, Shin

    2009-12-01

    It has recently been reported that a 4-wk high-fat diet gradually increases skeletal muscle peroxisome proliferator activated receptor (PPAR) gamma coactivator-1alpha (PGC-1alpha) protein content, which has been suggested to regulate GLUT-4 gene transcription. However, it has not been reported that a high-fat diet enhances GLUT-4 mRNA expression and protein content in skeletal muscle, suggesting that an increase in PGC-1alpha protein content is not sufficient to induce muscle GLUT-4 biogenesis in a high-fat fed animal. Therefore, we first evaluated the relationship between PGC-1alpha and GLUT-4 expression in skeletal muscle of rats fed a high-fat diet for 4 wk. The PGC-1alpha protein content in rat epitrochlearis muscle significantly increased by twofold after the 4-wk high-fat diet feeding. However, the high-fat diet had no effect on GLUT-4 protein content and induced a 30% decrease in GLUT-4 mRNA expression in rat skeletal muscle (p<0.05). To clarify the mechanism by which a high-fat diet downregulates GLUT-4 mRNA expression, we next examined the effect of PPARdelta activation, which is known to occur in response to a high-fat diet, on GLUT-4 mRNA expression in L6 myotubes. Incubation with 500 nM GW501516 (PPARdelta activator) for 24 h significantly decreased GLUT-4 mRNA in L6 myotubes. Taken together, these findings suggest that a high-fat diet downregulates GLUT-4 mRNA, possibly through the activation of PPARdelta, despite an increase in PGC-1alpha protein content in rat skeletal muscle, and that a posttranscriptional regulatory mechanism maintains GLUT-4 protein content in skeletal muscle of rats fed a high-fat diet.

  5. Combination of FACS and homologous recombination for the generation of stable and high-expression engineered cell lines.

    Directory of Open Access Journals (Sweden)

    Lei Shi

    Full Text Available Traditionally, cell line generation requires several months and involves screening of over several hundred cell clones for high productivity before dozens are selected as candidate cell lines. Here, we have designed a new strategy for the generation of stable and high-expression cell lines by combining homologous recombination (HR and fluorescence-activated cell sorting (FACS. High expression was indicated by the expression of secreted green fluorescent protein (SEGFP. Parental cell lines with the highest expression of SEGFP were then selected by FACS and identified by stability analysis. Consequently, HR vectors were constructed using the cassette for SEGFP as the HR region. After transfecting the HR vector, the cells with negative SEGFP expression were enriched by FACS. The complete exchange between SEGFP and target gene (TNFR-Fc cassettes was demonstrated by DNA analysis. Compared with the traditional method, by integrating the cassette containing the gene of interest into the pre-selected site, the highest producing cells secreted a more than 8-fold higher titer of target protein. Hence, this new strategy can be applied to isolated stable cell lines with desirable expression of any gene of interest. The stable cell lines can rapidly produce proteins for researching protein structure and function and are even applicable in drug discovery.

  6. Glioma-associated microglia/macrophages display an expression profile different from M1 and M2 polarization and highly express Gpnmb and Spp1.

    Directory of Open Access Journals (Sweden)

    Frank Szulzewsky

    Full Text Available Malignant glioma belong to the most aggressive neoplasms in humans with no successful treatment available. Patients suffering from glioblastoma multiforme (GBM, the highest-grade glioma, have an average survival time of only around one year after diagnosis. Both microglia and peripheral macrophages/monocytes accumulate within and around glioma, but fail to exert effective anti-tumor activity and even support tumor growth. Here we use microarray analysis to compare the expression profiles of glioma-associated microglia/macrophages and naive control cells. Samples were generated from CD11b+ MACS-isolated cells from naïve and GL261-implanted C57BL/6 mouse brains. Around 1000 genes were more than 2-fold up- or downregulated in glioma-associated microglia/macrophages when compared to control cells. A comparison with published data sets of M1, M2a,b,c-polarized macrophages revealed a gene expression pattern that has only partial overlap with any of the M1 or M2 gene expression patterns. Samples for the qRT-PCR validation of selected M1 and M2a,b,c-specific genes were generated from two different glioma mouse models and isolated by flow cytometry to distinguish between resident microglia and invading macrophages. We confirmed in both models the unique glioma-associated microglia/macrophage phenotype including a mixture of M1 and M2a,b,c-specific genes. To validate the expression of these genes in human we MACS-isolated CD11b+ microglia/macrophages from GBM, lower grade brain tumors and control specimens. Apart from the M1/M2 gene analysis, we demonstrate that the expression of Gpnmb and Spp1 is highly upregulated in both murine and human glioma-associated microglia/macrophages. High expression of these genes has been associated with poor prognosis in human GBM, as indicated by patient survival data linked to gene expression data. We also show that microglia/macrophages are the predominant source of these transcripts in murine and human GBM. Our

  7. Emotional expression of high school students with visual impairments and their typically developing peers

    OpenAIRE

    Vučinić Vesna; Jablan Branka; Stanimirović Dragana; Drinčić Natalija

    2015-01-01

    Emotions are adaptive reaction to events from the environment and they represent the central part of every person's life. Willing emotional expression influences people from the environment according to our expectations if they recognize our emotions. Studies on expressing emotions in persons with visual impairments indicate the existence of the same type of spontaneous emotional expressions as in typically developing population. Also, these studies point to difficulties in presenting willing...

  8. Propofol protects against high glucose-induced endothelial adhesion molecules expression in human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Zhu Minmin

    2013-01-01

    Full Text Available Abstract Background Hyperglycemia could induce oxidative stress, activate transcription factor nuclear factor kappa B (NF-κB, up-regulate expression of endothelial adhesion molecules, and lead to endothelial injury. Studies have indicated that propofol could attenuate oxidative stress and suppress NF-κB activation in some situations. In the present study, we examined whether and how propofol improved high glucose-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs. Methods Protein expression of endothelial adhesion molecules, NF-κB, inhibitory subunit of NF-κBα (IκBα, protein kinase Cβ2 (PKCβ2, and phosphorylation of PKCβ2 (Ser660 were measured by Western blot. NF-κB activity was measured by electrophoretic mobility shift assay. PKC activity was measured with SignaTECT PKC assay system. Superoxide anion (O2.- accumulation was measured with the reduction of ferricytochrome c assay. Human peripheral mononuclear cells were prepared with Histopaque-1077 solution. Results High glucose induced the expression of endothelial selectin (E-selectin, intercellular adhesion molecule 1 (ICAM-1, vascular cell adhesion molecule 1 (VCAM-1, and increased mononuclear-endothelial adhesion. High glucose induced O2.- accumulation, PKCβ2 phosphorylation and PKC activation. Further, high glucose decreased IκBα expression in cytoplasm, increased the translocation of NF-κB from cytoplasm to nuclear, and induced NF-κB activation. Importantly, we found these high glucose-mediated effects were attenuated by propofol pretreatment. Moreover, CGP53353, a selective PKCβ2 inhibitor, decreased high glucose-induced NF-κB activation, adhesion molecules expression, and mononuclear-endothelial adhesion. Conclusion Propofol, via decreasing O2.- accumulation, down-regulating PKCβ2 Ser660 phosphorylation and PKC as well as NF-κB activity, attenuated high glucose-induced endothelial adhesion molecules expression

  9. High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana

    DEFF Research Database (Denmark)

    Bach, Søren Spanner; Bassard, Jean-Étienne André; Andersen-Ranberg, Johan

    2014-01-01

    describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly...... making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps...

  10. Prognostic significance of L1CAM expression and its association with mutant p53 expression in high-risk endometrial cancer.

    Science.gov (United States)

    Van Gool, Inge C; Stelloo, Ellen; Nout, Remi A; Nijman, Hans W; Edmondson, Richard J; Church, David N; MacKay, Helen J; Leary, Alexandra; Powell, Melanie E; Mileshkin, Linda; Creutzberg, Carien L; Smit, Vincent T H B M; Bosse, Tjalling

    2016-02-01

    Studies in early-stage, predominantly low- and intermediate-risk endometrial cancer have demonstrated that L1 cell adhesion molecule (L1CAM) overexpression identifies patients at increased risk of recurrence, yet its prognostic significance in high-risk endometrial cancer is unclear. To evaluate this, its frequency, and the relationship of L1CAM with the established endometrial cancer biomarker p53, we analyzed the expression of both markers by immunohistochemistry in a pilot series of 116 endometrial cancers (86 endometrioid, 30 non-endometrioid subtype) with high-risk features (such as high tumor grade and deep myometrial invasion) and correlated results with clinical outcome. We used The Cancer Genome Atlas (TCGA) endometrial cancer series to validate our findings. Using the previously reported cutoff of 10% positive staining, 51/116 (44%) tumors were classified as L1CAM-positive, with no significant association between L1CAM positivity and the rate of distant metastasis (P=0.195). However, increasing the threshold for L1CAM positivity to 50% resulted in a reduction of the frequency of L1CAM-positive tumors to 24% (28/116), and a significant association with the rate of distant metastasis (P=0.018). L1CAM expression was strongly associated with mutant p53 in the high-risk and TCGA series (Pp53-mutant endometrial cancers displayed p53-wild-type non-endometrioid endometrial cancers demonstrated diffuse L1CAM staining, suggesting p53-independent mechanisms of L1CAM overexpression. In conclusion, the previously proposed threshold for L1CAM positivity of >10% does not predict prognosis in high-risk endometrial cancer, whereas an alternative threshold (>50%) does. L1CAM expression is strongly, but not universally, associated with mutant p53, and may be strong enough for clinical implementation as prognostic marker in combination with p53. The high frequency of L1CAM expression in high-risk endometrial cancers suggests that it may also be a promising therapeutic target

  11. Analytical Expressions of the Efficiency of Standard and High Contact Ratio Involute Spur Gears

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    Miguel Pleguezuelos

    2013-01-01

    Full Text Available Simple, traditional methods for computation of the efficiency of spur gears are based on the hypotheses of constant friction coefficient and uniform load sharing along the path of contact. However, none of them is accurate. The friction coefficient is variable along the path of contact, though average values can be often considered for preliminary calculations. Nevertheless, the nonuniform load sharing produced by the changing rigidity of the pair of teeth has significant influence on the friction losses, due to the different relative sliding at any contact point. In previous works, the authors obtained a nonuniform model of load distribution based on the minimum elastic potential criterion, which was applied to compute the efficiency of standard gears. In this work, this model of load sharing is applied to study the efficiency of both standard and high contact ratio involute spur gears (with contact ratio between 1 and 2 and greater than 2, resp.. Approximate expressions for the friction power losses and for the efficiency are presented assuming the friction coefficient to be constant along the path of contact. A study of the influence of some transmission parameters (as the gear ratio, pressure angle, etc. on the efficiency is also presented.

  12. The LARK/RBM4a protein is highly expressed in cerebellum as compared to cerebrum.

    Science.gov (United States)

    Pfuhl, Thorsten; Mamiani, Alfredo; Dürr, Matthias; Welter, Susanne; Stieber, Johanna; Ankara, Jasmin; Liss, Michael; Dobner, Thomas; Schmitt, Andrea; Falkai, Peter; Kremmer, Elisabeth; Jung, Volker; Barth, Stephanie; Grässer, Friedrich A

    2008-10-17

    The RNA binding motif protein 4 genes RBM4a and RBM4b are located on human chromosome 11q13.2 and encode highly similar proteins of 363 and 359 amino acids, respectively. They contain two RNA recognition motifs (RRMs) and a retroviral-type Zn-finger. RBM4a binds RNA, is involved in alternative splicing and is also a part of the microRNA-processing RISC complex. In particular, RBM4a is involved in exon 10 inclusion of the tau protein. The function of RBM4b is unknown. With new monoclonal antibodies we show that RBM4a is detectable in virtually all tissues and cell lines tested while RBM4b was only found in kidney and liver. Both RBM4a and RBM4b are nuclear phosphoproteins with half-lives of 2.5h and 4.5h, respectively. To our knowledge, this is the first description of RBM4b protein in human tissue. In human brain, expression of RBM4a was strongly up-regulated in cerebellum as compared to forebrain.

  13. High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris.

    Science.gov (United States)

    Brunel, Laetitia; Neugnot, Virginie; Landucci, Laure; Boze, Hélène; Moulin, Guy; Bigey, Frédéric; Dubreucq, Eric

    2004-07-01

    Candida parapsilosis has been previously shown to produce a lipase/acyltransferase (EC 3.1.1.3) that preferentially catalyses transfer reactions such as alcoholysis over hydrolysis in the presence of suitable nucleophiles other than water, even in aqueous media (aw > 0.9 ). This enzyme has been shown to belong to a new family of lipases. The present work describes the cloning of the gene coding for this lipase/acyltransferase in the yeast Pichia pastoris and the heterologous high-level expression of the recombinant enzyme. The lipase/acyltransferase gene, in which the sequence encoding the signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was placed under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). A transformed P. pastoris clone, containing five copies of the lipase/acyltransferase gene, was selected for the production of recombinant enzyme. The fed-batch culture supernatant contained 5.8 gl(-1) (weighted) of almost pure recombinant lipase/acyltransferase displaying the same catalytic behavior as the original enzyme.

  14. A locus on chromosome 20 encompassing genes that are highly expressed in the epididymis

    Institute of Scientific and Technical Information of China (English)

    (A)ke Lundwall

    2007-01-01

    During liquefaction of the ejaculate, the semen coagulum proteins semenogelin Ⅰ (SEMG1) and semenogelin Ⅱ (SEMG2) are degraded to low molecular mass fragments by kallikrein-related peptidase 3 (KLK3), also known as prostate-specific antigen. Semenogelin molecules initiate their own destruction by chelating Zn2+ that normally would completely inhibit the proteolytic activity of KLK3. In a similar way, semenogelins might regulate the activity of kallikrein-related peptidases in the epididymis, something that might be of importance for the maturation of spermatozoa or generation of anti-bacterial peptides. Studies on the evolution of semen coagulum proteins have revealed that most of them carry an exon that displays a rapid and unusual evolution. As a consequence, homologous proteins in rodents and primates show almost no conservation in primary structure. Further studies on their evolution suggest that the progenitor of the semen coagulum proteins probably was a protease inhibitor that might have displayed antimicrobial activity. The semenogelin locus on chromosome 20 contains at least 17 homologous genes encoding probable protease inhibitors with homology to semen coagulum proteins. All of these are highly expressed in the epididymis where they, similar to the semenogelins, could affect the maturation of spermatozoa or display antibacterial properties.

  15. Heightened serotonin influences contest outcome and enhances expression of high-intensity aggressive behaviors.

    Science.gov (United States)

    Bubak, Andrew N; Renner, Kenneth J; Swallow, John G

    2014-02-01

    The outcome of behavioral interactions between organisms can have significant fitness implications. Therefore, it is of great theoretical and practical importance to understand the mechanisms that modify different agonistic behaviors. Changes in central monoamines, such as serotonin (5-HT), contribute to modifying the expression of aggressive encounters in both vertebrates and invertebrates. In several invertebrate groups, neural 5-HT has been linked to heightened aggression and conflict escalation. The male stalk-eyed fly (Teleopsis dalmanni) competes with conspecifics daily over access to resources such as food and mates. Because encounters escalate in a stereotypical manner, stalk-eyed flies provide an excellent model system to study behavioral syndromes. We hypothesized that noninvasive, pharmacological augmentation of brain 5-HT by administration of the precursor, 5-hydroxytryptophan (5-HTP), would increase stereotypic behavioral escalation and the probability of winning a conflict over food. Size-matched male 5-HTP-treated and untreated flies were placed in a forced-fight paradigm and their aggressive behaviors scored. Individuals with higher brain 5-HT levels had a markedly higher probability of winning the contests, displayed greater levels of high-intensity aggressive behaviors and fewer retreats. Pretreatment with 5-HTP did not significantly alter octopamine or tyramine, suggesting that central 5-HT may modulate aggression in these organisms and play a role in determining reproductive success and resource attainment.

  16. Elastogenic protein expression of a highly elastic murine spinal ligament: the ligamentum flavum.

    Directory of Open Access Journals (Sweden)

    Jeffrey P Brown

    Full Text Available Spinal ligaments, such as the ligamentum flavum (LF, are prone to degeneration and iatrogenic injury that can lead to back pain and nerve dysfunction. Repair and regeneration strategies for these tissues are lacking, perhaps due to limited understanding of spinal ligament formation, the elaboration of its elastic fibers, maturation and homeostasis. Using immunohistochemistry and histology, we investigated murine LF elastogenesis and tissue formation from embryonic to mature postnatal stages. We characterized the spatiotemporal distribution of the key elastogenic proteins tropoelastin, fibrillin-1, fibulin-4 and lysyl oxidase. We found that elastogenesis begins in utero with the microfibril constituent fibrillin-1 staining intensely just before birth. Elastic fibers were first detected histologically at postnatal day (P 7, the earliest stage at which tropoelastin and fibulin-4 stained intensely. From P7 to P28, elastic fibers grew in diameter and became straighter along the axis. The growth of elastic fibers coincided with intense staining of tropoelastin and fibulin-4 staining, possibly supporting a chaperone role for fibulin-4. These expression patterns correlated with reported skeletal and behavioral changes during murine development. This immunohistochemical characterization of elastogenesis of the LF will be useful for future studies investigating mechanisms for elastogenesis and developing new strategies for treatment or regeneration of spinal ligaments and other highly elastic tissues.

  17. High mobility group protein 1: A collaborator in nucleosome dynamics and estrogen-responsive gene expression

    Institute of Scientific and Technical Information of China (English)

    William M Scovell

    2016-01-01

    High mobility group protein 1(HMGB1) is a multifunctional protein that interacts with DNA and chromatin to influence the regulation of transcription, DNA replication and repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome(N) in a nonenzymatic,adenosine triphosphate-independent manner. As a result, the canonical nucleosome is converted to two stable, physically distinct nucleosome conformers. Although estrogen receptor(ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes(N’ and N’’) remain stable and exhibit a number of characteristics that are distinctly different from the canonical nucleosome. These findings complement previous studies that showed(1) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and(2) knock down of HMGB1 expression by siR NA precipitously reduced transcriptional activation. The findings indicate that a major facet of the mechanism of HMGB1 action involves a restructuring of aspects of the nucleosome that appear to relax structural constraints within the nucleosome. The findings are extended to reveal the differences between ER and the other steroid hormone receptors. A working proposal outlines mechanisms that highlight the multiple facets that HMGB1 may utilize in restructuring the nucleosome.

  18. Cellular effect of high doses of silica-coated quantum dot profiled with high throughput gene expression analysis and high content cellomics measurements.

    Science.gov (United States)

    Zhang, Tingting; Stilwell, Jackie L; Gerion, Daniele; Ding, Lianghao; Elboudwarej, Omeed; Cooke, Patrick A; Gray, Joe W; Alivisatos, A Paul; Chen, Fanqing Frank

    2006-04-01

    Quantum dots (Qdots) are now used extensively for labeling in biomedical research, and this use is predicted to grow because of their many advantages over alternative labeling methods. Uncoated Qdots made of core/shell CdSe/ZnS are toxic to cells because of the release of Cd2+ ions into the cellular environment. This problem has been partially overcome by coating Qdots with polymers, poly(ethylene glycol) (PEG), or other inert molecules. The most promising coating to date, for reducing toxicity, appears to be PEG. When PEG-coated silanized Qdots (PEG-silane-Qdots) are used to treat cells, toxicity is not observed, even at dosages above 10-20 nM, a concentration inducing death when cells are treated with polymer or mercaptoacid coated Qdots. Because of the importance of Qdots in current and future biomedical and clinical applications, we believe it is essential to more completely understand and verify this negative global response from cells treated with PEG-silane-Qdots. Consequently, we examined the molecular and cellular response of cells treated with two different dosages of PEG-silane-Qdots. Human fibroblasts were exposed to 8 and 80 nM of these Qdots, and both phenotypic as well as whole genome expression measurements were made. PEG-silane-Qdots did not induce any statistically significant cell cycle changes and minimal apoptosis/necrosis in lung fibroblasts (IMR-90) as measured by high content image analysis, regardless of the treatment dosage. A slight increase in apoptosis/necrosis was observed in treated human skin fibroblasts (HSF-42) at both the low and the high dosages. We performed genome-wide expression array analysis of HSF-42 exposed to doses 8 and 80 nM to link the global cell response to a molecular and genetic phenotype. We used a gene array containing approximately 22,000 total probe sets, containing 18,400 probe sets from known genes. Only approximately 50 genes (approximately 0.2% of all the genes tested) exhibited a statistically significant

  19. High-level expression of nonglycosylated human pancreatic lipase-related protein 2 in Pichia pastoris.

    Science.gov (United States)

    Sebban-Kreuzer, Corinne; Deprez-Beauclair, Paule; Berton, Amelie; Crenon, Isabelle

    2006-10-01

    The human pancreatic lipase-related protein 2 (HPLRP2) was produced in the methylotrophic yeast Pichia pastoris. The HPLRP2 cDNA corresponding to the protein coding sequence including the native signal sequence, was cloned into the pPIC9K vector and integrated into the genome of P. pastoris. P. pastoris transformants secreting high-level rHPLRP2 were obtained and the expression level into the liquid culture medium reached about 40mg/L after 4 days of culture. rHPLRP2 was purified by a single anion-exchange step after an overnight dialysis. N-terminal sequence analysis showed that the purified rHPLRP2 mature protein possessed a correct N-terminal amino acid sequence indicating that its signal peptide was properly processed. Mass spectrometry analysis showed that the recombinant HPLRP2 molecular weight was 52,532Da which was 2451Da greater than the mass calculated from the sequence of the protein (50,081Da) and 1536Da greater than the mass of the native human protein (50,996Da). In vitro deglycosylation experiments by peptide:N-glycosidase F (PNGase F) indicated that rHPLRP2 secreted from P. pastoris was N-glycosylated. Specific conditions were setup in order to obtain a recombinant protein free of glycan chain. We observed that blocking glycosylation in vivo by addition of tunicamycin in the culture medium during the production resulted in a correct processing of the rHPLRP2 mature protein. The lipase activity of glycosylated or nonglycosylated rHPLRP2, which was about 800U/mg on tributyrin, was inhibited by the presence of bile salts and not restored by adding colipase. In conclusion, the experimental procedure which we have developed will allow us to get a high-level production in P. pastoris of glycosylated and nonglycosylated rHPLRP2, suitable for subsequent biophysical and structural studies.

  20. Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae

    Science.gov (United States)

    Tavakoli, Minoo; Bouzari, Saeed; Siadat, Seyed Davar; Najar Peerayeh, Shahin; Jafari, Anis

    2015-01-01

    Background: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis. Objectives: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study. Materials and Methods: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting. Results: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration. Conclusions: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway. PMID:26034537

  1. [A hydroponic cultivation system for rapid high-yield transient protein expression in Nicotiana plants under laboratory conditions].

    Science.gov (United States)

    Mo, Qianzhen; Mai, Rongjia; Yang, Zhixiao; Chen, Minfang; Yang, Tiezhao; Lai, Huafang; Yang, Peiliang; Chen, Qiang; Zhou, Xiaohong

    2012-06-01

    To develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions. To establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA. Our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration. We have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

  2. SERPINE2, an inhibitor of plasminogen activators, is highly expressed in the human endometrium during the secretory phase

    Directory of Open Access Journals (Sweden)

    Hwu Yuh-Ming

    2011-03-01

    Full Text Available Abstract Background SERPINE2, also known as protease nexin-1, belongs to the serine protease inhibitor (SERPIN superfamily. It is one of the potent SERPINs that modulates the activity of plasminogen activators (PAs. PAs and their SERPIN inhibitors, such as SERPINB2 and SERPINE1, were expressed in the human endometrium and were implicated in implantation. However, expression data about SERPINE2 in the human endometrium is still unknown. Thus, we conducted an investigation to reveal the spatiotemporal and cellular expression of SERPINE2 in the human uterus during the menstrual cycle. Methods Seven patients who underwent a hysterectomy and samples of 120 archived patients' endometrial curettage or parts of the uterus that were formalin-fixed and embedded in paraffin. Western blotting was performed to evaluate the specificity and sensitivity of the antibody. Immunohistochemistry was conducted to localize the SERPINE2 expression site. Quantitative analysis was conducted to evaluate expression levels of SERPINE2 in various sub-phases of the menstrual cycle. Results The SERPINE2 protein was primarily detected in the uterine fluid during the mid- and late-secretory phases of the menstrual cycle. It was predominantly expressed in the luminal and glandular epithelium, less in the myometrium, and only dispersedly in certain stromal cells throughout the menstrual cycle. A quantitative analysis of expression levels of SERPINE2 in the glandular epithelium revealed that it was highly expressed in the endometrium during the secretory phase compared to the proliferative phase. Conclusions The SERPINE2 protein is highly expressed in the endometrium during the secretory phase, indicating that it may participate in tissue remodeling involved in implantation.

  3. High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects.

    Science.gov (United States)

    Yu, Jingwen; Wu, Yanqing; Yang, Peixin

    2016-05-01

    Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses

  4. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    Science.gov (United States)

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

  5. High glucose decreases the expression of ATP-binding cassette transporter G1 in human vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Jiahong Xue; Zuyi Yuan; Yue Wu; Yan Zhao; Zhaofei Wan

    2008-01-01

    Objective:ATP-binding cassette transporters(ABC) A1 and G1 play an important role in mediating cholesterol efflux and preventing macrophage foam cell formation. In this study, we examined the regulation of ABC transporters by high glucose in human vascular smooth muscle cells(VSMCs), the other precursor of foam cells. Methods:Incubation of human VSMCs with D-ghicose(5 to 30 mM) for 1 to 7 days in the presence or absence of antioxidant and nuclear factor(NF)-kB inhibitors, the expressions of ABCA1 and ABCG1 were analyzed by real time PCR and Western blotting. Results:High glucose decreased ABCG1 mRNA and protein expression in cultured VSMCs, whereas the expression of ABCA1 was not significantly decreased. Down-regulation of ABCG1 mRNA expression by high glucose was abolished by antioxidant N-acetyl-L-cysteine(NAC) and NF-kB inhibitors, BAY 11-7085 and tosyl-phenylalanine chloromethyl-ketone(TPCK). Conclusion:High glucose suppresses the expression of ABCG1 in VSMCs, which is the possible mechanism of VSMC derived foam cell transformation.

  6. The AFT1 transcriptional factor is differentially required for expression of high-affinity iron uptake genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Casas, C; Aldea, M; Espinet, C; Gallego, C; Gil, R; Herrero, E

    1997-06-15

    High-affinity iron uptake in Saccharomyces cerevisiae involves the extracytoplasmic reduction of ferric ions by FRE1 and FRE2 reductases. Ferrous ions are then transported across the plasma membrane through the FET3 oxidase-FTR1 permease complex. Expression of the high-affinity iron uptake genes is induced upon iron deprivation. We demonstrate that AFT1 is differentially involved in such regulation. Aft1 protein is required for maintaining detectable non-induced level of FET3 expression and for induction of FRE2 in iron starvation conditions. On the contrary, FRE1 mRNA induction is normal in the absence of Aft1, although the existence of AFT1 point mutations causing constitutive expression of FRE1 (Yamaguchi-Iwai et al., EMBO J. 14: 1231-1239, 1995) indicates that Aft1 may also participate in FRE1 expression in a dispensable way. The alterations in the basal levels of expression of the high-affinity iron uptake genes may explain why the AFT1 mutant is unable to grow on respirable carbon sources. Overexpression of AFT1 leads to growth arrest of the G1 stage of the cell cycle. Aft1 is a transcriptional activator that would be part of the different transcriptional complexes interacting with the promoter of the high-affinity iron uptake genes. Aft1 displays phosphorylation modifications depending on the growth stage of the cells, and it might link induction of genes for iron uptake to other metabolically dominant requirement for cell growth.

  7. High expression of transcriptional coactivator p300 correlates with aggressive features and poor prognosis of hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Cao Yun

    2011-01-01

    Full Text Available Abstract Background It has been suggested that p300 participates in the regulation of a wide range of cell biological processes and mutation of p300 has been identified in certain types of human cancers. However, the expression dynamics of p300 in hepatocellular carcinoma (HCC and its clinical/prognostic significance are unclear. Methods In this study, the methods of reverse transcription-polymerase chain reaction (RT-PCR, Western blotting and immunohistochemistry (IHC were utilized to investigate protein/mRNA expression of p300 in HCCs. Receiver operating characteristic (ROC curve analysis, spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the data. Results Up-regulated expression of p300 mRNA and protein was observed in the majority of HCCs by RT-PCR and Western blotting, when compared with their adjacent non-malignant liver tissues. According to the ROC curves, the cutoff score for p300 high expression was defined when more than 60% of the tumor cells were positively stained. High expression of p300 was examined in 60/123 (48.8% of HCCs and in 8/123 (6.5% of adjacent non-malignant liver tissues. High expression of p300 was correlated with higher AFP level, larger tumor size, multiplicity, poorer differentiation and later stage (P P = 0.001. In different subsets of HCC patients, p300 expression was also a prognostic indicator in patients with stage II (P = 0.007 and stage III (P = 0.011. Importantly, p300 expression was evaluated as an independent prognostic factor in multivariate analysis (P = 0.021. Consequently, a new clinicopathologic prognostic model with three poor prognostic factors (p300 expression, AFP level and vascular invasion was constructed. The model could significantly stratify risk (low, intermediate and high for overall survival (P Conclusions Our findings provide a basis for the concept that high expression of p300 in HCC may be important in the acquisition of

  8. High expression of Sonic Hedgehog signaling pathway genes indicates a risk of recurrence of breast carcinoma

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    Jeng KS

    2013-12-01

    Full Text Available Kuo-Shyang Jeng,1 I-Shyan Sheen,2 Wen-Juei Jeng,2 Ming-Che Yu,3 Hsin-I Hsiau,3 Fang-Yu Chang31Department of Surgery, Far Eastern Memorial Hospital, Taipei, 2Department of Internal Medicine, Chang-Gung Memorial Hospital, Linkou Medical Center, Chang-Gung University, Tao-Yuan, 3Department of Medical Research, Far Eastern Memorial Hospital, Taipei, TaiwanBackground: Abnormal activation of the Sonic Hedgehog (SHH signaling pathway contributing to carcinogenesis of some organs has been reported in the literature. We hypothesize that activation of the SHH pathway contributes to the recurrence of breast carcinoma.Methods: Fifty consecutive patients with invasive breast carcinoma following curative resection were enrolled in this prospective study. The ratios of messenger RNA (mRNA expression for Sonic Hedgehog (SHH, patched homolog-1 (PTCH-1, glioma-associated oncogene-1 (GLI-1, and smoothened (SMOH were measured between breast carcinoma tissue and paired noncancerous breast tissue. These ratios were compared with their clinicopathologic characteristics. These factors and the mRNA ratios were compared between patients with recurrence and those without recurrence.Results: The size of the invasive cancer correlated significantly with the ratio of SHH mRNA (P=0.001, that of PTCH-1 mRNA (P=0.005, and that of SMOH mRNA (P=0.021. Lymph node involvement correlated significantly with the ratio of SMOH mRNA (P=0.041. The correlation between Her-2 neu and the ratio of GLI-1 mRNA was statistically significant (P=0.012. Each ratio of mRNA of SHH, PTCH-1, GLI-1, and SMOH correlated significantly with cancer recurrence (P<0.001 for each.Conclusion: We suggest that high expression of SHH mRNA, PTCH-1 mRNA, GLI-1 mRNA, and SMOH mRNA in breast cancer tissue correlates with invasiveness and is a potential biomarker to predict postoperative recurrence.Keywords: SHH pathway, breast carcinoma, prediction, recurrence

  9. Endothelial lipase is highly expressed in macrophages in advanced human atherosclerotic lesions

    DEFF Research Database (Denmark)

    Bartels, Emil D; Nielsen, John E; Lindegaard, Marie Louise Skakkebæk;

    2007-01-01

    Endothelial lipase (EL) is expressed in endothelial cells, and affects plasma lipoprotein metabolism by hydrolyzing phospholipids in HDL. To determine the cellular expression of EL mRNA and protein in human atherosclerotic lesions, we performed in situ hybridization and immunohistochemical studies...

  10. Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration( hALR), Expression and Purification of hALR

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Experimental evidence has been presented to suggest that the human augmenter of liver regeneration (hALR)serves as a hepatotrophic growth factor during liver regeneration and as a generalized growth factor during pancreas transplant/regeneration. A prokaryotic expression plasmid, pRSET/6his-c-myc-hALR was constructed, by cloning synthesized hALR cDNA into pRSET/6his-c-myc that was improved on the basis of pRSET B by the group. As a result, the protein was highly expressed in E. coli BL21. The recombinant hALR was over 60% of the total protein in E. coli. Its validity was confirmed by meansof Western Blotting. The protein was purified by Ni-NTA affinity chromatography and this FAD-dependent sulfhydryl oxidase activity was measured.

  11. High-throughput in vivo analysis of gene expression in Caenorhabditis elegans.

    Science.gov (United States)

    Hunt-Newbury, Rebecca; Viveiros, Ryan; Johnsen, Robert; Mah, Allan; Anastas, Dina; Fang, Lily; Halfnight, Erin; Lee, David; Lin, John; Lorch, Adam; McKay, Sheldon; Okada, H Mark; Pan, Jie; Schulz, Ana K; Tu, Domena; Wong, Kim; Zhao, Z; Alexeyenko, Andrey; Burglin, Thomas; Sonnhammer, Eric; Schnabel, Ralf; Jones, Steven J; Marra, Marco A; Baillie, David L; Moerman, Donald G

    2007-09-01

    Using DNA sequences 5' to open reading frames, we have constructed green fluorescent protein (GFP) fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5' DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index) and through WormBase (http://www.wormbase.org).

  12. High-throughput in vivo analysis of gene expression in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Rebecca Hunt-Newbury

    2007-09-01

    Full Text Available Using DNA sequences 5' to open reading frames, we have constructed green fluorescent protein (GFP fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5' DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index and through WormBase (http://www.wormbase.org.

  13. High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

    Science.gov (United States)

    Iglesias-Figueroa, Blanca; Valdiviezo-Godina, Norberto; Siqueiros-Cendón, Tania; Sinagawa-García, Sugey; Arévalo-Gallegos, Sigifredo; Rascón-Cruz, Quintín

    2016-01-01

    In this study, bovine lactoferrin (bLf), an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf) in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin) demonstrated antibacterial activity against Escherichia coli (E. coli) BL21DE3, Staphylococcus aureus (S. aureus) FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa) ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly. PMID:27294912

  14. High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

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    Blanca Iglesias-Figueroa

    2016-06-01

    Full Text Available In this study, bovine lactoferrin (bLf, an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin demonstrated antibacterial activity against Escherichia coli (E. coli BL21DE3, Staphylococcus aureus (S. aureus FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly.

  15. High-Throughput In Vivo Analysis of Gene Expression in Caenorhabditis elegans

    Science.gov (United States)

    Mah, Allan; Anastas, Dina; Fang, Lily; Halfnight, Erin; Lee, David; Lin, John; Lorch, Adam; McKay, Sheldon; Okada, H. Mark; Pan, Jie; Schulz, Ana K; Tu, Domena; Wong, Kim; Zhao, Z; Alexeyenko, Andrey; Burglin, Thomas; Sonnhammer, Eric; Schnabel, Ralf; Jones, Steven J; Marra, Marco A; Baillie, David L; Moerman, Donald G

    2007-01-01

    Using DNA sequences 5′ to open reading frames, we have constructed green fluorescent protein (GFP) fusions and generated spatial and temporal tissue expression profiles for 1,886 specific genes in the nematode Caenorhabditis elegans. This effort encompasses about 10% of all genes identified in this organism. GFP-expressing wild-type animals were analyzed at each stage of development from embryo to adult. We have identified 5′ DNA regions regulating expression at all developmental stages and in 38 different cell and tissue types in this organism. Among the regulatory regions identified are sequences that regulate expression in all cells, in specific tissues, in combinations of tissues, and in single cells. Most of the genes we have examined in C. elegans have human orthologs. All the images and expression pattern data generated by this project are available at WormAtlas (http://gfpweb.aecom.yu.edu/index) and through WormBase (http://www.wormbase.org). PMID:17850180

  16. Expression of CRM1 and CDK5 shows high prognostic accuracy for gastric cancer

    Science.gov (United States)

    Sun, Yu-Qin; Xie, Jian-Wei; Xie, Hong-Teng; Chen, Peng-Chen; Zhang, Xiu-Li; Zheng, Chao-Hui; Li, Ping; Wang, Jia-Bin; Lin, Jian-Xian; Cao, Long-Long; Huang, Chang-Ming; Lin, Yao

    2017-01-01

    AIM To evaluate the predictive value of the expression of chromosomal maintenance (CRM)1 and cyclin-dependent kinase (CDK)5 in gastric cancer (GC) patients after gastrectomy. METHODS A total of 240 GC patients who received standard gastrectomy were enrolled in the study. The expression level of CRM1 and CDK5 was detected by immunohistochemistry. The correlations between CRM1 and CDK5 expression and clinicopathological factors were explored. Univariate and multivariate survival analyses were used to identify prognostic factors for GC. Receiver operating characteristic analysis was used to compare the accuracy of the prediction of clinical outcome by the parameters. RESULTS The expression of CRM1 was significantly related to size of primary tumor (P = 0.005), Borrmann type (P = 0.006), degree of differentiation (P = 0.004), depth of invasion (P = 0.008), lymph node metastasis (P = 0.013), TNM stage (P = 0.002) and distant metastasis (P = 0.015). The expression of CDK5 was significantly related to sex (P = 0.048) and Lauren’s classification (P = 0.011). Multivariate Cox regression analysis identified that CRM1 and CDK5 co-expression status was an independent prognostic factor for overall survival (OS) of patients with GC. Integration of CRM1 and CDK5 expression could provide additional prognostic value for OS compared with CRM1 or CDK5 expression alone (P = 0.001). CONCLUSION CRM1 and CDK5 co-expression was an independent prognostic factors for GC. Combined CRM1 and CDK5 expression could provide a prognostic model for OS of GC. PMID:28373767

  17. Gene Structure and Expression of the High-affinity Nitrate Transport System in Rice Roots

    Institute of Scientific and Technical Information of China (English)

    Chao Cai; Jun-Yi Wang; Yong-Guan Zhu; Qi-Rong Shen; Bin Li; Yi-Ping Tong; Zhen-Sheng Li

    2008-01-01

    Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.

  18. Pathway-Based Factor Analysis of Gene Expression Data Produces Highly Heritable Phenotypes That Associate with Age

    Science.gov (United States)

    Anand Brown, Andrew; Ding, Zhihao; Viñuela, Ana; Glass, Dan; Parts, Leopold; Spector, Tim; Winn, John; Durbin, Richard

    2015-01-01

    Statistical factor analysis methods have previously been used to remove noise components from high-dimensional data prior to genetic association mapping and, in a guided fashion, to summarize biologically relevant sources of variation. Here, we show how the derived factors summarizing pathway expression can be used to analyze the relationships between expression, heritability, and aging. We used skin gene expression data from 647 twins from the MuTHER Consortium and applied factor analysis to concisely summarize patterns of gene expression to remove broad confounding influences and to produce concise pathway-level phenotypes. We derived 930 “pathway phenotypes” that summarized patterns of variation across 186 KEGG pathways (five phenotypes per pathway). We identified 69 significant associations of age with phenotype from 57 distinct KEGG pathways at a stringent Bonferroni threshold (P<5.38×10−5). These phenotypes are more heritable (h2=0.32) than gene expression levels. On average, expression levels of 16% of genes within these pathways are associated with age. Several significant pathways relate to metabolizing sugars and fatty acids; others relate to insulin signaling. We have demonstrated that factor analysis methods combined with biological knowledge can produce more reliable phenotypes with less stochastic noise than the individual gene expression levels, which increases our power to discover biologically relevant associations. These phenotypes could also be applied to discover associations with other environmental factors. PMID:25758824

  19. Multiple ribosomal proteins are expressed at high levels in developing zebrafish endoderm and are required for normal exocrine pancreas development.

    Science.gov (United States)

    Provost, Elayne; Weier, Christopher A; Leach, Steven D

    2013-06-01

    Ribosomal protein L (rpl) genes are essential for assembly of the 60S subunit of the eukaryotic ribosome and may also carry out additional extra-ribosomal functions. We have identified a common expression pattern for rpl genes in developing zebrafish larvae. After initially widespread expression in early embryos, the expression of multiple rpl genes becomes increasingly restricted to the endoderm. With respect to the pancreas, rpl genes are highly expressed in ptf1a-expressing pancreatic progenitors at 48 hpf, suggesting possible functional roles in pancreatic morphogenesis and/or differentiation. Utilizing two available mutant lines, rpl23a(hi2582) and rpl6(hi3655b), we found that ptf1a-expressing pancreatic progenitors fail to properly expand in embryos homozygous for either of these genes. In addition to these durable homozygous phenotypes, we also demonstrated recoverable delays in ptf1a-expressing pancreatic progenitor expansion in rpl23a(hi2582) and rpl6(hi3655b) heterozygotes. Disruptions in ribosome assembly are generally understood to initiate a p53-dependent cellular stress response. However, concomitant p53 knockdown was unable to rescue normal pancreatic progenitor expansion in either rpl23a(hi2582) or rpl6(hi3655b) mutant embryos, suggesting required and p53-independent roles for rpl23a and rpl6 in pancreas development.

  20. Regulation of the high-affinity copper transporter (hCtr1) expression by cisplatin and heavy metals.

    Science.gov (United States)

    Liang, Zheng Dong; Long, Yan; Chen, Helen H W; Savaraj, Niramol; Kuo, Macus Tien

    2014-01-01

    Platinum-based antitumor agents have been the mainstay in cancer chemotherapy for many human malignancies. Drug resistance is an important obstacle to achieving the maximal therapeutic efficacy of these drugs. Understanding how platinum drugs enter cells is of great importance in improving therapeutic efficacy. It has been demonstrated that human high-affinity copper transporter 1 (hCtr1) is involved in transporting cisplatin into cells to elicit cytotoxic effects, although other mechanisms may exist. In this communication, we demonstrate that cisplatin transcriptionally induces the expression of hCtr1 in time- and concentration-dependent manners. Cisplatin functions as a competitor for hCtr1-mediated copper transport, resulting in reduced cellular copper levels and leading to upregulated expression of Sp1, which is a positive regulator for hCtr1 expression. Thus, regulation of hCtr1 expression by cisplatin is an integral part of the copper homeostasis regulation system. We also demonstrate that Ag(I) and Zn(II), which are known to suppress hCtr1-mediated copper transport, can also induce hCtr1/Sp1 expression. In contrast, Cd(II), another inhibitor of copper transport, downregulates hCtr1 expression by suppressing Sp1 expression. Collectively, our results demonstrate diverse mechanisms of regulating copper metabolism by these heavy metals.

  1. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray

    Institute of Scientific and Technical Information of China (English)

    Xiao-Jing Liu; Li Yang; Feng-Ming Luo; Hong-Bin Wu; Qu-Qiang

    2004-01-01

    AIM: To characterize the gene expression profiles associated with activation of mouse hepatic stellate cell (HSC) and provide novel insights into the pathogenesis of hepatic fibrosis.METHODS: Mice HSCs were isolated from BALB/c mice by in situ perfusion of collagenase and pronase and singlestep density Nycodenz gradient. Total RNA and mRNA of quiescent HSC and culture-activated HSC were extracted,quantified and reversely transcripted into cDNA. cDNAs from activated HSC were labeled with Cy5 and cDNAs from the quiescent HSC were labeled with Cy3, which were mixed with equal quantity, then hybridized with cDNA chips containing 4 000 genes. Chips were washed, scanned and analyzed. Increased expression of 4 genes and decreased expression of one gene in activated HSC were confirmed by reverse transcription- polymerase chain reaction (RT-PCR).RESULTS: A total of 835 differentially expressed genes were identified by cDNA chip between activated and quiescent HSC, and 465 genes were highly expressed in activated HSC. The differentially expressed genes included those involved in protein synthesis, cell-cycle regulation,apoptosis, and DNA damage response.CONCLUSION: Many genes implicated in intrahepatic inflammation, fibrosis and proliferation were up-regulated in activated HSC. cDNA microarray is an effective technique in screening for differentially expressed genes between two different situations of the HSC. Further analysis of the obtained genes will help understand the molecular mechanism of activation of HSC and hepatic fibrosis.

  2. Effect of heparin on high glucose induced proliferation and expression of matrix metalloproteinases in normal human mesangial cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Qiao-ling; Arima Terukatsu; Yasumoto Yuichiro; Tsukamoto Masatoshi; Nozaki Tsuyoshi; Sogabe Atsushi; Harada Kouji; ZHANG Yi-xiang; LIN Xiao-yan; ZHANG Yang-de

    2005-01-01

    Background The pathogenesis of diabetic nephropathy (DN) is a complex pathophysiological process.Its precise mechanism is not fully known. In recent years it has been recognized that synthesis of various extracelluar matrix (ECM) components may increase, and that degradation of ECM may decrease in DN. It was reported heparin could inhibit mesangial cells proliferation in vitro. The main aim of this study is to explore whether heparin inhibits proliferation of mesangial cells grown in high glucose concentration and to measure the effect of heparin on matrix metalloproteinases (MMPs) expression in mesangial cells. Methods The medium contained either low glucose (5 mmol/L) or high glucose (25 mmol/L). The concentrations of heparin in the culture medium were 0, 25, 50,100, 200 or 400 μg/mL. A metabolic (WST-1) assay was used to measure mesangial cell proliferation and Western blot analysis was used to measure MMPs expression of mesangial cells. Results Normal human mesangial cell (NHMC) proliferation was higher in high glucose (HG) medium than in low glucose (LG) medium. They showed a 1.93 fold expansion after 72 h in high glucose in contrast to a 1.63 fold expansion in low glucose. In the presence of heparin, mesangial cells proliferation was inhibited, which was more obvious at high glucose concentrations than at low glucose concentrations. In high glucose, with heparin concentration of 50, 100, 200 and 400 μg/mL, the mesangial cells showed a 0. 61 fold, 0.52 fold, 0.52 fold and 0.41 fold reductions in cell number compared to cells grown without heparin. In low glucose, only concentrations of 200 μg/mL and 400 μg/mL showed reduction in cell number, namely 0.54 fold and 0.45 fold, when compared to cells grown without heparin. In Western blot analysis,MMP1, MMP2, MMP3 and MMP9 was expressed by mesangial cells expressed in both high and low glucose concentrations, which was more prominent in high glucose medium. Incubation of heparin further increased expression of

  3. Macroscopic Expressions of Molecular Adiabatic Compressibility of Methyl and Ethyl Caprate under High Pressure and High Temperature

    Directory of Open Access Journals (Sweden)

    Fuxi Shi

    2014-01-01

    Full Text Available The molecular compressibility, which is a macroscopic quantity to reveal the microcompressibility by additivity of molecular constitutions, is considered as a fixed value for specific organic liquids. In this study, we introduced two calculated expressions of molecular adiabatic compressibility to demonstrate its pressure and temperature dependency. The first one was developed from Wada’s constant expression based on experimental data of density and sound velocity. Secondly, by introducing the 2D fitting expressions and their partial derivative of pressure and temperature, molecular compressibility dependency was analyzed further, and a 3D fitting expression was obtained from the calculated data of the first one. The third was derived with introducing the pressure and temperature correction factors based on analogy to Lennard-Jones potential function and energy equipartition theorem. In wide range of temperatures (293

  4. Phenotypic and gene expression changes between low (glucose-responsive) and High (glucose non-responsive) MIN-6 beta cells

    DEFF Research Database (Denmark)

    O´Driscoll, L.; Gammell, p.; McKierman, E.

    2006-01-01

    that the glucose-stimulated insulin secretion (GSIS) phenotype is relatively unstable, in long-term culture. This study aimed to investigate phenotypic and gene expression changes associated with this loss of GSIS, using the MIN-6 cell line as model. Phenotypic differences between MIN-6(L, low passage) and MIN-6(H......, high passage) were determined by ELISA (assessing GSIS and cellular (pro)insulin content), proliferation assays, phase contrast light microscopy and analysis of alkaline phosphatase expression. Differential mRNA expression was investigated using microarray, bioinformatics and real-time PCR technologies....... Long-term culture was found to be associated with many phenotypic changes, including changes in growth rate and cellular morphology, as well as loss of GSIS. Microarray analyses indicate expression of many mRNAs, including many involved in regulated secretion, adhesion and proliferation...

  5. Exploring the molecular pathogenesis and biomarkers of high risk oral premalignant lesions on the basis of long noncoding RNA expression profiling by serial analysis of gene expression.

    Science.gov (United States)

    Jia, Hongcheng; Wang, Xuan; Sun, Zheng

    2017-04-24

    Oral premalignant lesions (OPLs) have malignant transformation potential, with no reliable markers available. This study aimed to assess molecular events to identify biomarkers that can reflect high-risk lesions as predictive factors to tailor clinical decision for patients on the basis of long noncoding RNAs (lncRNA) expression profiling by serial analysis of gene expression. The GSE31021 and GSE8127 datasets were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and lncRNAs were identified using the LIMMA package in R language. The genes targeted by lncRNAs were predicted among screened DEGs using Pearson's correlation. Gene ontology function and Kyoto Encyclopedia of Genes and Genomes pathway analyses were carried out for genes targeted by lncRNAs using the Database for Annotation, Visualization, and Integrated Discovery online tool. A total of 674 DEGs and differentially expressed lncRNAs were screened. Thirty-two interactions of 10 lncRNAs and 524 target genes were predicted. The lncRNA NEAT1 was among the top 10 lncRNAs. The coregulated target genes RP4-684O24, RP11-283I3, and RP11-350G8 were significantly enriched in the immune response and mannosyl-oligosaccharide mannosidase activity. The target genes coregulated by LINC00665 and MIR378D2 were significantly enriched in the ubiquitin-dependent protein catabolic process, ubiquitin-protein ligase activity, and neurotrophin signaling. The lncRNA NEAT1 may play an important role in high-risk lesions. The novel lncRNAs and DEGs identified in OPLs may mediate the immune response and neurotrophin signaling and show ubiquitin ligase activity. These results improve our understanding of the molecular pathogenesis of OPLs and identify some potential targets for early diagnosis of high risk OPLs.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to

  6. Effect of high temperature on the expressions of genes encoding starch synthesis enzymes in developing rice endosperms

    Institute of Scientific and Technical Information of China (English)

    CAO Zhen-zhen; PAN Gang; WANG Fu-biao; WEI Ke-su; LI Zhao-wei; SHI Chun-hai; GENG Wei; CHENG Fang-min

    2015-01-01

    High temperature is the major environmental factor affecting grain starch properties of cooking rice cultivars. In this study, two non-waxy indica rice genotypes, cv. 9311 and its mutant with extremely high amylose phenotype (9311eha) were used to study the differential expressions of genes in starch synthesis and their responses to high temperature (HT). Signiifcant increase in apparent amylose content and hot-water-soluble starch content in mutant 9311eha were genetical y caused by a substitution from AGTTATA to AGGTATA at the leader intron 5´ splice site in Wx gene. This mutation resulted in different mRNA transcript levels, mRNA splicing efifciencies and protein levels of Wx between the two rice genotypes, which also lead to the genotype-dependent alteration in the temporal pattern of Wx transcription and granule-bound starch synthase (GBSS) activity in response to HT. However, changes in the activities of other starch synthesizing enzymes and their expressions of distinct isoform genes were not signiifcant with the Wx gene mutation, thus only minor difference in the particle size of starch granule, chain-length distribution and gelatinization enthalpy were found between the two genotypes. The tempo-ral-speciifc expression of multiple isoform genes responsive to different temperature regiments indicated that the reduction of GBSS transcript expression under HT was general y accompanied by the decreased expressions of SSSIIa, SSSIIIa and SBEIIb. Consequently, high temperature-ripened grains in 9311eha showed high proportion of intermediate and long B chains and somewhat lower level of short A chain compared to the wildtype. The temperature-dependent alteration of amylose content was not only attributed to the reduced expression of GBSS, but also associated with the complimentary effect of SSSIIa and SBEIIb.

  7. High expressions of LDHA and AMPK as prognostic biomarkers for breast cancer.

    Science.gov (United States)

    Huang, Xiaojia; Li, Xing; Xie, Xinhua; Ye, Feng; Chen, Bo; Song, Cailu; Tang, Hailin; Xie, Xiaoming

    2016-12-01

    The purpose of this study was to investigate the potential correlation between lactate dehydrogenase A (LDHA) and AMP-activated protein kinase (AMPK) and their clinicopathologic significance in breast cancer. Western blot and qRT-PCR were used to detect the expression levels of LDHA and AMPK in eight breast cancer lines and eight breast cancer tissues. In addition, LDHA and AMPK were detected by immunohistochemistry (IHC) using breast cancer tissue microarrays (TMAs) of 112 patients. The association between LDHA and AMPK expression levels was statistically analyzed. So were the prognostic roles and clinicopathologic significances in breast cancer. The expression levels of LDHA and AMPK were relatively higher in triple-negative breast cancer (TNBC) cell lines than in non-triple-negative breast cancer (NTNBC) cell lines. LDHA and AMPK were also further up-regulated in TNBC tissues than in NTNBC tissues. Correlation analysis showed a positive correlation between LDHA and AMPK expression levels. Expression of LDHA and AMPK were significantly correlated with TNM stage, distant metastasis, Ki67 status and survival outcomes of patients. Patients with both positive expression of LDHA and AMPK showed shorter overall survival (OS) and disease-free survival (DFS). These findings improve our understanding of the expression pattern of LDHA and AMPK in breast cancer and clarify the role of LDHA and AMPK as promising prognostic biomarkers for breast cancer. Copyright © 2016. Published by Elsevier Ltd.

  8. Bone marrow-infiltrating human neuroblastoma cells express high levels of calprotectin and HLA-G proteins.

    Directory of Open Access Journals (Sweden)

    Fabio Morandi

    Full Text Available Metastases in the bone marrow (BM are grim prognostic factors in patients with neuroblastoma (NB. In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5 y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin, CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.

  9. The expression of high-mobility group box protein-1 in temporomandibular joint osteoarthritis with disc perforation.

    Science.gov (United States)

    Feng, Yaping; Fang, Wei; Li, Cheng; Guo, Huilin; Li, Yingjie; Long, Xing

    2016-02-01

    High-mobility group box protein-1 (HMGB-1), a potent promoter of inflammation, was believed to be a potential trigger of osteoarthritis (OA). Only a few studies have investigated the role of HMGB-1 in temporomandibular joint (TMJ) OA, especially in disc perforation cases. But in this study, not only the expression of HMGB-1 in TMJ OA with disc perforation was investigated but also the possible role of HMGB-1 in TMJ OA was discussed. Synovial membrane and disc specimens were collected from patients with TMJ OA, and the expression of HMGB-1 was detected using immunohistochemistry, real-time quantitative PCR and Western blotting. High-mobility group box protein-1 expressed strongly in cytoplasm and nucleus of lining layer cells and endothelial cells in osteoarthritic synovium. Staining of HMGB-1 was found intensive in the frontier tissue of the perforation in the perforated discs. HMGB-1 expression was also upregulated in osteoarthritic synovial cells and disc cells according to real-time quantitative PCR and Western blotting analysis. High-mobility group box protein-1 expression was upregulated in TMJ OA and might promote the progression of TMJ OA. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. MAGE-A is More Highly Expressed Than NY-ESO-1 in a Systematic Immunohistochemical Analysis of 3668 Cases.

    Science.gov (United States)

    Kerkar, Sid P; Wang, Zeng-Feng; Lasota, Jerzy; Park, Tristen; Patel, Krishna; Groh, Eric; Rosenberg, Steven A; Miettinen, Markku M

    2016-05-01

    Two cancer testis antigens, the New York esophageal squamous cell carcinoma-1 (NY-ESO-1) and the melanoma-antigen family A (MAGE-A), represent promising immunotherapy targets due to the low expression of these antigens in nonmalignant tissue. To assess overexpression patterns in various cancers, we performed a systematic immunohistochemical analysis for NY-ESO-1 and MAGE-A on tissue array samples of 3668 common epithelial carcinomas (CA) and germ cell tumors of high prevalence and mortality. Here, we find significantly higher expression of MAGE-A (>50% on tumor cells) compared with NY-ESO-1 in several CAs including cutaneous squamous cell carcinomas (SCC) (52.8%/2.8%), esophageal SCC (50%/0%), head and neck SCC (41.1%/ESO-1, was seen in whole slide evaluations of an independent cohort of metastatic SCC (45.5%/3.6%) and metastatic CA (13.5%/0%) of various primaries with significantly higher expression of MAGE-A in metastatic SCC compared with other metastatic CA. MAGE-A is also more highly expressed in germ cell tumors, seminomas (69%/3.5%) and nonseminomas (40.1%/4.7%). In summary, MAGE-A is more highly expressed than NY-ESO-1 in a majority of human malignancies, and targeting MAGE-A may benefit a large number of patients.

  11. Isoflavone Regulates Lipid Metabolism via Expression of Related Genes in OVX Rats Fed on a High-fat Diet

    Institute of Scientific and Technical Information of China (English)

    XIAO-LIN NA; JUNKO EZAKI; FUMIE SUGIYAMA; HONG-BIN CUI; YOSHIKO ISHIMI

    2008-01-01

    Objective To investigate the effects of isoflavone on body weight, fat mass, and gene expression in relation to lipid metabolism. Methods Thirty-six female SD rats were variectomized or sham-operated and fed on a high-fat diet. Two months later, abdominal incision was made, blood was collected to separate serum, and the liver and adipose tissue were immediately collected and weighed. Some portions of these tissues were frozen in liquid nitrogen and stored at -80℃. Results Ovariectomy (OVX) with a high-fat diet could induce obesity in rats, while treatment with isoflavone significantly inhibited the increase in body weight and fat mass in abdomen. Serum total cholesterol and leptin were significantly decreased in isoflavone group, compared with the OVX group. The mRNA expression of liver fatty acid synthase (FAS) in the OVX group was significantly higher than that in sham-operated group, while this difference was not observed in the isoflavone group. The mRNA expression of liver hormone-sensitive lipase (HSL) in the OVX rats tended to be lower than that in the sham-operated rats. Furthermore, a large amount of isoflavone maintained the mRNA expression at a sham level. Conclusion lsoflavone may prevent obesity induced by ovariectomy with a high-fat diet, in part by modulating gene expression related to lipid metabolism.

  12. Genome wide expression analysis suggests perturbation of vascular homeostasis during high altitude pulmonary edema.

    Directory of Open Access Journals (Sweden)

    Manish Sharma

    Full Text Available BACKGROUND: High altitude pulmonary edema (HAPE is a life-threatening form of non-cardiogenic edema which occurs in unacclimatized but otherwise normal individuals within two to four days after rapid ascent to altitude beyond 3000 m. The precise pathoetiology and inciting mechanisms regulating HAPE remain unclear. METHODOLOGY/PRINCIPLE FINDINGS: We performed global gene expression profiling in individuals with established HAPE compared to acclimatized individuals. Our data suggests concurrent modulation of multiple pathways which regulate vascular homeostasis and consequently lung fluid dynamics. These pathways included those which regulate vasoconstriction through smooth muscle contraction, cellular actin cytoskeleton rearrangements and endothelial permeability/dysfunction. Some notable genes within these pathways included MYLK; rho family members ARGEF11, ARHGAP24; cell adhesion molecules such as CLDN6, CLDN23, PXN and VCAM1 besides other signaling intermediates. Further, several important regulators of systemic/pulmonary hypertension including ADRA1D, ECE1, and EDNRA were upregulated in HAPE. We also observed significant upregulation of genes involved in paracrine signaling through chemokines and lymphocyte activation pathways during HAPE represented by transcripts of TNF, JAK2, MAP2K2, MAP2K7, MAPK10, PLCB1, ARAF, SOS1, PAK3 and RELA amongst others. Perturbation of such pathways can potentially skew vascular homeostatic equilibrium towards altered vascular permeability. Additionally, differential regulation of hypoxia-sensing, hypoxia-response and OXPHOS pathway genes in individuals with HAPE were also observed. CONCLUSIONS/SIGNIFICANCE: Our data reveals specific components of the complex molecular circuitry underlying HAPE. We show concurrent perturbation of multiple pathways regulating vascular homeostasis and suggest multi-genic nature of regulation of HAPE.

  13. A Highly Efficient Gene Expression Programming (GEP Model for Auxiliary Diagnosis of Small Cell Lung Cancer.

    Directory of Open Access Journals (Sweden)

    Zhuang Yu

    Full Text Available Lung cancer is an important and common cancer that constitutes a major public health problem, but early detection of small cell lung cancer can significantly improve the survival rate of cancer patients. A number of serum biomarkers have been used in the diagnosis of lung cancers; however, they exhibit low sensitivity and specificity.We used biochemical methods to measure blood levels of lactate dehydrogenase (LDH, C-reactive protein (CRP, Na+, Cl-, carcino-embryonic antigen (CEA, and neuron specific enolase (NSE in 145 small cell lung cancer (SCLC patients and 155 non-small cell lung cancer and 155 normal controls. A gene expression programming (GEP model and Receiver Operating Characteristic (ROC curves incorporating these biomarkers was developed for the auxiliary diagnosis of SCLC.After appropriate modification of the parameters, the GEP model was initially set up based on a training set of 115 SCLC patients and 125 normal controls for GEP model generation. Then the GEP was applied to the remaining 60 subjects (the test set for model validation. GEP successfully discriminated 281 out of 300 cases, showing a correct classification rate for lung cancer patients of 93.75% (225/240 and 93.33% (56/60 for the training and test sets, respectively. Another GEP model incorporating four biomarkers, including CEA, NSE, LDH, and CRP, exhibited slightly lower detection sensitivity than the GEP model, including six biomarkers. We repeat the models on artificial neural network (ANN, and our results showed that the accuracy of GEP models were higher than that in ANN. GEP model incorporating six serum biomarkers performed by NSCLC patients and normal controls showed low accuracy than SCLC patients and was enough to prove that the GEP model is suitable for the SCLC patients.We have developed a GEP model with high sensitivity and specificity for the auxiliary diagnosis of SCLC. This GEP model has the potential for the wide use for detection of SCLC in less

  14. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

    Science.gov (United States)

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

    2015-01-01

    Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

  15. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

    Directory of Open Access Journals (Sweden)

    Federico Abascal

    2015-06-01

    Full Text Available Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved--all the homologous exons we identified evolved over 460 million years ago--and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles.

  16. Altered Expression of High Molecular Weight Heat Shock Proteins after OCT4B1 Suppression in Human Tumor Cell Lines

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Mirzaei

    2016-02-01

    Full Text Available Objective: OCT4B1, a novel variant of OCT4, is expressed in cancer cell lines and tissues. Based on our previous reports, OCT4B1 appears to have a crucial role in regulating apoptosis as well as stress response [heat shock proteins (HSPs] pathways. The aim of the present study was to determine the effects of OCT4B1 silencing on the expression of high molecular weight HSPs in three different human tumor cell lines. Materials and Methods: In this experimental study, OCT4B1 expression was suppressed in AGS (gastric adenocarcinoma, 5637 (bladder tumor and U-87MG (brain tumor cell lines using RNAi strategy. Real-time polymerase chain reaction (PCR array was employed for expression level analysis and the fold changes were calculated using RT2 Profiler PCR array data analysis software version 3.5. Results: Our data revealed up-regulation of HSPD1 (from HSP60 family as well as HSPA14, HSPA1L, HSPA4, HSPA5 and HSPA8 (from HSP70 family following OCT4B1 knock-down in all three cell lines. In contrast, the expression of HSP90AA1 and HSP- 90AB1 (from HSP90 family as well as HSPA1B and HSPA6 (from HSP70 family was down-regulated under similar conditions. Other stress-related genes showed varying expression pattern in the examined tumor cell lines. Conclusion: Our data suggest a direct or indirect correlation between the expression of OCT4B1 and HSP90 gene family. However, OCT4B1 expression was not strongly correlated with the expression of HSP70 and HSP60 gene families.

  17. The Long Noncoding RNA MALAT-1 Is Highly Expressed in Ovarian Cancer and Induces Cell Growth and Migration.

    Directory of Open Access Journals (Sweden)

    Yanqing Zhou

    Full Text Available Metastasis associated in lung adenocarcinoma transcript-1 (MALAT-1 is overexpressed during cancer progression and promotes cell migration and invasion in many solid tumors. However, its role in ovarian cancer remains poorly understood.Expressions of MALAT-1 were detected in 37 normal ovarian tissues and 45 ovarian cancer tissues by reverse transcription polymerase chain reaction (RT-PCR. Cell proliferation was observed by CCK-8 assay; Flow cytometry was used to measure cell cycle and apoptosis; Cell migration was detected by transwell migration and invasion assay. In order to evaluate the function of MALAT-1, shRNA combined with DNA microarray and Functional enrichment analysis were performed to determine the transcriptional effects of MALAT-1 silencing in OVCAR3 cells. RNA and protein expression were measured by qRT-PCR and Western blotting, respectively.We found that upregulation of MALAT-1 mRNA in ovarian cancer tissues and enhanced MALAT-1 expression was associated with FIGO stage. Knockdown of MALAT-1 expression in OVCAR3 cells inhibited cell proliferation, migration, and invasion, leading to G0/G1 cell cycle arrest and apoptosis. Overexpressed MALAT-1 expression in SKOV3 cells promoted cell proliferation, migration and invasion. Downregulation of MALAT-1 resulted in significant change of gene expression (at least 2-fold in 449 genes, which regulate proliferation, cell cycle, and adhesion. As a consequence of MALAT-1 knockdown, MMP13 protein expression decreased, while the expression of MMP19 and ADAMTS1 was increased.The present study found that MALAT-1 is highly expressed in ovarian tumors. MALAT-1 promotes the growth and migration of ovarian cancer cells, suggesting that MALAT-1 may be an important contributor to ovarian cancer development.

  18. High CD10 expression in lymph node metastases from surgically treated prostate cancer independently predicts early death.

    Science.gov (United States)

    Fleischmann, Achim; Rocha, Carla; Saxer-Sekulic, Nikolina; Zlobec, Inti; Sauter, Guido; Thalmann, George N

    2011-06-01

    Patients with nodal positive prostate cancers are an important cohort with poorly defined risk factors. CD10 is a cell surface metallopeptidase that has been suggested to play a role in prostate cancer progression. CD10 expression was evaluated in 119 nodal positive prostate cancer patients using tissue microarrays constructed from primary tumors and lymph node metastases. All patients underwent radical prostatectomy and standardized extended lymphadenectomy. They had no neoadjuvant therapy and received deferred androgen deprivation. In the primary tumor, high CD10 expression was significantly associated with earlier death from disease when compared with low CD10 expression (5-year survival 73.7% vs. 91.8%; p = 0.043). In the metastases, a high CD10 expression was significantly associated with larger total size of metastases (median 11.4 vs. 6.5 mm; p = 0.015), earlier death of disease (5-year survival 71.5% vs. 87.3%; p = 0.017), and death of any cause (5-year survival 70.0% vs. 87.2%; p = 0.001) when compared with low CD10 expression. CD10 expression in the metastases added independent prognostic information for overall survival (p = 0.029) after adjustment for Gleason score of the primary tumor, nodal tumor burden, and resection margins. In conclusion, a high CD10 expression in prostate cancer predicts early death. This information is inherent in the primary tumors and in the lymph node metastases and might help to personalize patient management.

  19. Treadmill exercise promotes interleukin 15 expression in skeletal muscle and interleukin 15 receptor alpha expression in adipose tissue of high-fat diet rats.

    Science.gov (United States)

    Yang, Hongtao; Chang, Jinrui; Chen, Wenjia; Zhao, Lei; Qu, Bo; Tang, Chaoshu; Qi, Yongfen; Zhang, Jing

    2013-06-01

    Interleukin 15 (IL-15) has recently been proposed as a myokine involved in regulating lipid metabolism. We investigated the effect of exercise training on IL-15 content in skeletal muscle and expression of IL-15 receptor (IL-15R) in adipose tissue of obese rats. After 12 weeks of a high-fat diet, obese rats underwent treadmill running at 26 m/min (60 min each, 5 days/week for 8 weeks). High-fat diet induced obesity, with increased body weight, body fat, and lipid profile. The level of IL-15 immunoreactivity (IL-15-ir) in plasma and gastrocnemius muscle was lower in obese than control rats, and the mRNA level of IL-15 in gastrocnemius muscle was markedly decreased. The mRNA and protein levels of IL-15R in adipose tissue were markedly lower in obese rats. Compared with sedentary obese rats, treadmill running showed decreased body weight and elevated mRNA expression of IL-15 in muscle and elevated IL-15-ir level in plasma and muscle. The mRNA and protein level of IL-15R were increased in adipose tissue in treadmill running obese rats. Our results showed that exercise training improve obesity and reversed the downregulation of the IL-15 in muscle and IL-15R in adipose tissue induced by high-fat diet.

  20. Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH

    DEFF Research Database (Denmark)

    Sørensen, Brita Singers; Alsner, Jan; Overgaard, Jens

    2007-01-01

    BACKGROUND: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression......Ha and FaDu(DD) cells Ca9 and LOX reached the highest level of expression at 1% oxygen. In FaDu(DD) cells, a pH of 6.5 had a medium suppression effect on the hypoxia induced expression of Ca9. pH 6.3 resulted in severe suppression of expression for Ca9 and LOX in both SiHa and FaDu(DD). Glut1 and LDH-A had...

  1. Construction of a high-EGFR expression cell line and its biological ...

    African Journals Online (AJOL)

    USER

    African Journal of Biotechnology Vol. 9(30), pp. 4674-4680, 26 July, 2010 ... cancer cells and plays an important role in regulating cellular proliferation, differentiation and .... Construction of a EGFR eukaryotic expression vector. The plasmid ...

  2. Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH

    DEFF Research Database (Denmark)

    Sørensen, Brita Singers; Alsner, Jan; Overgaard, Jens;

    2007-01-01

    BACKGROUND: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression......Ha and FaDu(DD) cells Ca9 and LOX reached the highest level of expression at 1% oxygen. In FaDu(DD) cells, a pH of 6.5 had a medium suppression effect on the hypoxia induced expression of Ca9. pH 6.3 resulted in severe suppression of expression for Ca9 and LOX in both SiHa and FaDu(DD). Glut1 and LDH-A had...

  3. Gene expression identifies heterogeneity of metastatic propensity in high-grade soft tissue sarcomas

    DEFF Research Database (Denmark)

    Skubitz, Keith M; Francis, Princy; Skubitz, Amy P N;

    2012-01-01

    Metastatic propensity of soft tissue sarcoma (STS) is heterogeneous and may be determined by gene expression patterns that do not correlate well with morphology. The authors have reported gene expression patterns that distinguish 2 broad classes of clear cell renal carcinoma (ccRCC-gene set......), and other patterns that can distinguish heterogeneity of serous ovarian carcinoma (OVCA-gene set) and aggressive fibromatosis (AF-gene set); however, clinical follow-up data were not available for these samples....

  4. Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice.

    Science.gov (United States)

    Püschel, A W; Gruss, P; Westerfield, M

    1992-03-01

    Despite obvious differences in the patterns of early embryonic development, vertebrates share a number of developmental mechanisms and control genes, suggesting that they use similar genetic programs at some stages of development. To examine this idea, we isolated and characterized one such gene, pax-6, a member of the pax gene family, from the zebrafish Brachydanio rerio and determined the evolutionary conservation in the structure and expression of this gene by comparison to its homolog in mice. We found two alternatively spliced forms of the zebrafish pax-6 message. Sequence and expression pattern of the zebrafish pax-6 gene are remarkably similar to its murine homolog. pax-6 expression begins during early neurulation. A stripe of cells in the neuroectoderm, including the prospective diencephalon and a part of the telencephalon, expresses pax-6 as well as the hindbrain and the ventral spinal cord extending from the level of the first rhombomere to the posterior end of the CNS. During later development more limited regions of the brain including the eye, the olfactory bulb and the pituitary gland express pax-6. Cells at the midbrain-hindbrain junction express eng genes and are separated from the neighboring pax-6 regions by several cells that express neither gene, indicating a complex subdivision of this region. pax-6 expression appears during processes when cell-to-cell signalling is thought to be important, for example during induction of the eye and regionalization of the spinal cord and brain, suggesting that it may be one component mediating the response to inductive interactions.

  5. High-level expression and purification of soluble bioactive recombinant human heparin-binding epidermal growth factor in Escherichia coli.

    Science.gov (United States)

    Khalili, Mostafa; Soleyman, Mohammad Reza; Baazm, Maryam; Beyer, Cordian

    2015-07-01

    Heparin-binding epidermal growth factor (HB-EGF) is a member of highly conserved superfamily of proteins that has potential mitogenic activity and stimulates differentiation and migration of various cell types. Since HB-EGF has three intra-molecular disulfide bonds, a high expression pattern of active HB-EGF in an E. coli expression system was not successfully established. The aim of this study was to increase production of soluble bioactive recombinant human HB-EGF in E. coli by modifying growth conditions and codon optimization. The open reading frame codons of human HB-EGF were optimized to achieve high level expression in E. coli. The optimized codon was amplified, cloned into plasmid pET-32a, and transformed into E. coli BL21 for further expression. The cultivation parameters (temperature and inducer) were optimized to produce a high yield of soluble HB-EGF. The fusion protein was purified by Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Amethylthiazole tetrazolium assay was used to evaluate the bioactivity of the produced recombinant protein. After codon optimization, the codon adaptation index (CAI) was increased from 0.255 in native gene to 0.829 using the optimized sequence. By lowering the temperature to 22°C and the inducer to 0.4 μM, we obtained 35% soluble expression of recombinant and biologically active human HB-EGF. Our data demonstrate that codon optimization increases the yield of HB-EGF in an E. coli expression system. Furthermore, the chosen modifications in cell culturing increase the solubility of recombinant human HB-EGF.

  6. High levels of protein expression using different mammalian CMV promoters in several cell lines.

    Science.gov (United States)

    Xia, Wei; Bringmann, Peter; McClary, John; Jones, Patrick P; Manzana, Warren; Zhu, Ying; Wang, Soujuan; Liu, Yi; Harvey, Susan; Madlansacay, Mary Rose; McLean, Kirk; Rosser, Mary P; MacRobbie, Jean; Olsen, Catherine L; Cobb, Ronald R

    2006-01-01

    With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.

  7. Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker.

    Science.gov (United States)

    Bradley, Luke H; Bricken, Michael L; Randle, Charlotte

    2011-02-01

    Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: all library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.

  8. Chinese High School Students' Perceptions of Freedom of Expression: Implications for Researching Emerging Civil Liberties in Global Educational Contexts

    Science.gov (United States)

    Torres, Mario S.; Qin, Lixia

    2017-01-01

    This study explored attitudes and perceptions of Chinese high school students regarding freedom of expression in their country. A survey capturing perceptions over various forms of free speech (e.g., student publication, dress code) was administered to a sample of 838, which included students from both urban and rural areas within Shaanxi Province…

  9. Effect of high-intensity training on exercise-induced gene expression specific to ion homeostasis and metabolism

    DEFF Research Database (Denmark)

    Nordsborg, Nikolai; Bangsbo, Jens; Pilegaard, Henriette

    2003-01-01

    Changes in gene expression during recovery from high-intensity, intermittent, one-legged exercise were studied before and after 5.5 wk of training. Genes related to metabolism, as well as Na+, K+, and pH homeostasis, were selected for analyses. After the same work was performed before and after...

  10. Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Assing, K; Jensen, Lone Hummelshøj;

    2006-01-01

    Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells...

  11. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    Science.gov (United States)

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  12. A cholesterol-free, high-fat diet suppresses gene expression of cholesterol transporters in murine small intestine

    NARCIS (Netherlands)

    den Bosch, Heleen M. de Vogel-van; de Wit, Nicole J. W.; Hooiveld, Guido J. E. J.; Vermeulen, Hanneke; van der Veen, Jelske N.; Houten, Sander M.; Kuipers, Folkert; Mueller, Michael; van der Meer, Roelof

    2008-01-01

    A cholesterol-free, high-fat diet suppresses gene expression of cholesterol transporters in murine small intestine. Am J Physiol Gastrointest Liver Physiol 294: G1171-G1180, 2008. First published March 20, 2008; doi:10.1152/ajpgi.00360.2007.-Transporters present in the epithelium of the small intest

  13. Effect of metformin on proliferation and related genes expression of human osteoblast MG63 under high glucose

    Institute of Scientific and Technical Information of China (English)

    曹小俊

    2013-01-01

    Objective To study the effect of metformin on proliferation and related genes expression of human osteoblast.Methods The proliferation of MG63 cells under high glucose intervened with metformin was measured by CCK-8 assay. The activity of intracellular alkaline phosphatase

  14. High-affinity glucose transport in Aspergillus nidulans is mediated by the products of two related but differentially expressed genes.

    Directory of Open Access Journals (Sweden)

    Josep V Forment

    Full Text Available Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation.

  15. Martian Valley Networks and Associated Fluvial Features as Seen by the Mars Express High Resolution Stereo Camera (HRSC)

    Science.gov (United States)

    Jaumann, R.; Reiss, D.; Frei, S.; Scholten, F.; Gwinner, K.; Roatsch, T.; Matz, K.-D.; Hauber, E.; Mertens, V.; Hoffmann, H.; Head, J. W., III; Hiesinger, H.; Carr, M. H.; Neukum, G.; HRSC Co-Investigator Team

    2005-03-01

    In High Resolution Stereo Camera (HRSC) images of the Mars Express Mission a 130 km long inner channel is Lybia Montes. Based on HRSC stereo information we were able to determine the depth of this inner structure and thus we could estimate the discharge in the inner channel.

  16. Comparative analysis of codon usage patterns and identification of predicted highly expressed genes in five Salmonella genomes

    Directory of Open Access Journals (Sweden)

    Mondal U

    2008-01-01

    Full Text Available Purpose: To anlyse codon usage patterns of five complete genomes of Salmonella , predict highly expressed genes, examine horizontally transferred pathogenicity-related genes to detect their presence in the strains, and scrutinize the nature of highly expressed genes to infer upon their lifestyle. Methods: Protein coding genes, ribosomal protein genes, and pathogenicity-related genes were analysed with Codon W and CAI (codon adaptation index Calculator. Results: Translational efficiency plays a role in codon usage variation in Salmonella genes. Low bias was noticed in most of the genes. GC3 (guanine cytosine at third position composition does not influence codon usage variation in the genes of these Salmonella strains. Among the cluster of orthologous groups (COGs, translation, ribosomal structure biogenesis [J], and energy production and conversion [C] contained the highest number of potentially highly expressed (PHX genes. Correspondence analysis reveals the conserved nature of the genes. Highly expressed genes were detected. Conclusions: Selection for translational efficiency is the major source of variation of codon usage in the genes of Salmonella . Evolution of pathogenicity-related genes as a unit suggests their ability to infect and exist as a pathogen. Presence of a lot of PHX genes in the information and storage-processing category of COGs indicated their lifestyle and revealed that they were not subjected to genome reduction.

  17. High mobility group A1 protein expression reduces the sensitivity of colon and thyroid cancer cells to antineoplastic drugs.

    Science.gov (United States)

    D'Angelo, Daniela; Mussnich, Paula; Rosa, Roberta; Bianco, Roberto; Tortora, Giampaolo; Fusco, Alfredo

    2014-11-20

    Development of resistance to conventional drugs and novel biological agents often impair long-term chemotherapy. HMGA gene overexpression is often associated with antineoplastic drug resistance and reduced survival. Inhibition of HMGA expression in thyroid cancer cells reduces levels of ATM protein, the main cellular sensor of DNA damage, and enhances cellular sensitivity to DNA-damaging agents. HMGA1 overexpression promotes chemoresistance to gemcitabine in pancreatic adenocarcinoma cells through an Akt-dependent mechanism. To elucidate the role of HMGA1 proteins in chemoresistance we analyzed resistance to conventional drugs and targeted therapies of human colon carcinoma cells (GEO) that are sensitive to the epidermal growth factor receptor inhibitor cetuximab, and express minimal levels of HMGA1 and cetuximab-resistant (GEO CR) cells expressing high HMGA1 protein levels. GEO CR cells were less sensitive than GEO cells to cetuximab and 5-fluorouracil. GEO CR cells silenced for HMGA1 expression were more susceptible than empty vector-transfected cells to the drugs' cytotoxicity. Similar results were obtained with anaplastic thyroid carcinoma cells expressing or not HMGA1 proteins, treated with doxorubicin or the HDAC inhibitor LBH589. Finally, HMGA1 overexpression promoted the DNA-damage response and stimulated Akt phosphorylation and prosurvival signaling. Our findings suggest that the blockage of HMGA1 expression is a promising approach to enhance cancer cell chemosensitivity, since it could increase the sensitivity of cancer cells to antineoplastic drugs by inhibiting the survival signal and DNA damage repair pathways.

  18. High-frequency stimulation induces gradual immediate early gene expression in maturing adult-generated hippocampal granule cells.

    Science.gov (United States)

    Jungenitz, Tassilo; Radic, Tijana; Jedlicka, Peter; Schwarzacher, Stephan W

    2014-07-01

    Increasing evidence shows that adult neurogenesis of hippocampal granule cells is advantageous for learning and memory. We examined at which stage of structural maturation and age new granule cells can be activated by strong synaptic stimulation. High-frequency stimulation of the perforant pathway in urethane-anesthetized rats elicited expression of the immediate early genes c-fos, Arc, zif268 and pCREB133 in almost 100% of mature, calbindin-positive granule cells. In contrast, it failed to induce immediate early gene expression in immature doublecortin-positive granule cells. Furthermore, doublecortin-positive neurons did not react with c-fos or Arc expression to mild theta-burst stimulation or novel environment exposure. Endogenous expression of pCREB133 was increasingly present in young cells with more elaborated dendrites, revealing a close correlation to structural maturation. Labeling with bromodeoxyuridine revealed cell age dependence of stimulation-induced c-fos, Arc and zif268 expression, with only a few cells reacting at 21 days, but with up to 75% of cells activated at 35-77 days of cell age. Our results indicate an increasing synaptic integration of maturing granule cells, starting at 21 days of cell age, but suggest a lack of ability to respond to activation with synaptic potentiation on the transcriptional level as long as immature cells express doublecortin. © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli.

    Science.gov (United States)

    Liu, Xinxin; Huang, Anliang; Luo, Dan; Liu, Haipeng; Han, Huzi; Xu, Yang; Liang, Peng

    2015-05-01

    The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification.

  20. Directed engineering of a high-expression chimeric transgene as a strategy for gene therapy of hemophilia A.

    Science.gov (United States)

    Doering, Christopher B; Denning, Gabriela; Dooriss, Kerry; Gangadharan, Bagirath; Johnston, Jennifer M; Kerstann, Keith W; McCarty, David A; Spencer, H Trent

    2009-07-01

    Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

  1. Multiple cancer/testis antigens are preferentially expressed in hormone-receptor negative and high-grade breast cancers.

    Directory of Open Access Journals (Sweden)

    Yao-Tseng Chen

    Full Text Available BACKGROUND: Cancer/testis (CT antigens are protein antigens normally expressed only in germ cells of testis, and yet are expressed in a proportion of a wide variety of human cancers. CT antigens can elicit spontaneous immune responses in cancer patients with CT-positive cancers, and CT antigen-based therapeutic cancer vaccine trials are ongoing for "CT-rich" tumors. Although some previous studies found breast cancer to be "CT-poor", our recent analysis identified increased CT mRNA transcripts in the ER-negative subset of breast cancer. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we performed a comprehensive immunohistochemical study to investigate the protein expression of eight CT genes in 454 invasive ductal carcinomas, including 225 ER/PR/HER2-negative (triple-negative carcinomas. We found significantly more frequent expression of all eight CT antigens in ER-negative cancers, and five of them--MAGEA, CT7, NY-ESO-1, CT10 and CT45, were expressed in 12-24% of ER-negative cancers, versus 2-6% of ER-positive cancers (p2 cm. CONCLUSIONS/SIGNIFICANCE: CT antigens are preferentially expressed in hormone receptor-negative and high-grade breast cancer. Considering the limited treatment options for ER/PR/HER2 triple-negative breast cancer, the potential of CT-based immunotherapy should be explored.

  2. Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study.

    Science.gov (United States)

    Marusic, Carla; Vitale, Alessandro; Pedrazzini, Emanuela; Donini, Marcello; Frigerio, Lorenzo; Bock, Ralph; Dix, Philip J; McCabe, Matthew S; Bellucci, Michele; Benvenuto, Eugenio

    2009-08-01

    The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10 years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to boost gene expression, and protein fusions to improve recombinant protein stability and accumulation. Thus, several HIV/SIV antigens and neutralizing anti-HIV antibodies have recently been successfully expressed in plants by stable nuclear or plastid transformation, and by transient expression systems based on plant virus vectors or Agrobacterium-mediated infection. The current article gives an overview of plant expressed HIV antigens and antibodies and provides an account of the use of different strategies aimed at increasing the expression of the accessory multifunctional HIV-1 Nef protein in transgenic plants.

  3. Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    Wen-Song Ge; Jian-Xin Wu; Jian-Gao Fan; Yao-Jun Wang; Ying-Wei Chen

    2011-01-01

    AIM: To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA. METHODS: Hepatic fibrosis in rats was induced through serial subcutaneous injections of dimethylnitrosamine, and expression of HMGB1 was detected by immunohistochemistry. HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000. HMGB1 expression was evaluated by real-time polymerase chain reaction (PCR) and Western blotting analysis. Expression of α-smooth muscle actin (α-SMA) and collagen types Ⅰ and Ⅲ was evaluated by real-time PCR. Cell proliferation and the cell cycle were determined using the methyl thiazolyl tetrazolium method. Finally, collagen content in HSC supernatant was evaluated by an enzyme-linked immunosorbent assay. RESULTS: The results showed that HMGB1 was upregulated during liver fibrosis and that its expression was closely correlated with the deposition of collagen. siRNA molecules were successfully transfected into HSCs and induced inhibition of HMGB1 expression in a time-dependent manner. Moreover, HMGB1 siRNA treatment inhibited synthesis of α-SMA and collagen types Ⅰ and Ⅲ in transfected HSCs. CONCLUSION: This study suggests a significant functional role for HMGB1 in the development of liver fibrosis. It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.

  4. Identification of differentially expressed genes in esophageal cancer through SSH in com- bination with high throughput reverse Northern screening

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To understand the molecular mechanisms of carcinogenesis of esophagus and to isolate genes with different expression levels in esophageal cancer, suppression subtractive hybridization (SSH) was combined with PCR-based cDNA synthesis and reverse Northern on the cancer tissues and matched almost normal mucosa using 5 microgram of total RNA as starting marterial. Eight genes were found expressed differentially in esophageal cancer, in which 5 were known genes and 3 were novel ones; and 6 were down-regulated in cancer tissues, while 2 were up-regulated; 6 were of mid-high abundance and 2 were of low abundance in esophagus. The results revealed that alteration in expression level of multiple genes underlied the initiation and development of esophageal cancer. The differentially expressed genes identified in this study such as liporcotinⅠ, cystatin A, cystatin B, cytokeratin 13 may play roles in dedifferentiation, transformation and malignant proliferation of esophageal cancer. The combination of SSH with PCR-based double- strand cDNA synthesis and high throughput reverse Northern screening is an efficient way to isolate differentially expressed genes from microgram of total RNA.

  5. The Myocyte Expression of Adiponectin Receptors and PPARδ Is Highly Coordinated and Reflects Lipid Metabolism of the Human Donors

    Directory of Open Access Journals (Sweden)

    Anna-Maria Ordelheide

    2011-01-01

    Full Text Available Muscle lipid oxidation is stimulated by peroxisome proliferator-activated receptor (PPAR δ or adiponectin receptor signalling. We studied human myocyte expression of the PPARδ and adiponectin receptor genes and their relationship to lipid parameters of the donors. The mRNA levels of the three adiponectin receptors, AdipoR1, AdipoR2, and T-cadherin, were highly interrelated (r≥0.91. However, they were not associated with GPBAR1, an unrelated membrane receptor. In addition, the adiponectin receptors were positively associated with PPARδ expression (r≥0.75. However, they were not associated with PPARα. Using stepwise multiple linear regression analysis, PPARδ was a significant determinant of T-cadherin (P=.0002. However, pharmacological PPARδ activation did not increase T-cadherin expression. The myocyte expression levels of AdipoR1 and T-cadherin were inversely associated with the donors' fasting plasma triglycerides (P<.03. In conclusion, myocyte expression of PPARδ and the adiponectin receptors are highly coordinated, and this might be of relevance for human lipid metabolism in vivo.

  6. Efficient and reproducible generation of high-expressing, stable human cell lines without need for antibiotic selection

    Directory of Open Access Journals (Sweden)

    Kewes Helmut

    2008-02-01

    Full Text Available Abstract Background Human cell lines are the most innovative choice of host cell for production of biopharmaceuticals since they allow for authentic posttranslational modification of therapeutic proteins. We present a new method for generating high and stable protein expressing cell lines based on human amniocytes without the requirement of antibiotic selection. Results Primary amniocytes from routine amniocentesis samples can be efficiently transformed with adenoviral functions resulting in stable human cell lines. Cotransfection of the primary human amniocytes with a plasmid expressing adenoviral E1 functions plus a second plasmid containing a gene of interest resulted in permanent cell lines expressing up to 30 pg/cell/day of a fully glycosylated and sialylated protein. Expression of the gene of interest is very stable for more than 90 passages and, importantly, was achieved in the absence of any antibiotic selection. Conclusion We describe an improved method for developing high protein expressing stable human cell lines. These cell lines are of non-tumor origin, they are immortalized by a function not oncogenic in human and they are from an ethically accepted and easily accessible cell source. Since the cell can be easily adapted to growth in serum-free and chemically defined medium they fulfill the requirements of biopharmaceutical production processes.

  7. High-level diterpene production by transient expression in Nicotiana benthamiana

    Science.gov (United States)

    2013-01-01

    Background Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis. Results Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2. Conclusion With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants. PMID:24330621

  8. Locus coeruleus galanin expression is enhanced after exercise in rats selectively bred for high capacity for aerobic activity.

    Science.gov (United States)

    Murray, Patrick S; Groves, Jessica L; Pettett, Brett J; Britton, Steven L; Koch, Lauren G; Dishman, Rod K; Holmes, Philip V

    2010-12-01

    The neuropeptide galanin extensively coexists with norepinephrine in locus coeruleus (LC) neurons. Previous research in this laboratory has demonstrated that unlimited access to activity wheels in the home cage increases mRNA for galanin (GAL) in the LC, and that GAL mediates some of the beneficial effects of exercise on brain function. To assess whether capacity for aerobic exercise modulates this upregulation in galanin mRNA, three heterogeneous rat models were tested: rats selectively bred for (1) high intrinsic (untrained) aerobic capacity (High Capacity Runners, HCR) and (2) low intrinsic aerobic capacity (Low Capacity Runners, LCR) and (3) unselected Sprague-Dawley (SD) rats with and without free access to running wheels for 3 weeks. Following this exercise protocol, mRNA for tyrosine hydroxylase (TH) and GAL was measured in the LC. The wheel running distances between the three models were significantly different, and age contributed as a significant covariate. Both selection and wheel access condition significantly affected GAL mRNA expression, but not TH mRNA expression. GAL was elevated in exercising HCR and SD rats compared to sedentary rats while LCR rats did not differ between conditions. Overall running distance significantly correlated with GAL mRNA expression, but not with TH mRNA expression. No strain differences in GAL or TH gene expression were observed in sedentary rats. Thus, intrinsic aerobic running capacity influences GAL gene expression in the LC only insofar as actual running behavior is concerned; aerobic capacity does not influence GAL expression in addition to changes associated with running.

  9. High expression of hexokinase domain containing 1 is associated with poor prognosis and aggressive phenotype in hepatocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zijian; Huang, Shanzhou [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Wang, Huanyu [Department of Thyroid and Breast Surgery, Nanshan District People’s Hospital, Shenzhen, 518000 (China); Wu, Jian [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Chen, Dong [Department of Biliopancreatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Peng, Baogang [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China); Zhou, Qi, E-mail: hnzhouqi@163.com [Department of Hepatic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080 (China)

    2016-06-10

    Rapid progress and metastasis remain the major treatment failure modes of hepatocarcinoma (HCC). Unfortunately, the underlying molecular mechanisms of hepatoma cell proliferation and migration are poorly understood. Metabolic abnormalities play critical roles in tumorigenesis and progression. Hexokinase domain containing 1 (HKDC1) catalyzes the phosphorylation of glucose. However, the functions and mechanisms of HKDC1 in cancer remain unknown. In this study, real-time RT-PCR and Western blotting assays were used to detect the HKDC1 expression levels in HCC tissues and cell lines. The Oncomine™ Cancer Microarray Database was applied to analysis the correlations between HKDC1 expression and HCC clinical characteristics. MTT and Transwell migration assays were performed to determine the functions of HKDC1 in HCC cells. The effect of HKDC1 on Wnt/β-catenin signaling pathway was assessed using Western blotting assay. In this study, we found that HKDC1 expression levels were elevated in HCC tissues compared with the adjacent tissues. HCC patients with high expression levels of HKDC1 had poor overall survival (OS). Furthermore, higher HKDC1 levels also predicted a worse OS of patients within solitary, elevated pre-operated serum alpha fetoprotein (AFP) level and higher tumor diameter. Moreover, silencing HKDC1 suppressed HCC cells proliferation and migration in vitro. Downregulated HKDC1 expression repressed β-Catenin and c-Myc expression, which indicates that silencing HKDC1 may reduce proliferation and migration via inhibiting the Wnt/β-catenin signaling pathway in HCC. In summary, HKDC1 provides further insight into HCC tumor progression and may provide a novel prognostic biomarker and therapeutic target for HCC treatment. -- Highlights: •HKDC1 is upregulated in HCC. •Patients with high HKDC1 expressions perform worse OS. •Silencing HKDC1 suppresses proliferation and migration. •Silencing HKDC1 represses Wnt/β-catenin signaling pathway.

  10. Gene expression profile differences in high and low metastatic human ovarian cancer cell lines by gene chip

    Institute of Scientific and Technical Information of China (English)

    许沈华; 牟瀚舟; 吕桂泉; 朱赤红; 羊正炎; 高永良; 楼洪坤; 刘祥麟; 程勇; 杨文

    2002-01-01

    Objectives To study the difference between gene expressions of high (H0-8910PM) and low (HO-8910) metastatic human ovarian carcinoma cell lines and screen novel associated genes by cDNA microarray. Methods cDNA retro-transcribed from equal quantities of mRNA derived from high and low metastatic tumor cells or normal ovarian tissues were labeled with Cy5 and Cy3 fluorescein as probes. The mixed probe was hybridized with two pieces of BioDoor 4096 double dot human whole gene chip and scanned with a ScanArray 3000 laser scanner. The acquired image was analyzed by ImaGene 3.0 software. Results A total of 355 genes with expression levels more than 3 times larger were found by comparing the HO-8910 cell with normal ovarian epithelial cells. A total of 323 genes with expression levels more than 3 times larger in HO-8910PM cells compared to normal ovarian epithelium cells were also detected. A total of 165 genes whose expression levels were more than two times those of HO-8910PM cells compared to their mother cell line (HO-8910) were detected. Twenty-one genes with expression levels >3 times were found from comparison of these two tumor cell lines.Conclusions cDNA microarray techniques are effective in screening differential gene expression between two human ovarian cancer cell lines (H0-8910PM; HO-8910) and normal ovarian epithelial cells. These genes may be related to the genesis and development of ovarian carcinoma. Analysis of the human ovarian cancer gene expression profile with cDNA microarray may help in gene diagnosis, treatment and prevention.

  11. Engineering of a wheat germ expression system to provide compatibility with a high throughput pET-based cloning platform.

    Science.gov (United States)

    Zhao, Li; Zhao, Kate Q; Hurst, Robin; Slater, Michael R; Acton, Thomas B; Swapna, G V T; Shastry, Ritu; Kornhaber, Gregory J; Montelione, Gaetano T

    2010-09-01

    Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT((R)) SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354-359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two (15)N-, (13)C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline.

  12. High ambient temperature increases 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy")-induced Fos expression in a region-specific manner.

    Science.gov (United States)

    Hargreaves, G A; Hunt, G E; Cornish, J L; McGregor, I S

    2007-03-16

    3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") is a popular drug that is often taken under hot conditions at dance clubs. High ambient temperature increases MDMA-induced hyperthermia and recent studies suggest that high temperatures may also enhance the rewarding and prosocial effects of MDMA in rats. The present study investigated whether ambient temperature influences MDMA-induced expression of Fos, a marker of neural activation. Male Wistar rats received either MDMA (10 mg/kg i.p.) or saline, and were placed in test chambers for 2 h at either 19 or 30 degrees C. MDMA caused significant hyperthermia at 30 degrees C and a modest hypothermia at 19 degrees C. The 30 degrees C ambient temperature had little effect on Fos expression in vehicle-treated rats. However MDMA-induced Fos expression was augmented in 15 of 30 brain regions at the high temperature. These regions included (1) sites associated with thermoregulation such as the median preoptic nucleus, dorsomedial hypothalamus and raphe pallidus, (2) the supraoptic nucleus, a region important for osmoregulation and a key mediator of oxytocin and vasopressin release, (3) the medial and central nuclei of the amygdala, important in the regulation of social and emotional behaviors, and (4) the shell of the nucleus accumbens and (anterior) ventral tegmental area, regions associated with the reinforcing effects of MDMA. MDMA-induced Fos expression was unaffected by ambient temperature at many other sites, and was diminished at high temperature at one site (the islands of Calleja), suggesting that the effect of temperature on MDMA-induced Fos expression was not a general pharmacokinetic effect. Overall, these results indicate that high temperatures accentuate key neural effects of MDMA and this may help explain the widespread use of the drug under hot conditions at dance parties as well as the more hazardous nature of MDMA taken under such conditions.

  13. Preferential cytotoxicity of bortezomib toward highly malignant human liposarcoma cells via suppression of MDR1 expression and function

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Yamei; Wang, Lingxian; Wang, Lu; Wu, Xuefeng; Wu, Xudong [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093 (China); Gu, Yanhong; Shu, Yongqian [Department of Clinical Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029 (China); Sun, Yang [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093 (China); Shen, Yan, E-mail: shenyan@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093 (China); Xu, Qiang, E-mail: molpharm@163.com [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093 (China)

    2015-02-15

    Liposarcoma is the most common soft tissue sarcoma with a high risk of relapse. Few therapeutic options are available for the aggressive local or metastatic disease. Here, we report that the clinically used proteasome inhibitor bortezomib exhibits significantly stronger cytotoxicity toward highly malignant human liposarcoma SW872-S cells compared with its parental SW872 cells, which is accompanied by enhanced activation of apoptotic signaling both in vitro and in vivo. Treatment of cells with Jun-N-terminal kinase (JNK) inhibitor SP60015 or the translation inhibitor cycloheximide ameliorated this enhanced apoptosis. Bortezomib inhibited MDR1 expression and function more effectively in SW872-S cells than in SW872 cells, indicating that the increased cytotoxicity relies on the degree of proteasome inhibition. Furthermore, the pharmacological or genetic inhibition of sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2, which is highly expressed in SW872-S cells, resulted in partial reversal of cell growth inhibition and increase of MDR1 expression in bortezomib-treated SW872-S cells. These results show that bortezomib exhibits preferential cytotoxicity toward SW872-S cells possibly via highly expressed SERCA2-associated MDR1 suppression and suggest that bortezomib may serve as a potent agent for treating advanced liposarcoma. - Highlights: • We compare the cytotoxicity of different drugs between SW872-S and SW872 cells. • Highly malignant liposarcoma cells SW872-S show hypersensitivity to bortezomib. • Apoptotic signaling is robustly enhanced in bortezomib-treated SW872-S cells. • Bortezomib has strong suppression on MDR1 expression and function in SW872-S cells. • Inhibition of SERCA2 protects SW872-S cells from bortezomib.

  14. Upregulation of URI/RMP gene expression in cervical cancer by high-throughput tissue microarray analysis.

    Science.gov (United States)

    Gu, Junxia; Li, Xiaoyun; Liang, Yuting; Qiao, Longwei; Ran, Deyuan; Lu, Yaojuan; Li, Xingang; Wei, Wenxiang; Zheng, Qiping

    2013-01-01

    URI, or RMP, is a RNA polymerase II subunit RPB5-associated protein known to play essential roles in ubiquitination and transcription. Recently, we and others have shown that URI/RMP is also important for progression of hepatocellular carcinoma, ovarian, and prostate cancers. To identify the mechanistic basis of URI/RMP during multiple cellular processes, we investigated URI/RMP expression in a tissue microarray (TMA) containing multiple normal human tissues. The results showed that URI/RMP is ubiquitously but differentially expressed in these human tissues which partially explains its multiple cellular functions. To elucidate the role of URI/RMP during oncogenesis of multiple malignancies, especially the tumors of reproductive system, we analyzed URI/RMP expression in a TMA containing multiple reproductive system tumors. We did not observe significant difference of URI/RMP expression between cancerous and adjacent tissues of the prostate, breast, ovarian, and endometrial cancers. However, increased URI/RMP expression was observed in two of the three cases of cervical SCC (squamous cell carcinoma) cells compared to their adjacent epithelial cells. Moreover, we detected significantly upregulated URI/RMP expression not only in cervical cancers but also in pre-cancerous CINs (cervical intra-epithelial neoplasias) in a TMA that covers the whole spectrum of normal cervix, CINs, and cervical cancers. No difference of URI/RMP expression was observed between CINs and cervical cancers. Given the high risk of CINs (especially CIN3) turning into cervical cancer if left untreated, the increased URI/RMP expression in CINs as well as in cervical cancers suggest a clinical relevance of URI/RMP upon cervical cancer tumorigenesis and worth further investigation.

  15. Immunohistochemical LRIG3 expression in cervical intraepithelial neoplasia and invasive squamous cell cervical cancer: association with expression of tumor markers, hormones, high-risk HPV-infection, smoking and patient outcome

    Directory of Open Access Journals (Sweden)

    A.K. Lindström

    2014-04-01

    Full Text Available The novel biomarker LRIG3 is a member of the LRIG family (LRIG1-3. While LRIG1 has been associated with favorable prognosis and LRIG2 with poor prognosis in invasive cervical cancer, little is known about the role of LRIG3. The aim of this study was to investigate the expression of LRIG3 in invasive cancer and cervical intraepithelial neoplasia (CIN for possible correlation with other tumor markers, to hormones and smoking, as a diagnostic adjunct in CIN, and prognostic value in invasive cancer. Cervical biopsies from 129 patients with invasive squamous cell carcinoma and 170 biopsies showing low grade and high grade CIN, or normal epithelium were stained for LRIG3 and 17 additional tumor markers. Among other variables the following were included: smoking habits, hormonal contraceptive use, serum progesterone, serum estradiol, high-risk HPV-infection, menopausal status and ten-year survival. In CIN, high expression of the tumor suppressors retinoblastoma protein, p53, and p16, and E-cadherin (cell-cell interaction, or low expression of CK10, correlated to LRIG3 expression. In addition, progestogenic contraceptive use correlated to high expression of LRIG3. In invasive cancer there was a correlation between expression of the major tumor promoter c-myc and high LRIG3 expression. High LRIG3 expression correlated significantly to presence of high-risk HPV infection in patients with normal epithelium and CIN. There was no correlation between LRIG3 expression and 10-year survival in patients with invasive cell cervical cancer. LRIG3 expression is associated with a number of molecular events in CIN. Expression also correlates to hormonal contraceptive use. The results on expression of other tumor markers suggest that LRIG3 is influenced by or influences a pattern of tumor markers in cancer and precancerous cells. Further studies are needed to elucidate if LRIG3 expression might be clinically useful.

  16. Characterization of a bZIP gene highly expressed during ripening of the peach fruit.

    Science.gov (United States)

    Lovisetto, Alessandro; Guzzo, Flavia; Tadiello, Alice; Confortin, Enrico; Pavanello, Anna; Botton, Alessandro; Casadoro, Giorgio

    2013-09-01

    A ripening specific bZIP gene of peach was studied by ectopically expressing it in tomato. Two lines, with either a mild or a strong phenotype, respectively, were analyzed in detail. Transgenic fruit morphology was normal, yet the time spent to proceed through the various ripening stages was longer compared to wild type. In agreement with this finding the transgenic berries produced less ethylene, and also had a modified expression of some ripening-related genes that was particularly evident in berries with a strong phenotype. In particular, in the latter fruits polygalacturonase and lipoxygenase genes, but also genes coding for transcription factors (TFs) important for tomato ripening (i.e. TAGL1, CNR, APETALA2a, NOR) did not show the expected decreased expression in the red berries. As regards the RIN gene, its expression continued to increase in both mild and strong lines, and this is in agreement with the dilated ripening times. Interestingly, a metabolomic analysis of berries at various stages of ripening showed that the longer time spent by the transgenic berries to proceed from a stage to another was not due to a slackened metabolism. In fact, the differences in amount of stage-specific marker metabolites indicated that the transgenic berries had a very active metabolism. Therefore, the dilated ripening and the enhanced metabolism of the berries over-expressing the bZIP gene suggest that such gene might regulate ripening by acting as a pacemaker for some of the ripening metabolic pathways.

  17. High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

    Institute of Scientific and Technical Information of China (English)

    Lan-lan ZHANG; Jin-yu SHEN; Cheng-feng LEI; Xiao-ming LI; Qin FANG

    2008-01-01

    Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.

  18. High RBM3 expression in prostate cancer independently predicts a reduced risk of biochemical recurrence and disease progression

    Directory of Open Access Journals (Sweden)

    Bjartell Anders

    2011-09-01

    Full Text Available Abstract Background High expression of the RNA-binding protein RBM3 has previously been found to be associated with good prognosis in breast cancer, ovarian cancer, malignant melanoma and colorectal cancer. The aim of this study was to examine the prognostic impact of immunohistochemical RBM3 expression in prostate cancer. Findings Immunohistochemical RBM3 expression was examined in a tissue microarray with malignant and benign prostatic specimens from 88 patients treated with radical prostatectomy for localized disease. While rarely expressed in benign prostate gland epithelium, RBM3 was found to be up-regulated in prostate intraepithelial neoplasia and present in various fractions and intensities in invasive prostate cancer. High nuclear RBM3 expression was significantly associated with a prolonged time to biochemical recurrence (BCR (HR 0.56, 95% CI: 0.34-0.93, p = 0.024 and clinical progression (HR 0.09, 95% CI: 0.01-0.71, p = 0.021. These associations remained significant in multivariate analysis, adjusted for preoperative PSA level in blood, pathological Gleason score and presence or absence of extracapsular extension, seminal vesicle invasion and positive surgical margin (HR 0.41, 95% CI: 0.19-0.89, p = 0.024 for BCR and HR 0.06, 95% CI: 0.01-0.50, p = 0.009 for clinical progression. Conclusion Our results demonstrate that high nuclear expression of RBM3 in prostate cancer is associated with a prolonged time to disease progression and, thus, a potential biomarker of favourable prognosis. The value of RBM3 for prognostication, treatment stratification and follow-up of prostate cancer patients should be further validated in larger studies.

  19. Change in mRNA expression of sirtuin 1 and sirtuin 3 in cats fed on high fat diet.

    Science.gov (United States)

    Ishikawa, Shingo; Li, Gebin; Takemitsu, Hiroshi; Fujiwara, Megumi; Mori, Nobuko; Yamamoto, Ichiro; Arai, Toshiro

    2013-09-27

    Mammalian sirtuins are homologs to the yeast silent information regulator 2 (Sir2), which is an NAD-dependent deacetylase. Sirtuins are comprised of 7 proteins, and each has different target proteins. Sirtuin 1 (SIRT1) plays important roles in maintaining metabolic functions and immune responses, and SIRT3 protects cells from oxidative stress-induced cell death. Both SIRT1 and SIRT3 are regulated by metabolic status and aging. Hence, SIRT1 and SIRT3 have been researched in metabolic diseases, such as type 2 diabetes mellitus (DM), fatty liver, and heart diseases. Although these diseases have been increasing, there is little information about relation between the diseases and SIRT1 and SIRT3 in cats. Therefore we cloned SIRT1 and SIRT3 cDNA, examined mRNA expression in cat tissues, and investigated the changes in SIRT1 and SIRT3 mRNA expression in peripheral blood leukocyte of cats fed on HFD for 6 weeks. Cat SIRT1 and SIRT3 contained a catalytic core region and showed high sequence homology with other vertebrate SIRT1 (>61.3%) and SIRT3 (>65.9%) amino acids. Real-time polymerase chain reaction analyses revealed that high expression levels were observed in the liver and skeletal muscle for SIRT1 and in the heart for SIRT3 in cats. In addition, both cat SIRT1 and SIRT3 expression levels in the pancreas were different between individuals. Cat SIRT1 mRNA expression in peripheral blood leukocytes was significantly elevated in obese cats fed on HFD (P Cat SIRT1 and SIRT3 genes are highly conserved among vertebrates, and HFD feeding may be related to SIRT1 mRNA expression mechanisms in cat peripheral blood leukocytes.

  20. High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.

    Directory of Open Access Journals (Sweden)

    Yao Nie

    successful expression of pullulanase with lac operator regulation provides an efficient way for enhancement of expression stability and hence high-level production of target protein in recombinant E. coli.

  1. Gene Expression of Stress Proteins and Identification of Molecular Markers of Plant Resistance to High Temperatures and Drought

    OpenAIRE

    L.P. Khokhlova

    2016-01-01

    Molecular biomarkers of plant resistance to both individual and combined action of high tempera-tures (42 °C) and drought have been identified. For this purpose, correlation between gene expression of four stress proteins (non-photosynthetic malic enzyme (TaNADP-ME2), serine-threonine kinase (W55a), dehydrin (DHN14), and lipocalin (TaTIL)) and resistance of eight spring wheat cultivars has been determined for the first time. Gene expression has been studied using the RT-PCR method based on th...

  2. High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Christensen, Søren Tvorup; Voss, Jesper W.; Teilmann, Stefan C.

    2005-01-01

    Taurine, present in high concentrations in various mammalian cells, is essential for regulation of cell volume, cellular oxidative status as well as the cellular Ca2+ homeostasis. Cellular taurine content is a balance between active uptake through the saturable, Na+-dependent taurine transporter...... TauT expression and (iii) long-term exposure to hypertonic taurine medium, i.e., growth medium supplemented with 100 mM taurine, reduces ciliary TauT expression. These results point to an important role of taurine in the regulation of physiological processes located to the primary cilium....

  3. Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays.

    Directory of Open Access Journals (Sweden)

    Jianjun Guo

    Full Text Available BACKGROUND: Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. METHODOLOGY/PRINCIPAL FINDINGS: We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. CONCLUSIONS/SIGNIFICANCE: This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

  4. Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jianjun [ORNL; Morrell-Falvey, Jennifer L [ORNL; Labbe, Jessy L [ORNL; Muchero, Wellington [ORNL; Kalluri, Udaya C [ORNL; Tuskan, Gerald A [ORNL; Chen, Jay [ORNL

    2012-01-01

    Background: Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. Methodology/Principal Findings: We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. Conclusions/Significance: This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

  5. Synthesis and high expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris GS115.

    Science.gov (United States)

    Kang, Lixin; Chen, Xiaomei; Zhai, Chao; Ma, Lixin

    2012-09-01

    A gene, ClCDA, encoding chitin deacetylase from Colletotrichum lindemuthianum, was optimized according to the codon usage bias of Pichia pastoris and synthesized in vitro by overlap extension PCR. It was secretorily expressed in P. pastoris GS115 using the constitutive expression vector pHMB905A. The expression level reached the highest with 110 mg/l culture supernatant after 72 h of methanol induction, which comprised 77.27 U/mg chitin deacetylase activity. SDS-PAGE, mass spectrometry, and deglycosylation assays demonstrated that partial recombinant protein was glycosylated with an apparent molecular mass of 33 kDa. The amino acid sequences of recombinant proteins were confirmed by mass spectrometry.

  6. The expansion of thymopoiesis in neonatal mice is dependent on expression of high mobility group a 2 protein (Hmga2).

    Science.gov (United States)

    Berent-Maoz, Beata; Montecino-Rodriguez, Encarnacion; Fice, Michael; Casero, David; Seet, Christopher S; Crooks, Gay M; Lowry, William; Dorshkind, Kenneth

    2015-01-01

    Cell number in the mouse thymus increases steadily during the first two weeks after birth. It then plateaus and begins to decline by seven weeks after birth. The factors governing these dramatic changes in cell production are not well understood. The data herein correlate levels of High mobility group A 2 protein (Hmga2) expression with these temporal changes in thymopoiesis. Hmga2 is expressed at high levels in murine fetal and neonatal early T cell progenitors (ETP), which are the most immature intrathymic precursors, and becomes almost undetectable in these progenitors after 5 weeks of age. Hmga2 expression is critical for the initial, exponential expansion of thymopoiesis, as Hmga2 deficient mice have a deficit of ETPs within days after birth, and total thymocyte number is repressed compared to wild type littermates. Finally, our data raise the possibility that similar events occur in humans, because Hmga2 expression is high in human fetal thymic progenitors and falls in these cells during early infancy.

  7. The expansion of thymopoiesis in neonatal mice is dependent on expression of high mobility group a 2 protein (Hmga2.

    Directory of Open Access Journals (Sweden)

    Beata Berent-Maoz

    Full Text Available Cell number in the mouse thymus increases steadily during the first two weeks after birth. It then plateaus and begins to decline by seven weeks after birth. The factors governing these dramatic changes in cell production are not well understood. The data herein correlate levels of High mobility group A 2 protein (Hmga2 expression with these temporal changes in thymopoiesis. Hmga2 is expressed at high levels in murine fetal and neonatal early T cell progenitors (ETP, which are the most immature intrathymic precursors, and becomes almost undetectable in these progenitors after 5 weeks of age. Hmga2 expression is critical for the initial, exponential expansion of thymopoiesis, as Hmga2 deficient mice have a deficit of ETPs within days after birth, and total thymocyte number is repressed compared to wild type littermates. Finally, our data raise the possibility that similar events occur in humans, because Hmga2 expression is high in human fetal thymic progenitors and falls in these cells during early infancy.

  8. Assessment of multi-enzyme operon engineering of tobacco chloroplast genome for high-level simultaneous expression of cellulolytic enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Kolotilin, I. [Agriculture and Agri-Food Canada, London, ON (Canada); Pereira, E.O.; Menassa, R. [Western Ontario Univ., London, ON (Canada). Dept. of Biology; Agriculture and Agri-Food Canada, London, ON (Canada)

    2009-07-01

    The use of biofuels as an environmentally-sound substitute for depleting fossil fuels was discussed. Commercially produced biofuels are generated primarily from starch or sugar and supply only a small fraction of global fuel requirements. Although cellulosic biomass can serve as an abundant and renewable source of fermentable sugars, the cost of converting biomass to fuel is too high. Plant genetic engineering techniques are more economical for producing recombinant proteins because of the low-cost of the growing bioreactors. The transformation of the tobacco chloroplast genome has proven to be very prolific in terms of recombinant protein yield, which typically reaches 10 to 20 per cent of total soluble protein. In addition, plastid transcription-translation machinery allows for the simultaneous expression of several genes from artificial operons, providing the potential to engineer several proteins in one transformation step. The purpose of this study was to produce transplastomic tobacco plants bearing single genes as well as operons of cell wall-degrading enzymes for high-level expression. An attempt was made to reproduce an engineering approach in tobacco chloroplasts to generate a potent mini-cellulosome. The resulting enzymes were evaluated for their ability to degrade biomass. The study also examined the feasibility of using crude extracts of highly-expressing plants as an additive in the biomass fermentation process. The productivity of transplastomic plants was compared with plants transiently expressing cellulolytic enzymes directed to other cellular compartments.

  9. MicroRNA expression profiling in skeletal muscle reveals different regulatory patterns in high and low responders to resistance training.

    Science.gov (United States)

    Ogasawara, Riki; Akimoto, Takayuki; Umeno, Tokushi; Sawada, Shuji; Hamaoka, Takafumi; Fujita, Satoshi

    2016-04-01

    Large variability exists in muscle adaptive response to resistance exercise (RE) training between individuals. Recent studies have revealed a significant role for microRNAs (miRNAs) in skeletal muscle plasticity. In this study, we investigated how RE affects miRNA expression and whether the variability of muscle hypertrophy to RE training may be attributed to differential miRNA regulation in the skeletal muscle. To screen high and low responders to RE, we had 18 young men perform arm curl exercise training. After screening, all the men performed 12 wk of lower body RE training, but only the high or low responders participated in the acute RE test before training. Muscle biopsies were obtained from the vastus lateralis muscle at baseline, 3 h after acute RE, and after the training period. Total RNA was extracted from the skeletal muscle, and miRNA expression (800 miRNAs) was analyzed. RE training increased the cross-sectional area of the biceps brachii (-1.7-26.1%), quadriceps (2.2-16.8%), and hamstrings (1.6-18.4%). Eighty-five and 102 miRNAs were differentially expressed after acute and chronic RE, respectively (P muscle between high and low responders, indicating that the expression patterns of several miRNAs are altered by acute or chronic RE, and that miRNAs are involved in skeletal muscle adaptation to RE training.

  10. High-Level HOOK3 Expression Is an Independent Predictor of Poor Prognosis Associated with Genomic Instability in Prostate Cancer.

    Directory of Open Access Journals (Sweden)

    Nathaniel Melling

    Full Text Available Hook microtubule-tethering protein 3 (HOOK3 is an adaptor protein for microtubule-dependent intracellular vesicle and protein trafficking. In order to assess the role of HOOK3 in prostate cancer we analyzed HOOK3 expression by immunohistochemistry on a TMA containing more than 12,400 prostate cancers. Results were compared to tumor phenotype and PSA recurrence as well as aberrations possibly defining relevant molecular subtypes such as ERG status and deletions of 3p13, 5q21, 6q15 and PTEN. HOOK3 immunostaining was negative in normal luminal cells of prostate epithelium, whereas 53.3% of 10,572 interpretable cancers showed HOOK3 expression, which was considered low in 36.4% and high in 16.9% of cases. High-level HOOK3 expression was linked to advanced tumor stage, high Gleason score, high proliferation index, positive lymph node stage, and PSA recurrence (p<0.0001 each. The prognostic role of HOOK3 expression was independent of established clinico-pathological parameters both in preoperative and postoperative settings. Comparisons with molecular features were performed to draw conclusions on the potential function of HOOK3 in the prostate. A strong association with all examined deletions is consistent with a role of HOOK3 for maintaining genomic integrity by contributing to proper centrosome assembly. Finding HOOK3 expression in 74% of ERG positive but in only 38% of ERG negative cancers (p<0.0001 further suggests functional interactions between these genes. In conclusion, the results of our study identify HOOK3 as a strong candidate prognostic marker with a possible role in maintaining genomic integrity in prostate cancer, which may have potential for inclusion into clinical routine assays.

  11. Insertional activation of cepA leads to high-level beta-lactamase expression in Bacteroides fragilis clinical isolates.

    Science.gov (United States)

    Rogers, M. B.; Bennett, T. K.; Payne, C. M.; Smith, C. J.

    1994-01-01

    Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infections, including beta-lactams. A strain set comprised of B. fragilis isolates producing either low or high levels of the endogenous cephalosporinase activity, CepA, has been described previously (M. B. Rogers, A. C. Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400, 1993). Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleotide sequence comparisons revealed that there were few differences between the cepA coding sequences of the low- and high-activity strains. The cepA coding sequences were cloned into an expression vector, pFD340, and analyzed in a B. fragilis 638 cepA mutant. The results of beta-lactamase assays and ampicillin MICs showed that there was no significant difference in the enzymatic activity of structural genes from the high- or low-activity strains. Comparison of sequences upstream of the cepA coding region revealed that 50 bp prior to the translation start codon, the sequence for high-activity strains change dramatically. This region of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expression of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differential cepA expression in low- and high-activity strains was controlled at the level of transcription. However, the insertion did not alter the cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mechanisms of cepA activation are discussed. Images PMID:7517394

  12. Anopheles gambiae P450 reductase is highly expressed in oenocytes and in vivo knockdown increases permethrin susceptibility.

    Science.gov (United States)

    Lycett, G J; McLaughlin, L A; Ranson, H; Hemingway, J; Kafatos, F C; Loukeris, T G; Paine, M J I

    2006-06-01

    We describe an in vivo model for investigation of detoxification mechanisms of the mosquito Anopheles gambiae, important for the development of malaria control programmes. Cytochrome P450s are involved in metabolic insecticide resistance and require NADPH cytochrome P450 reductase (CPR) to function. Here we demonstrate that the major sites of adult mosquito CPR expression are oenocytes, mid-gut epithelia and head appendages. High CPR expression was also evident in Drosophila oenocytes indicating a general functional role in these insect cells. RNAi mediated knockdown drastically reduced CPR expression in oenocytes, and to a lesser extent in mid-gut epithelia; the head was unaffected. These flies showed enhanced sensitivity to permethrin, demonstrating a key role for abdominal/mid-gut P450s in pyrethroid metabolism, aiding the development of insecticides.

  13. Effects of High-Fat Feeding on Skeletal Muscle Gene Expression in Diabetic Goto-Kakizaki Rats

    Directory of Open Access Journals (Sweden)

    Jing Nie

    2017-05-01

    Full Text Available In the present report, we examined the responses of diabetic Goto-Kakizaki (GK rats and control Wistar-Kyoto (WKY rats fed either a standard chow or high-fat diet (HFD from weaning to 20 weeks of age. This comparison included gene expression profiling of skeletal muscle using Affymetrix gene array chips. The expression profiling is interpreted within the context of a wide array of physiological measurements. Genes whose expressions are different between the 2 strains regardless of diet, as well as genes that differ between strains only with HFD, were identified. In addition, genes that were regulated by diet in 1 or both strains were identified. The results suggest that both strains respond to HFD by an increased capacity to oxidize lipid fuels in the musculature but that this adaptation occurs more rapidly in WKY rats. The results also demonstrated an impaired cytokine signalling and heightened inflammatory status in the GK rats.

  14. High expression of the circadian gene mPer2 diminishes the radiosensitivity of NIH 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chang, L.; Liu, Y.Y.; Zhu, B.; Li, Y.; Hua, H.; Wang, Y.H.; Zhang, J.; Jiang, Z.; Wang, Z.R. [Sichuan University, Chengdu (China). West China Medical Center. Health Ministry Key Lab. of Chronobiology], e-mail: wangzhengrong@126.com

    2009-10-15

    Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to {sup 60}Co-{gamma}-rays. NIH 3T3 cells were treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with {sup 6}0Co-{gamma}-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 {+-} 6.51 vs 66.0 {+-} 3.51 and 67.7 {+-} 7.37; transfection: 121.7 {+-} 6.50 vs 65.3 {+-} 3.51 and 69.0 {+-} 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, cmyc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes. (author)

  15. Gene expression responses to highly pathogenic avian influenza H5N1 virus infections in ducks

    Science.gov (United States)

    Differences in host response to infection with avian influenza (AI) viruses were investigated by identifying genes differentially expressed in tissues of infected ducks. Clear differences in pathogenicity were observed among ducks inoculated with five H5N1 HPAI viruses. Virus titers in tissues cor...

  16. NEC1, a novel gene, highly expressed in the nectary tissue of Petunia hybrida

    NARCIS (Netherlands)

    Ge, X.Y.; Angenent, G.C.; Wittich, P.E.; Peters, J.; Franken, J.; Busscher, M.; Zhang Lanying,; Dahlhaus, E.; Kater, M.M.; Wullems, G.J.; Creemers-Molenaar, T.

    2000-01-01

    To study the molecular regulation of nectary development, we cloned NEC1, a gene predominantly expressed in the nectaries of Petunia hybrida, by using the differential display RTPCR technique. The secondary structure of the putative NEC1 protein is reminiscent of a transmembrane protein, indicating

  17. High-level soluble expression of Serratia marcescens H30 lipase in Escherichia coli.

    Science.gov (United States)

    Su, Erzheng; Xu, Jingjing; Wu, Xiangping

    2015-01-01

    Serratia marcescens lipase (SmL) is an important biocatalyst used to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphynyl) glycidic acid methyl ester. However, the economically justified level recombinant soluble expression of SmL in Escherichia coli has not been established. Thus, fusion genes of lipase from S. marcescens H30 with different fusion tags were constructed and expressed in E. coli. The effects of fusion tags were revealed. A significant increase in recombinant lipase solubility showed that E. coli BL21 (DE3)/pET32a-SmL was a suitable choice for SmL production. To optimize the performance of recombinant SmL production, changes in culture medium compositions and induction conditions were systematically tested. Finally, the recombinant SmL activity and productivity reached approximately 23,000 U/L and 1,278 U/L/H in shake flasks, respectively. This value is the highest SmL activity attained by heterogeneous recombinant expression in E. coli. Lipase activity and productivity reached 19,650 U/L and 1,228 U/L/H, respectively, by scaling up SmL production in a 7.0 L fermenter. The existence of the Trx tag did not influence the chiral selectivity of recombinant SmL. These findings indicate a possibility for soluble and economical SmL expression in E. coli to meet industrial needs.

  18. VEGF expression and microvascular density in relation to high-risk ...

    African Journals Online (AJOL)

    Bassma M. El Sabaa

    2012-01-13

    Jan 13, 2012 ... types in relation to tumor type, grade and HPV antigen expression was performed ... VEGF density was significantly lower in squamous cell carcinomas compared to adenocarcinomas ... intraepithelial lesions and invasive cervical carcinoma.9 ..... many levels including estimation of serum HPV virus load,58.

  19. High energy particles at Mars and Venus: Phobos-2, Mars Express and Venus Express observations and their interpretation by hybrid model simulations

    Science.gov (United States)

    McKenna-Lawlor, Susan; Kallio, Esa; Fram, Rudy A.; Alho, Markku; Jarvinen, Riku; Dyadechkin, Sergey; Wedlund, Cyril Simon; Zhang, Tielong; Collinson, Glyn A.; Futaana, Yoshifumi

    2013-04-01

    Mars and Venus can both be reached by Solar Energetic Particles (SEPs). Such high energy particles (protons, multiply charged heavy ions, electrons) penetrate the upper atmospheres of Mars and Venus because, in contrast to Earth, these bodies do not have a significant, global, intrinsic magnetic field to exclude them. One especially well documented, complex and prolonged SEP took in place in early 1989 (Solar Cycle 23) when the Phobos-2 spacecraft was orbiting Mars. This spacecraft had a dedicated high energy particle instrument onboard (SLED), which measured particles with energies in the keV range up to a few tens of MeV. There was in addition a magnetometer as well as solar wind plasma detectors onboard which together provided complementary data to support contemporaneous studies of the background SEP environment. Currently, while the Sun is displaying maximum activity (Solar Cycle 24), Mars and Venus are being individually monitored by instrumentation flown onboard the Mars Express (MEX) and Venus Express (VEX) spacecraft. Neither of these spacecraft carry a high energy particle instrument but their Analyzer of Space Plasmas and Energetic Atoms (ASPERA) experiments (ASPERA-3 on MEX and ASPERA-4 on VEX), can be used to study SEPs integrated over E ≥ ~30 MeV which penetrate the instrument hardware and form background counts in the plasma data. In the present work we present SEP events measured at Mars and Venus based on Phobos-2, 1989 data and on, more recent, MEX and VEX (identified from particle background) observations. We further introduce numerical global SEP simulations of the measured events based on 3-D self-consistent hybrid models (HYB-Mars and HYB-Venus). Through comparing the in situ SEP observations with these simulations, new insights are provided into the properties of the measured SEPs as well as into how their individual planetary bow shocks and magnetospheres affect the characteristics of their ambient Martian and Venusian SEP environments.

  20. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

    Energy Technology Data Exchange (ETDEWEB)

    Sustarsic, Elahu G. [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Department of Biological Sciences, Ohio University, Athens, OH (United States); Junnila, Riia K. [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Kopchick, John J., E-mail: kopchick@ohio.edu [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Department of Biological Sciences, Ohio University, Athens, OH (United States); Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH (United States)

    2013-11-08

    Highlights: •Most cancer types of the NCI60 have sub-sets of cell lines with high GHR expression. •GHR is highly expressed in melanoma cell lines. •GHR is elevated in advanced stage IV metastatic tumors vs. stage III. •GH treatment of metastatic melanoma cell lines alters growth and cell signaling. -- Abstract: Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on

  1. SOX11 expression is highly specific for mantle cell lymphoma and identifies the cyclin D1-negative subtype

    Science.gov (United States)

    Mozos, Ana; Royo, Cristina; Hartmann, Elena; De Jong, Daphne; Baró, Cristina; Valera, Alexandra; Fu, Kai; Weisenburger, Dennis D.; Delabie, Jan; Chuang, Shih-Sung; Jaffe, Elaine S.; Ruiz-Marcellan, Carmen; Dave, Sandeep; Rimsza, Lisa; Braziel, Rita; Gascoyne, Randy D.; Solé, Francisco; López-Guillermo, Armando; Colomer, Dolors; Staudt, Louis M.; Rosenwald, Andreas; Ott, German; Jares, Pedro; Campo, Elias

    2009-01-01

    Background Cyclin D1-negative mantle cell lymphoma is difficult to distinguish from other small B-cell lymphomas. The clinical and pathological characteristics of patients with this form of lymphoma have not been well defined. Overexpression of the transcription factor SOX11 has been observed in conventional mantle cell lymphoma. The aim of this study was to determine whether this gene is expressed in cyclin D1-negative mantle cell lymphoma and whether its detection may be useful to identify these tumors. Design and Methods The microarray database of 238 mature B-cell neoplasms was re-examined. SOX11 protein expression was investigated immunohistochemically in 12 cases of cyclin D1-negative mantle cell lymphoma, 54 cases of conventional mantle cell lymphoma, and 209 additional lymphoid neoplasms. Results SOX11 mRNA was highly expressed in conventional and cyclin D1-negative mantle cell lymphoma and in 33% of the cases of Burkitt’s lymphoma but not in any other mature lymphoid neoplasm. SOX11 nuclear protein was detected in 50 cases (93%) of conventional mantle cell lymphoma and also in the 12 cyclin D1-negative cases of mantle cell lymphoma, the six cases of lymphoblastic lymphomas, in two of eight cases of Burkitt’s lymphoma, and in two of three T-prolymphocytic leukemias but was negative in the remaining lymphoid neoplasms. Cyclin D2 and D3 mRNA levels were significantly higher in cyclin D1-negative mantle cell lymphoma than in conventional mantle cell lymphoma but the protein expression was not discriminative. The clinico-pathological features and outcomes of the patients with cyclin D1-negative mantle cell lymphoma identified by SOX11 expression were similar to those of patients with conventional mantle cell lymphoma. Conclusions SOX11 mRNA and nuclear protein expression is a highly specific marker for both cyclin D1-positive and negative mantle cell lymphoma. PMID:19880778

  2. Differential Expression of Non-Shelterin Genes Associated with High Telomerase Levels and Telomere Shortening in Plasma Cell Disorders

    Science.gov (United States)

    Panero, Julieta; Stella, Flavia; Schutz, Natalia; Fantl, Dorotea Beatriz; Slavutsky, Irma

    2015-01-01

    Telomerase, shelterin proteins and various interacting factors, named non-shelterin proteins, are involved in the regulation of telomere length (TL). Altered expression of any of these telomere-associated genes can lead to telomere dysfunction, causing genomic instability and disease development. In this study, we investigated the expression profile of a set of non-shelterin genes involved in essential processes such as replication (RPA1), DNA damage repair pathways (MRE11-RAD50-NBS1) and stabilization of telomerase complex (DKC1), in 35 patients with monoclonal gammopathy of undetermined significance (MGUS) and 40 cases with multiple myeloma (MM). Results were correlated with hTERT expression, TL and clinical parameters. Overall, a significant increase in DKC1, RAD50, MRE11, NBS1 and RPA1 expression along with an upregulation of hTERT in MM compared with MGUS was observed (p≤0.032). Interestingly, in both entities high mRNA levels of non-shelterin genes were associated with short TLs and increased hTERT expression. Significant differences were observed for DKC1 in MM (p ≤0.026), suggesting an important role for this gene in the maintenance of short telomeres by telomerase in myeloma plasma cells. With regard to clinical associations, we observed a significant increase in DKC1, RAD50, MRE11 and RPA1 expression in MM cases with high bone marrow infiltration (p≤0.03) and a tendency towards cases with advanced ISS stage, providing the first evidence of non-shelterin genes associated to risk factors in MM. Taken together, our findings bring new insights into the intricate mechanisms by which telomere-associated proteins collaborate in the maintenance of plasma cells immortalization and suggest a role for the upregulation of these genes in the progression of the disease. PMID:26366868

  3. Differential Expression of Non-Shelterin Genes Associated with High Telomerase Levels and Telomere Shortening in Plasma Cell Disorders.

    Directory of Open Access Journals (Sweden)

    Julieta Panero

    Full Text Available Telomerase, shelterin proteins and various interacting factors, named non-shelterin proteins, are involved in the regulation of telomere length (TL. Altered expression of any of these telomere-associated genes can lead to telomere dysfunction, causing genomic instability and disease development. In this study, we investigated the expression profile of a set of non-shelterin genes involved in essential processes such as replication (RPA1, DNA damage repair pathways (MRE11-RAD50-NBS1 and stabilization of telomerase complex (DKC1, in 35 patients with monoclonal gammopathy of undetermined significance (MGUS and 40 cases with multiple myeloma (MM. Results were correlated with hTERT expression, TL and clinical parameters. Overall, a significant increase in DKC1, RAD50, MRE11, NBS1 and RPA1 expression along with an upregulation of hTERT in MM compared with MGUS was observed (p≤0.032. Interestingly, in both entities high mRNA levels of non-shelterin genes were associated with short TLs and increased hTERT expression. Significant differences were observed for DKC1 in MM (p ≤0.026, suggesting an important role for this gene in the maintenance of short telomeres by telomerase in myeloma plasma cells. With regard to clinical associations, we observed a significant increase in DKC1, RAD50, MRE11 and RPA1 expression in MM cases with high bone marrow infiltration (p≤0.03 and a tendency towards cases with advanced ISS stage, providing the first evidence of non-shelterin genes associated to risk factors in MM. Taken together, our findings bring new insights into the intricate mechanisms by which telomere-associated proteins collaborate in the maintenance of plasma cells immortalization and suggest a role for the upregulation of these genes in the progression of the disease.

  4. A chronic high-cholesterol diet paradoxically suppresses hepatic CYP7A1 expression in FVB/NJ mice.

    Science.gov (United States)

    Henkel, Anne S; Anderson, Kristy A; Dewey, Amanda M; Kavesh, Mark H; Green, Richard M

    2011-02-01

    Cholesterol 7α-hydroxylase (CYP7A1) encodes for the rate-limiting step in the conversion of cholesterol to bile acids in the liver. In response to acute cholesterol feeding, mice upregulate CYP7A1 via stimulation of the liver X receptor (LXR) α. However, the effect of a chronic high-cholesterol diet on hepatic CYP7A1 expression in mice is unknown. We demonstrate that chronic cholesterol feeding (0.2% or 1.25% w/w cholesterol for 12 weeks) in FVB/NJ mice results in a >60% suppression of hepatic CYP7A1 expression associated with a >2-fold increase in hepatic cholesterol content. In contrast, acute cholesterol feeding induces a >3-fold upregulation of hepatic CYP7A1 expression. We show that chronic, but not acute, cholesterol feeding increases the expression of hepatic inflammatory cytokines, tumor necrosis factor (TNF)α, and interleukin (IL)-1β, which are known to suppress hepatic CYP7A1 expression. Chronic cholesterol feeding also results in activation of the mitogen activated protein (MAP) kinases, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Furthermore, we demonstrate in vitro that suppression of CYP7A1 by TNFα and IL-1β is dependent on JNK and ERK signaling. We conclude that chronic high-cholesterol feeding suppresses CYP7A1 expression in mice. We propose that chronic cholesterol feeding induces inflammatory cytokine activation and liver damage, which leads to suppression of CYP7A1 via activation of JNK and ERK signaling pathways.

  5. High Silicon Accumulation in the Shoot is Required for Down-Regulating the Expression of Si Transporter Genes in Rice.

    Science.gov (United States)

    Mitani-Ueno, Namiki; Yamaji, Naoki; Ma, Jian Feng

    2016-12-01

    Rice requires high silicon (Si) for its high and sustainable yield. The efficient uptake of Si in rice is mediated by two transporters OsLsi1 and OsLsi2, which function as influx and efflux transporters, respectively. Our previous studies showed that the mRNA expression levels of these transporter genes were down-regulated by Si. Herein we investigated the mechanism underlying regulation of OsLsi1 and OsLsi2 expression. There was a negative correlation between the expression level of OsLsi1 and OsLsi2 and shoot Si accumulation when the rice seedlings were exposed to different Si supply conditions. A split root experiment showed that the expression of both OsLsi1 and OsLsi2 was also down-regulated in half the roots without direct Si exposure when the other half of the roots were exposed to Si. Analysis with transgenic rice carrying different lengths of OsLsi1 promoter regions fused with green fluorescent protein (GFP) as a reporter gene revealed that the region responsible for the Si response of OsLsi1 expression is present between -327 to -292 in the promoter. However, this region was not associated with the tissue and cellular localization of OsLsi1. In conclusion, the Si-induced down-regulation of Si transporter genes is controlled by shoot Si, not root Si, and the region between -327 and -292 in the OsLsi1 promoter is involved in this regulation of OsLsi1 expression in rice.

  6. High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1.

    Science.gov (United States)

    Bovy, Arnaud; de Vos, Ric; Kemper, Mark; Schijlen, Elio; Almenar Pertejo, Maria; Muir, Shelagh; Collins, Geoff; Robinson, Sue; Verhoeyen, Martine; Hughes, Steve; Santos-Buelga, Celestino; van Tunen, Arjen

    2002-10-01

    Flavonoids are a group of polyphenolic plant secondary metabolites important for plant biology and human nutrition. In particular flavonols are potent antioxidants, and their dietary intake is correlated with a reduced risk of cardiovascular diseases. Tomato fruit contain only in their peel small amounts of flavonoids, mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. To increase flavonoid levels in tomato, we expressed the maize transcription factor genes LC and C1 in the fruit of genetically modified tomato plants. Expression of both genes was required and sufficient to upregulate the flavonoid pathway in tomato fruit flesh, a tissue that normally does not produce any flavonoids. These fruit accumulated high levels of the flavonol kaempferol and, to a lesser extent, the flavanone naringenin in their flesh. All flavonoids detected were present as glycosides. Anthocyanins, previously reported to accumulate upon LC expression in several plant species, were present in LC/C1 tomato leaves but could not be detected in ripe LC/C1 fruit. RNA expression analysis of ripening fruit revealed that, with the exception of chalcone isomerase, all of the structural genes required for the production of kaempferol-type flavonols and pelargonidin-type anthocyanins were induced strongly by the LC/C1 transcription factors. Expression of the genes encoding flavanone-3'-hydroxylase and flavanone-3'5'-hydroxylase, which are required for the modification of B-ring hydroxylation patterns, was not affected by LC/C1. Comparison of flavonoid profiles and gene expression data between tomato leaves and fruit indicates that the absence of anthocyanins in LC/C1 fruit is attributable primarily to an insufficient expression of the gene encoding flavanone-3'5'-hydroxylase, in combination with a strong preference of the tomato dihydroflavonol reductase enzyme to use the flavanone-3'5'-hydroxylase reaction product dihydromyricetin as a substrate.

  7. Identification of rat mammary tumor-1 gene (RMT-1), which is highly expressed in rat mammary tumors.

    Science.gov (United States)

    Chiou, S; Yoo, J; Loh, K C; Guzman, R C; Gopinath, G R; Rajkumar, L; Chou, Y C; Yang, J; Popescu, N C; Nandi, S

    2001-12-10

    Full-term pregnancy early in life results in a permanent reduction in lifetime breast cancer risk in women. Parous rats and mice are also refractory to chemical carcinogenesis. Therefore, investigation of the differences between mammary glands from virgin and parous rats would provide valuable information regarding the protective effects of early full-term pregnancy. In this report, we examined the gene expression patterns in mammary glands from virgin and parous Lewis rats. Using differential display technology, a novel 4.2 kb cDNA, designated rat mammary tumor-1 (RMT-1) was isolated. Northern blot analysis of RMT-1 showed that RMT-1 expression was higher in the pre-pubertal and pubertal stages during rat mammary gland development while it was down-regulated in mammary glands from mature virgin and parous rats. RMT-1 expression was highest in rat mammary cancers compared with either the mammary glands of virgin or parous rats. At the Northern blot sensitivity level, RMT-1 expression was found only in the mammary gland. Northern blot analysis also showed that the expression of this gene was found in 74% of N-methyl-nitrosourea (MNU)-induced mammary cancers while it was not found in MNU-induced cancers from other organs. The examination of the RMT-1 gene structure revealed that it consists of five exons spanning 5.9 kb. Using fluorescence in situ hybridization, the gene was localized on rat chromosome 1 band q 43-51. The present data show that there is a correlation between high RMT-1 expression and rat mammary carcinogenesis or decreased RMT-1 expression and parity associated refractoriness to chemically induced mammary carcinogenesis. However, whether or not RMT-1 gene has a functional role in these processes remains to be investigated.

  8. Gene expression profile of Jurkat cells exposed to high power terahertz radiation

    Science.gov (United States)

    Grundt, Jessica E.; Roth, Caleb C.; Rivest, Benjamin D.; Doroski, Michael L.; Payne, Jason; Ibey, Bennett L.; Wilmink, Gerald J.

    2011-03-01

    Terahertz (THz) radiation sources are now being used in a host of military, defense, and medical applications. Widespread employment of these applications has prompted concerns regarding the health effects associated with THz radiation. In this study, we examined the gene expression profile of mammalian cells exposed to THz radiation. We hypothesized that if THz radiation couples directly to cellular constituents, then exposed cells may express a specific gene expression profile indicative of ensuing damage. To test this hypothesis, Jurkat cells were irradiated with a molecular gas THz laser (2.52 THz, 636 mWcm-2, durations: 5, 10, 20, 30, 40, or 50 minutes). Viability was assessed 24 h post-exposure using MTT assays, and gene expression profiles were evaluated 4 h post-exposure using mRNA microarrays. Comparable analyses were also performed for hyperthermic positive controls (44°C for 40 minutes). We found that cellular temperatures increased by ~6 °C during THz exposures. We also found that cell death increased with exposure duration, and the median lethal dose (LD50) was calculated to be ~44 minutes. The microarray data showed that THz radiation induced the transcriptional activation of genes associated with cellular proliferation, differentiation, transcriptional activation, chaperone protein stabilization, and apoptosis. For most genes, we found that the magnitude of differential expression was comparable for both the THz and thermal exposure groups; however, several genes were specifically activated by the THz exposure. These results suggest that THz radiation may elicit effects that are not exclusively due to the temperature rise created during THz exposures (i.e. thermal effects). In future work, we plan to verify the results of our microarray experiments using qPCR techniques.

  9. PPARγ expression by rambutan peel extract in obesity rat model-induced high-calorie diet简

    Institute of Scientific and Technical Information of China (English)

    Sri; Rahayu; Lestari; Muhammad; Sasmito; Djati; Achmad; Rudijanto; Fatchiyah; Fatchiyah

    2015-01-01

    Objective: To monitor the physiological characteristics and genes expression of obesity rat model after rambutan peel extract(RPE) treatment.Methods: Twenty-four 12-week-old male rats were divided into 4 groups: normal,obesity, obesity treated with ellagic acid(O-EA) and obesity treated with RPE30(ORPE30). Physiological characteristics were monitored by measuring body weight, calorie intake, size of adipocyte and level of triglyceride. Peroxisome proliferator activated receptor gamma(PPARg), CCAAT/enhancer-binding proteins a and fatty acid binding protein 4(FABP4) expression were observed using immunohistochemistry, Western blotting and quantitative RT-PCR methods.Results: Body weight gain of O-EA and O-RPE30 rats were lower than obesity group and size of adipocyte cells were smaller than obesity group(P < 0.05), but when we compared to normal group, those groups had higher body weight gain and larger adipocyte cells. The level of triglycerides, protein expression of PPARg and m RNA level of FABP4 genes were significantly downregulated on O-EA and O-RPE30 compared to obesity group(P < 0.05). Our results indicated that RPE had potential substance as inhibitor of body weight gain, declining of size of adipocyte, level of triglycerides, PPARg expression and m RNA level of FABP4 gene on obesity rat model.Conclusions: RPE have anti-obesity activity by inhibiting body weight gain, declining size of adipocyte, decreasing triglyceride, PPARg expression and m RNA level of FABP4 gene on obesity rat model.

  10. Expression Analysis of Barrett’s Esophagus Associated High Grade Dysplasia in Laser Capture Microdissected Archival Tissue

    Science.gov (United States)

    Sabo, Edmond; Meitner, Patricia A; Tavares, Rosemarie; Corless, Christopher L; Lauwers, Gregory Y; Moss, Steven F; Resnick, Murray B

    2009-01-01

    Purpose Identifying genes differentially expressed in non-dysplastic Barrett’s esophagus (BE) from those expressed in high grade dysplasia (HGD) should be of value in improving our understanding of this transition and may yield new diagnostic and/or prognostic markers. The aim of this study was to determine the differential transcriptome of HGD compared with non-dysplastic BE through gene microarray analysis of epithelial cells microdissected from archival tissue specimens. Experimental Design Laser capture microdissection (LCM) was used to isolate epithelial cells from adjacent inflammatory and stromal cells. Epithelial mRNA was extracted from areas of non-dysplastic BE and HGD in matched biopsies from 11 patients. mRNA was reverse transcribed and applied on Affymetrix cDNA microarray chips customized for formalin-exposed tissue. For a subset of these genes, differential gene expression was confirmed by RT-PCR and immunohistochemistry. Results There were 131 genes over-expressed by at least 2.5-fold in HGD versus non-dysplastic BE and 16 genes that were under-expressed by at least 2.5-fold. Among the over-expressed genes are several previously demonstrated to be increased in the neoplastic progression of BE, as well as novel genes such as lipocalin-2, S100A9, matrix metallopeptidase 12, secernin 1 and topoisomerase IIα. Genes decreased in dysplastic epithelium include MUC5AC, trefoil factor1 (TFF1), meprin A and CD13. RT-PCR validated the changes in expression in 24 of 28 selected genes. Immunohistochemistry confirmed increased protein expression for topoisomerase IIα, S100A9 and lipocalin-2 and decreased expression of TFF1 across the spectrum of BE associated dysplasia from non-dysplastic BE through adenocarcinoma. Conclusions This is the first study to identify epithelial genes differentially expressed in HGD versus non-dysplastic BE in matched patient samples. The genes identified include several previously implicated in the pathogenesis of Barrett

  11. A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

    Directory of Open Access Journals (Sweden)

    Xiaodong Xiao

    Full Text Available High throughput screenings of single chain Fv (scFv antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins

  12. Identification of several soybean cytosolic glutamine synthetase transcripts highly or specifically expressed in nodules: expression studies using one of the corresponding genes in transgenic Lotus corniculatus.

    Science.gov (United States)

    Marsolier, M C; Debrosses, G; Hirel, B

    1995-01-01

    A DNA fragment containing sequences hybridizing to the 5' region of GS15, a gene encoding soybean cytosolic glutamine synthetase, was isolated from a soybean genomic library. Mapping and partial sequence analysis of the genomic clone revealed that it encodes a cytosolic GS gene, GS21, which is different from GS15. In parallel, a number of cDNA clones encoding cytosolic GS were isolated using the coding region of pGS20 as a probe (pGS20 is a cDNA clone which corresponds to a transcript of the GS15 gene). Two new full-length cDNAs designated pGS34 and pGS38 were isolated and sequenced. In the 5' non-coding region a strong homology was found between the two clones and the GS21 gene. However, none of these sequences were identical, which suggests that there are at least three members in this group of genes. In order to determine their relative levels of transcription, specific sequences from pGS34, pGS38 and GS21 were used in an RNAse protection assay. This experiment clearly showed that GS21 and the gene encoding pGS38 are specifically expressed in young or mature nodules, whereas the gene encoding pGS34 is highly transcribed in nodules and constitutively expressed at a lower level in other soybean organs. In order to further analyse the molecular mechanisms controlling GS21 transcription, different fragments of the promoter region were fused to the Escherichia coli reporter gene encoding beta-glucuronidase (GUS) and the constructs were introduced into Lotus corniculatus via Agrobacterium rhizogenes-mediated transformation. Analysis of GUS activity showed that the GS21 promoter-GUS constructs were expressed in the vasculature of all vegetative organs. This result is discussed in relation to species-specific metabolic and developmental characteristics of soybean and Lotus.

  13. Characterization of expression, cytokine regulation, and effector function of the high affinity IgG receptor Fc gamma RI (CD64) expressed on human blood dendritic cells.

    Science.gov (United States)

    Fanger, N A; Voigtlaender, D; Liu, C; Swink, S; Wardwell, K; Fisher, J; Graziano, R F; Pfefferkorn, L C; Guyre, P M

    1997-04-01

    The mechanisms responsible for efficient sequestration of Ag by cells of the dendritic cell (DC) lineage remain incompletely characterized. One pathway, internalization of Ag-IgG complexes via CD32 (the type II IgG FcR, Fc gamma RII) enhances Ag presentation 100-fold over noncomplexed Ag. Blood leukocytes differentially express two additional IgG FcR, Fc gamma RI (CD64) and Fc gamma RIII (CD16), which may also participate in leukocyte functions such as phagocytosis, Ab-dependent cellular cytotoxicity (ADCC), release of oxygen intermediates, and enhancement of Ag presentation. A phagocytically active form of CD64 was recently demonstrated on human blood DC, but complete functional potential of CD64 on the DC lineage remains undefined. Therefore, highly purified human blood DC (CD33(2)+, CD14-, CD11c2+, HLA-DR3+, CD64+ (CD83+ after overnight culture)) and monocytes (CD33(2)+, CD14(3)+, CD11c2+, HLA-DR+, CD64(2)+, CD83-) were compared for cytokine modulation and effector functions of CD64. Both DC and monocyte CD64 expression was increased by IFN-gamma and IL-10, but while monocyte CD64 was decreased by IL-4, DC CD64 remained unchanged. FcR-mediated functional differences were also evident between the DC and the monocytes. Monocytes generated robust Fc gamma R-dependent superoxide anion release and ADCC activity, while DC failed to release reactive oxygen intermediates and demonstrated minimal ADCC activity, despite apparently normal expression of the gamma-chain subunit and the signaling molecule Syk. In contrast, DC were more efficient than monocytes with respect to T cell activation when Ag was targeted specifically to CD64. These new findings suggest a previously unappreciated potential for CD64 to shape the immune response by dramatically increasing the efficiency with which DC sequester Ag prior to achieving full T cell stimulatory potential.

  14. Stable high-level expression of truncated human papillomavirus type 16 L1 protein in Drosophila Schneider-2 cells

    Institute of Scientific and Technical Information of China (English)

    Jin Zheng; Xiaofeng Yang; Ying Sun; Baochang Lai; Yili Wang

    2008-01-01

    To improve the existing human papillomavirus type 16 (HPV16) virus-like particle (VLP) preparation, the Drosophila inducible/secreted expression system, a highly efficient, economical method, was used to produce HPV16 VLPs. Drosophila Schneider-2 cells were cotransfected with pMT/BiP/V5-His expression vector containing the target gene encoding HPV16L1protein without nucleus localization sequence and the selection vector pCoHygro plasmids at the ratio of 4:1. The stabled hygromycin-resistant cell line was obtained 1 month later, and the protein expression was induced by copper sulfate. The molecular mass of expressed HPV16L1 protein was 66 kDa, as revealed by SDS-PAGE,and confirmed by Western blot analysis. The yield of HPV16L1 protein was 0.554 mg per lxl07 cells. The characteristics of HPV16L1 protein were further analyzed by mouse erythrocyte hemagglutination assay, hemagglutination inhibition assay, and transmission electron microscopy. Results showed that the truncated protein was as biologically active as natural HPVLI protein, inducing murine erythrocyte agglutination and VLP formation. These findings indicate that the Drosophila inducible/secreted expression system is promising as a convenient and economical method for the preparation of HPV 16 VLP vaccine.

  15. Differential microRNA expression between shoots and rhizomes in Oryza longistaminata using high-throughput RNA sequencing

    Institute of Scientific and Technical Information of China (English)

    Ying; Zong; Liyu; Huang; Ting; Zhang; Qiao; Qin; Wensheng; Wang; Xiuqin; Zhao; Fengyi; Hu; Binying; Fu; Zhikang; Li

    2014-01-01

    Plant microRNAs(miRNAs)play important roles in biological processes such as development and stress responses.Although the diverse functions of miRNAs in model organisms have been well studied,their function in wild rice is poorly understood.In this study,high-throughput small RNA sequencing was performed to characterize tissue-specific transcriptomes in Oryza longistaminata.A total of 603 miRNAs,380 known rice miRNAs,72 conserved plant miRNAs,and151 predicted novel miRNAs were identified as being expressed in aerial shoots and rhizomes.Additionally,99 and 79 miRNAs were expressed exclusively or differentially,respectively,in the two tissues,and 144 potential targets were predicted for the differentially expressed miRNAs in the rhizomes.Functional annotation of these targets suggested that transcription factors,including squamosa promoter binding proteins and auxin response factors,function in rhizome growth and development.The expression levels of several miRNAs and target genes in the rhizomes were quantified by RT-PCR,and the results indicated the existence of complex regulatory mechanisms between the miRNAs and their targets.Eight target cleavage sites were verified by RNA ligase-mediated rapid 5′end amplification.These results provide valuable information on the composition,expression and function of miRNAs in O.longistaminata,and will aid in understanding the molecular mechanisms of rhizome development.

  16. Effect of high-fructose corn syrup on Streptococcus mutans virulence gene expression and on tooth demineralization.

    Science.gov (United States)

    Sun, Minmin; Kang, Qiongyi; Li, Tingting; Huang, Lili; Jiang, Yuntao; Xia, Wenwei

    2014-06-01

    High-fructose corn syrup-55 (HFCS-55) has been widely welcomed in recent years as a substitute for sucrose on the basis of its favourable properties and price. The objective of this study was to determine the influence of HFCS-55 on the expression of Streptococcus mutans UA159 virulence genes and on tooth demineralization. Real-time reverse-transcription PCR (real-time RT-PCR) and microhardness evaluations were performed to examine gene expression and enamel demineralization, respectively, after treatment with HFCS-55 and/or sucrose. Significant up-regulation of glucosyltransferase B (gtfB) by HFCS-55 was found. A mixture of HFCS-55 and sucrose could positively enhance expression of glucan-binding protein (gbp) genes. Regarding acidogenicity, expression of the lactate dehydrogenase (ldh) gene was unaffected by HFCS-55. A notable finding in this study was that 5% HFCS-55 significantly enhanced expression of the intracellular response gene of the two-component VicRK signal transduction system (vicR). Demineralization testing showed that the microhardness of teeth decreased by a greater extent in response to HFCS-55 than in response to sucrose. The results indicate that HFCS-55 can enhance S. mutans biofilm formation indirectly in the presence of sucrose and that HFCS-55 has a more acidogenic potential than does sucrose. Summing up the real-time PCR and demineralization results, HFCS-55 appears to be no less cariogenic than sucrose in vitro - at least, not under the conditions of our experiments.

  17. An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.

    Science.gov (United States)

    Long, Dingpei; Lu, Weijian; Zhang, Yuli; Bi, Lihui; Xiang, Zhonghuai; Zhao, Aichun

    2015-03-05

    We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs.

  18. Expression of high mobility group box 1 in inflamed dental pulp and its chemotactic effect on dental pulp cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xufang, E-mail: xufang.zhang@student.qut.edu.au [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD 4059 (Australia); Jiang, Hongwei, E-mail: jianghw@163.com [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Gong, Qimei, E-mail: gongqmei@gmail.com [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Fan, Chen, E-mail: c3.fan@student.qut.edu.au [Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD 4059 (Australia); Huang, Yihua, E-mail: enu0701@163.com [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China); Ling, Junqi, E-mail: lingjq@mail.sysu.edu.cn [Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Guangdong Province Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055 (China)

    2014-08-08

    Highlights: • HMGB1 translocated from nucleus to cytoplasm during dental pulp inflammation. • HMGB1and its receptor RAGE were up-regulated in hDPCs under LPS stimulation. • HMGB1 enhanced hDPCs migration and induces cytoskeleton reorganization. • HMGB1 may play a critical role in dental pulp repair during inflamed state. - Abstract: High mobility group box 1 protein (HMGB1) is a chromatin protein which can be released extracellularly, eliciting a pro-inflammatory response and promoting tissue repair process. This study aimed to examine the expression and distribution of HMGB1 and its receptor RAGE in inflamed dental pulp tissues, and to assess its effects on proliferation, migration and cytoskeleton of cultured human dental pulp cells (DPCs). Our data demonstrated that cytoplasmic expression of HMGB1 was observed in inflamed pulp tissues, while HMGB1 expression was confined in the nuclei in healthy dental pulp. The mRNA expression of HMGB1 and RAGE were significantly increased in inflamed pulps. In in vitro cultured DPCs, expression of HMGB1 in both protein and mRNA level was up-regulated after treated with lipopolysaccharide (LPS). Exogenous HMGB1 enhanced DPCs migration in a dose-dependent manner and induced the reorganization of f-actin in DPCs. Our results suggests that HMGB1 are not only involved in the process of dental pulp inflammation, but also play an important role in the recruitment of dental pulp stem cells, promoting pulp repair and regeneration.

  19. Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris.

    Science.gov (United States)

    Zhou, Wen-Jing; Yang, Jiang-Ke; Mao, Lin; Miao, Li-Hong

    2015-04-01

    Lipase (EC 3.1.1.3) stands amongst the most important and promising biocatalysts for industrial applications. In this study, in order to realize a high-level expression of the Yarrowia lipolytica lipase gene in Pichia pastoris, we optimized the codon of LIP2 by de novo gene design and synthesis, which significantly improved the lipase expression when compared to the native lip2 gene. We also comparatively analyzed the effects of the promoter types (PAOX1 and PFLD1) and the Pichia expression systems, including the newly developed PichiaPink system, on lipase production and obtained the optimal recombinants. Bench-top scale fermentation studies indicated that the recombinant carrying the codon-optimized lipase gene syn-lip under the control of promoter PAOX1 has a significantly higher lipase production capacity in the fermenter than other types of recombinants. After undergoing methanol inducible expression for 96h, the wet cell weight of Pichia, the lipase activity and the protein content in the fermentation broth reached their highest values of 262g/L, 38,500U/mL and 2.82g/L, respectively. This study has not only greatly facilitated the bioapplication of lipase in industrial fields but the strategies utilized, such as de novo gene design and synthesis, the comparative analysis among promoters and different generations of Pichia expression systems will also be useful as references for future work in this field.

  20. In vivo imaging of human breast cancer mouse model with high level expression of calcium sensing receptor at 3T.

    Science.gov (United States)

    Baio, Gabriella; Fabbi, Marina; Emionite, Laura; Cilli, Michele; Salvi, Sandra; Ghedin, Piero; Prato, Sabina; Carbotti, Grazia; Tagliafico, Alberto; Truini, Mauro; Neumaier, Carlo Emanuele

    2012-03-01

    To demonstrate that manganese can visualise calcium sensing receptor (CaSR)-expressing cells in a human breast cancer murine model, as assessed by clinical 3T magnetic resonance (MR). Human MDA-MB-231-Luc or MCF7-Luc breast cancer cells were orthotopically grown in NOD/SCID mice to a minimum mass of 5 mm. Mice were evaluated on T1-weighted sequences before and after intravenous injection of MnCl(2). To block the CaSR-activated Ca(2+) channels, verapamil was injected at the tumour site 5 min before Mn(2+) administration. CaSR expression in vivo was studied by immunohistochemistry. Contrast enhancement was observed at the tumour periphery 10 min after Mn(2+) administration, and further increased up to 40 min. In verapamil-treated mice, no contrast enhancement was observed. CaSR was strongly expressed at the tumour periphery. Manganese enhanced magnetic resonance imaging can visualise CaSR-expressing breast cancer cells in vivo, opening up possibilities for a new MR contrast agent. • Manganese contrast agents helped demonstrate breast cancer cells in an animal model. • Enhancement was most marked in cells with high calcium sensing receptor expression. • Manganese uptake was related to the distribution of CaSR within the tumour. • Manganese MRI may become useful to investigate human breast cancer.

  1. High Throughput Quantitative PCR Using Low-input Samples for mRNA and MicroRNA Gene Expression Analyses

    Science.gov (United States)

    Jang, Jinsung; Kolbert, Christopher; Jen, Jin; Simon, Vernadette

    2013-01-01

    Technical advancements in quantitative PCR (qPCR) instrumentation have made it possible to perform gene expression measurements using small sample input to support both basic and clinical research studies. As part of the strategic goals to assess new technologies and identify protocols that best fit the needs of the Mayo Clinic, we compared the Fluidigm BioMark system with standard Applied Biosystems (AB) instrumentation for mRNA and miRNA gene expression measurements. We also examined the performance of the BioMark system when using very low-input RNA. We evaluated a set of control samples using the same TaqMan assays with both systems. We observed that the BioMark-generated data routinely yields Ct values approximately 10 cycles lower than those obtained with AB instrumentation. The correlations between the two platforms were high (r = 0.96) for both mRNA and miRNA expression experiments. For miRNA expression, a similarly high correlation was observed between fresh frozen and formalin-fixed paraffin embedded (FFPE) samples. In an effort to accommodate our customer needs, we also evaluated the performance of the BioMark for evaluating gene expression in very low-input samples. Using six standard TaqMan control assays (having high, medium and low expression levels), we observed that high quality RNA samples as low as 10pg achieved linear amplification across four different pre-amplification cycles (10, 14, 18 and 22). At 10pg total RNA input, low-expression control assay IPO8 demonstrated a correlation of r = .999 among the four pre-amplification cycles. This linearity was also observed at higher RNA input levels, up to 10ng. The only control assay that did not perform in a linear fashion across all input amounts and all pre-amplification cycles was 18S ribosomal RNA. The highest correlation observed for 18S was r = 0.801, and this supports the vendor suggestion that 18S is not the best control assay option.

  2. Lower Female Genital Tract Tumors With Adenoid Cystic Differentiation: P16 Expression and High-risk HPV Detection.

    Science.gov (United States)

    Xing, Deyin; Schoolmeester, J Kenneth; Ren, Zhiyong; Isacson, Christina; Ronnett, Brigitte M

    2016-04-01

    Lower female genital tract tumors with adenoid cystic differentiation are rare, and data on their relationship with high-risk human papillomavirus (HPV) are limited. Here we report the clinicopathologic features from a case series. Tumors with adenoid cystic differentiation, either pure or as part of a carcinoma with mixed differentiation, arising in the lower female genital tract were evaluated by means of immunohistochemical analysis for p16 expression and in situ hybridization using 1 or more probes for high-risk HPV (a high-risk probe covering multiple types, a wide-spectrum probe, and separate type-specific probes for HPV16 and HPV18) and when possible by polymerase chain reaction for high-risk HPV. Six cervical carcinomas with adenoid cystic differentiation admixed with various combinations of at least 1 other pattern of differentiation, including adenoid basal tumor (epithelioma and/or carcinoma), squamous cell carcinoma (basaloid or keratinizing), and small cell carcinoma were identified in patients ranging in age from 50 to 86 years (mean, 73 y; median, 76 y). All of these tumors were characterized by diffuse p16 expression. High-risk HPV was detected in 5 of 6 tested cases: 4 cases by in situ hybridization (all positive for HPV-wide-spectrum and HPV16) and 1 by polymerase chain reaction (HPV45). Seven pure adenoid cystic carcinomas (6 vulvar and 1 cervical) were identified in patients ranging in age from 27 to 74 years (mean, 48 y; median, 48 y). All of these tumors were characterized by variable p16 expression ranging from very limited to more extensive but never diffuse. No high-risk HPV was detected in any of these pure tumors. Lower female genital tract carcinomas with adenoid cystic differentiation appear to comprise 2 pathogenetically distinct groups. Cervical carcinomas with mixed differentiation, including adenoid cystic, adenoid basal, squamous, and small cell components, are etiologically related to high-risk HPV and can be identified by diffuse

  3. High Laccase Expression by Trametes versicolor in a Simulated Textile Effluent with Different Carbon Sources and PHs

    Directory of Open Access Journals (Sweden)

    Cristiane Ottoni

    2016-08-01

    Full Text Available Textile effluents are highly polluting and have variable and complex compositions. They can be extremely complex, with high salt concentrations and alkaline pHs. A fixed-bed bioreactor was used in the present study to simulate a textile effluent treatment, where the white-rot fungus, Trametes versicolor, efficiently decolourised the azo dye Reactive Black 5 over 28 days. This occurred under high alkaline conditions, which is unusual, but advantageous, for successful decolourisation processes. Active dye decolourisation was maintained by operation in continuous culture. Colour was eliminated during the course of operation and maximum laccase (Lcc activity (80.2 U∙L−1 was detected after glycerol addition to the bioreactor. Lcc2 gene expression was evaluated with different carbon sources and pH values based on reverse transcriptase-PCR (polymerase chain reaction. Glycerol was shown to promote the highest lcc2 expression at pH 5.5, followed by sucrose and then glucose. The highest levels of expression occurred between three and four days, which corroborate the maximum Lcc activity observed for sucrose and glycerol on the bioreactor. These results give new insights into the use of T. versicolor in textile dye wastewater treatment with high pHs.

  4. Changes in polyphenols and expression levels of related genes in 'Duke' blueberries stored under high CO2 levels.

    Science.gov (United States)

    Harb, Jamil; Saleh, Omar; Kittemann, Dominikus; Neuwald, Daniel Alexandre; Hoffmann, Thomas; Reski, Ralf; Schwab, Wilfried

    2014-07-30

    Blueberries are highly perishable fruits, and consequently, storage under high CO2 and low O2 levels is recommended to preserve the highly appreciated polyphenols. However, high CO2 levels might be detrimental for certain cultivars. The aim of this study was to investigate the impact of storage conditions on various quality parameters, including polyphenol composition in 'Duke' berries. Results show that storage under 18 kPa CO2, coupled with 3 kPa O2, resulted in accelerated softening of berries, which was accompanied by lower levels compared to other conditions of hexosides and arabinosides of malvidin, petunidin, cyanidine, and delphinidin. However, this storage condition had no negative impact on chlorogenic acid levels. Expression data of key polyphenol-biosynthesis genes showed higher expression levels of all investigated genes at harvest time compared to all storage conditions. Of particular importance is the expression level of chalcone synthase (VcCHS), which is severely affected by storage at 18 kPa CO2.

  5. High Laccase Expression by Trametes versicolor in a Simulated Textile Effluent with Different Carbon Sources and PHs

    Science.gov (United States)

    Ottoni, Cristiane; Simões, Marta F.; Fernandes, Sara; Santos, Cledir R.; Lima, Nelson

    2016-01-01

    Textile effluents are highly polluting and have variable and complex compositions. They can be extremely complex, with high salt concentrations and alkaline pHs. A fixed-bed bioreactor was used in the present study to simulate a textile effluent treatment, where the white-rot fungus, Trametes versicolor, efficiently decolourised the azo dye Reactive Black 5 over 28 days. This occurred under high alkaline conditions, which is unusual, but advantageous, for successful decolourisation processes. Active dye decolourisation was maintained by operation in continuous culture. Colour was eliminated during the course of operation and maximum laccase (Lcc) activity (80.2 U∙L−1) was detected after glycerol addition to the bioreactor. Lcc2 gene expression was evaluated with different carbon sources and pH values based on reverse transcriptase-PCR (polymerase chain reaction). Glycerol was shown to promote the highest lcc2 expression at pH 5.5, followed by sucrose and then glucose. The highest levels of expression occurred between three and four days, which corroborate the maximum Lcc activity observed for sucrose and glycerol on the bioreactor. These results give new insights into the use of T. versicolor in textile dye wastewater treatment with high pHs. PMID:27490563

  6. The expression profile of phosphatidylinositol in high spatial resolution imaging mass spectrometry as a potential biomarker for prostate cancer.

    Directory of Open Access Journals (Sweden)

    Takayuki Goto

    Full Text Available High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS is an emerging application for the comprehensive and detailed analysis of the spatial distribution of ionized molecules in situ on tissue slides. HR-MALDI-IMS in negative mode in a mass range of m/z 500-1000 was performed on optimal cutting temperature (OCT compound-embedded human prostate tissue samples obtained from patients with prostate cancer at the time of radical prostatectomy. HR-MALDI-IMS analysis of the 14 samples in the discovery set identified 26 molecules as highly expressed in the prostate. Tandem mass spectrometry (MS/MS showed that these molecules included 14 phosphatidylinositols (PIs, 3 phosphatidylethanolamines (PEs and 3 phosphatidic acids (PAs. Among the PIs, the expression of PI(18:0/18:1, PI(18:0/20:3 and PI(18:0/20:2 were significantly higher in cancer tissue than in benign epithelium. A biomarker algorithm for prostate cancer was formulated by analyzing the expression profiles of PIs in cancer tissue and benign epithelium of the discovery set using orthogonal partial least squares discriminant analysis (OPLS-DA. The sensitivity and specificity of this algorithm for prostate cancer diagnosis in the 24 validation set samples were 87.5 and 91.7%, respectively. In conclusion, HR-MALDI-IMS identified several PIs as being more highly expressed in prostate cancer than benign prostate epithelium. These differences in PI expression profiles may serve as a novel diagnostic tool for prostate cancer.

  7. Regulation of PD-L1 expression in a high-grade invasive human oral squamous cell carcinoma microenvironment

    Science.gov (United States)

    Hirai, Mariko; Kitahara, Hiroko; Kobayashi, Yutaka; Kato, Koroku; Bou-Gharios, George; Nakamura, Hiroyuki; Kawashiri, Shuichi

    2017-01-01

    Blockade of the programmed-death 1 receptor (PD-1)/programmed-death ligand (PD-L1) pathway efficiently reduces tumour growth and improves survival. Durable tumour regression with blockade of the PD-1/PD-L1 checkpoint has been demonstrated in recent clinical studies. Oral squamous cell carcinoma (OSCC) is highly immunosuppressive, and PD-L1 expression has been proposed as a potential mechanism responsible for this phenotype. Despite the fact that anti-PD-1 treatment can produce durable responses, such therapy appears to benefit only a subset of patients. Thus, it is important to understand the mechanisms underlying regulation of PD-L1 expression in the OSCC microenvironment. In this study, we showed that PD-L1 expression in high-grade invasive OSCC cell lines was lower than that in a low-grade invasive OSCC line and found a close correlation between PD-L1 expression and the epithelial-mesenchymal transition (EMT). PD-L1 expression was upregulated in macrophages and dendritic cells (DCs) in high-grade invasive human OSCC tissues or co-cultured with mesenchymal-phenotype OSCC cells in vitro. TLR4-inhibitory peptide successfully suppressed PD-L1 upregulation on macrophages and DCs co-cultured with mesenchymal-phenotype OSCC cells, suggesting that some EMT-induced tumour antigen is critical for PD-L1 induction on tumour-associated macrophages and DCs. Further studies are necessary to explore the impact of EMT on the tumour immune microenvironment and to identify potential biomarkers for selecting patients who might preferentially benefit from PD-1/PD-L1 blockade or immunotherapies more broadly. PMID:27922697

  8. EGFR High Expression, but not KRAS Status, Predicts Sensitivity of Pancreatic Cancer Cells to Nimotuzumab Treatment In Vivo.

    Science.gov (United States)

    Zhou, Chenfei; Zhu, Liangjun; Ji, Jun; Ding, Fangmi; Wang, Chao; Cai, Qu; Yu, Yingyan; Zhu, Zhenggang; Zhang, Jun

    2017-01-01

    Nimotuzumab is shown to be efficacious in advanced pancreatic cancer treatment, but its predictive marker has not been established. To investigate the impact of EGFR and KRAS status on antitumor efficacy of nimotuzumab and to explore its underlying mechanism. EGFR expressions of pancreatic cancer cell lines, BxPC3, Panc-1, and Patu-8988, were analyzed by Western blot and immunocytochemistry, and KRAS status was determined by gene sequencing. Anti-tumor effect of nimotuzumab were evaluated in vitro and in vivo. The expressions of related molecules in EGFR pathway and IL-6 was analyzed by Western blot, immunohistochemistry, and/or real-time PCR. BxPC3 cells had wild type KRAS and high-level EGFR; Panc-1 cells had mutant KRAS (G13A) and low-level EGFR; Patu-8988 cells had mutant KRAS (G12V) and high-level EGFR. Nimotuzumab did not affect cell proliferation or apoptosis in vitro. Growth of BxPC3 and Patu-8988 xenografts were significantly inhibited by nimotuzumab, but not Panc-1 xenografts, compared with that of the control group. Expression of EGFR in BxPC3 and Patu-8988 xenografts was significantly reduced by nimotuzumab. The IL-6 expression in BxPC3 and Patu-8988 xenografts was higher than that in Panc-1 xenografts in the control group, and was significantly reduced by nimotuzumab. Pancreatic cancer cells with EGFR high expression were more sensitive to nimotuzumab in vivo. KRAS status had no impact on anti-tumor efficacy of nimotuzumab in pancreatic cancer cells.

  9. High EVI1 expression predicts poor survival in acute myeloid leukemia: a study of 319 de novo AML patients.

    Science.gov (United States)

    Barjesteh van Waalwijk van Doorn-Khosrovani, Sahar; Erpelinck, Claudia; van Putten, Wim L J; Valk, Peter J M; van der Poel-van de Luytgaarde, Sonja; Hack, Ronald; Slater, Rosalyn; Smit, Elisabeth M E; Beverloo, H Berna; Verhoef, Gregor; Verdonck, Leo F; Ossenkoppele, Gert J; Sonneveld, Pieter; de Greef, Georgine E; Löwenberg, Bob; Delwel, Ruud

    2003-02-01

    The proto-oncogene EVI1 encodes a DNA binding protein and is located on chromosome 3q26. The gene is aberrantly expressed in acute myeloid leukemia (AML) patients carrying 3q26 abnormalities. Two mRNAs are transcribed from this locus: EVI1 and a fusion of EVI1 with MDS1 (MDS1-EVI1), a gene located 5' of EVI1. The purpose of this study was to investigate which of the 2 gene products is involved in transformation in human AML. To discriminate between EVI1 and MDS1-EVI1 transcripts, distinct real-time quantitative polymerase chain reaction (PCR) assays were developed. Patients with 3q26 abnormalities often showed high EVI1 and MDS1-EVI1 expression. In a cohort of 319 AML patients, 4 subgroups could be distinguished: EVI1(+) and MDS1-EVI1(-) (6 patients; group I), EVI1(+) and MDS1-EVI1(+) (26 patients; group II), EVI1(-) and MDS1-EVI1(+) (12 patients; group III), and EVI1(-) and MDS1-EVI1(-) (275 patients; group IV). The only 4 patients with a 3q26 aberration belonged to groups I and II. Interestingly, high EVI1 and not MDS1-EVI1 expression was associated with unfavorable karyotypes (eg, -7/7q-) or complex karyotypes. Moreover, a significant correlation was observed between EVI1 expression and 11q23 aberrations (mixed lineage leukemia [MLL] gene involvement). Patients from groups I and II had significantly shorter overall and event-free survival than patients in groups III and IV. Our data demonstrate that high EVI1 expression is an independent poor prognostic marker within the intermediate- risk karyotypic group.

  10. Gender-specific reduction of hepatic Mrp2 expression by high-fat diet protects female mice from ANIT toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Bo; Csanaky, Iván L. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Aleksunes, Lauren M. [Department of Pharmacology and Toxicology, School of Pharmacy and Environmental and Occupational Health Institute, Rutgers University, Piscataway, NJ (United States); Patni, Meghan; Chen, Qi; Ma, Xiaochao; Jaeschke, Hartmut [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Weir, Scott; Broward, Melinda; Klaassen, Curtis D. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); University of Kansas Cancer Center, Kansas City, KS (United States); Guo, Grace L., E-mail: lguo@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); University of Kansas Cancer Center, Kansas City, KS (United States)

    2012-06-01

    Emerging evidence suggests that feeding a high-fat diet (HFD) to rodents affects the expression of genes involved in drug transport. However, gender-specific effects of HFD on drug transport are not known. The multidrug resistance-associated protein 2 (Mrp2, Abcc2) is a transporter highly expressed in the hepatocyte canalicular membrane and is important for biliary excretion of glutathione-conjugated chemicals. The current study showed that hepatic Mrp2 expression was reduced by HFD feeding only in female, but not male, C57BL/6J mice. In order to determine whether down-regulation of Mrp2 in female mice altered chemical disposition and toxicity, the biliary excretion and hepatotoxicity of the Mrp2 substrate, α-naphthylisothiocyanate (ANIT), were assessed in male and female mice fed control diet or HFD for 4 weeks. ANIT-induced biliary injury is a commonly used model of experimental cholestasis and has been shown to be dependent upon Mrp2-mediated efflux of an ANIT glutathione conjugate that selectively injures biliary epithelial cells. Interestingly, HFD feeding significantly reduced early-phase biliary ANIT excretion in female mice and largely protected against ANIT-induced liver injury. In summary, the current study showed that, at least in mice, HFD feeding can differentially regulate Mrp2 expression and function and depending upon the chemical exposure may enhance or reduce susceptibility to toxicity. Taken together, these data provide a novel interaction between diet and gender in regulating hepatobiliary excretion and susceptibility to injury. -- Highlights: ► High-fat diet decreases hepatic Mrp2 expression only in female but not in male mice. ► HFD significantly reduces early-phase biliary ANIT excretion in female mice. ► HFD protects female mice against ANIT-induced liver injury.

  11. High cell density and high expression of recombinant human ApoA-Imilano in Eschenichia coli by twice temperature-shifted induction

    Institute of Scientific and Technical Information of China (English)

    ZHUANG Yingping; MA Wenfeng; GUO Meijin; DING Mansheng; CHU Ju; ZHANG Siliang

    2006-01-01

    The effect of temperature on the formation of recombinant protein,apolipoprotein A-IMilano was investigated in the present study.The temperature of the initial growth phase was set at 30℃,while temperature variation in induction phase was arranged in three modes.High cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried out.Experimental results showed that ApoA-IMilano reached 4.8 g/L with the final cell density of OD600,150.It was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density and the expression of the product.The present study provides a basic procedure for the production of recombinant ApoA-IMilano.

  12. Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.

    Science.gov (United States)

    Giritch, Anatoli; Marillonnet, Sylvestre; Engler, Carola; van Eldik, Gerben; Botterman, Johan; Klimyuk, Victor; Gleba, Yuri

    2006-10-03

    Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses that were found to be noncompeting. Unlike vectors derived from the same virus, noncompeting vectors effectively coexpress the heavy and light chains in the same cell throughout the plant body, resulting in yields of up to 0.5 g of assembled mAbs per kg of fresh-leaf biomass. This technology allows production of gram quantities of mAbs for research purposes in just several days, and the same protocol can be used on an industrial scale in situations requiring rapid response, such as pandemic or terrorism events.

  13. Mycobacterium tuberculosis RNA expression patterns in sputum bacteria indicate secreted Esx factors contributing to growth are highly expressed in active disease

    Directory of Open Access Journals (Sweden)

    Archana eBukka

    2012-01-01

    Full Text Available To identify factors contributing to the ability of tubercle bacilli to grow in the lung during active infection, we analyzed RNA expression patterns in bacteria present in patient sputum. Prominent among bacterial transcripts identified were those encoding secreted peptides of the Esat-6 subfamily that includes EsxK and EsxL (Rv1197 and Rv1198. H37Rv esxKL and esxJI transcripts were differentially expressed under different growth conditions, and disruption of these genes altered growth phase kinetics in typical laboratory batch broth cultures. These growth defects, including the reduced intracellular growth of an ΔesxKL mutant in primary human macrophages, were reversed by either low multiplicity co-infection or co-culture with wild-type bacteria, demonstrating the ability of the secreted factors to rescue isogenic mutants. Complementing either only esxL or esxI alone (Rv1198 or Rv1037c also reduced observed growth defects, indicating these genes encode factors capable of contributing to growth. Our studies indicate that the M. tuberculosis Mtb9.9 family secreted factors EsxL and EsxI can act in trans to modulate growth of intracellular bacteria, and are highly expressed during active human lung infection. EsxL (Rv1197 and Rv1198. The H37Rv genome contains 4 additional and nearly identical pairs of co-linear open reading frames designated esx JI, esx MN, esx PO, and esxWV. These ORFs show little sequence similarity to esxBA (Cfp10-Esat-6, other than encoding 2 short ~100 residue peptides with the 5’ ORF encoding a variant carboxyl-terminal 'QILSS' motif and the 3’ encoding the Mtb9.9 family of secreted T-cell antigens. All contain a central ‘WXG100’ esx family structural motif, and are thought to encode effectors of an uncharacterized ESX-5 transport system. esxKL and esxJI transcripts were differentially expressed under different growth conditions, and disruption of these genes altered different growth phase kinetics in typical

  14. High SINE RNA Expression Correlates with Post-Transcriptional Downregulation of BRCA1

    Directory of Open Access Journals (Sweden)

    Giovanni Bosco

    2013-04-01

    Full Text Available Short Interspersed Nuclear Elements (SINEs are non-autonomous retrotransposons that comprise a large fraction of the human genome. SINEs are demethylated in human disease, but whether SINEs become transcriptionally induced and how the resulting transcripts may affect the expression of protein coding genes is unknown. Here, we show that downregulation of the mRNA of the tumor suppressor gene BRCA1 is associated with increased transcription of SINEs and production of sense and antisense SINE small RNAs. We find that BRCA1 mRNA is post-transcriptionally down-regulated in a Dicer and Drosha dependent manner and that expression of a SINE inverted repeat with sequence identity to a BRCA1 intron is sufficient for downregulation of BRCA1 mRNA. These observations suggest that transcriptional activation of SINEs could contribute to a novel mechanism of RNA mediated post-transcriptional silencing of human genes.

  15. Endoplasmic reticulum aminopeptidase 2 is highly expressed in papillary thyroid microcarcinoma with cervical lymph node metastasis

    Directory of Open Access Journals (Sweden)

    Woo Young Kim

    2015-01-01

    Full Text Available Background: The cervical lymph node metastasis (CLNM of papillary thyroid microcarcinoma (PTMC is not uncommon. However, prophylactic cervical lymph node dissection in all PTMC is debatable. Molecular markers of predicting CLNM would help to decide to either do or not do cervical lymph node dissection which might increase morbidities. Aims: We aimed to characterize gene expression profiles and molecular markers of CLNM in PTMC. Settings and Design: The thyroid frozen tissues were obtained with from six PTMC patients, who underwent total thyroidectomy. Methods: We performed oligonucleotide microarray analysis with three PTMCs with CLNM and three without CLNM. Real-time quantitative reverse transcription-polymerase chain reaction was used to validate the gene. Statistical Analysis Used: We used linear models for microarray data. Results: We identified 12 differentially expressed gene, and most one is endoplasmic reticulum aminopeptidase 2 (ERAP2. Conclusion: ERAP2 might be associated with CLNM in PTMC.

  16. Functional expression of Rhizopus oryzae lipase in Pichia pastoris: high-level production and some properties.

    Science.gov (United States)

    Minning, S; Schmidt-Dannert, C; Schmid, R D

    1998-12-11

    The mature lipase of the fungus Rhizopus oryzae (ROL) was functionally expressed and secreted in the methylotrophic yeast Pichia pastoris. In a batch cultivation, where methanol feeding was linked to the dissolved oxygen content in the cultivation solution, a lipase activity of 500,000 units per liter (60 mg active lipase per liter) of culture was achieved after initial glycerol feeding of the culture. Recombinant ROL lipase was purified to homogeneity by a simple two-step purification procedure and had a specific activity of 8571 U mg-1 (triolein, 30 degrees C, pH 8.1) which is comparable with the purified native enzyme. The properties of the recombinant lipase were similar to those reported both for the native lipase and for the enzyme expressed in Escherichia coli and refolded from inactive inclusion bodies.

  17. High-level expression of a full-length Eph receptor.

    Science.gov (United States)

    Paavilainen, Sari; Grandy, David; Karelehto, Eveliina; Chang, Elizabeth; Susi, Petri; Erdjument-Bromage, Hediye; Nikolov, Dimitar; Himanen, Juha

    2013-11-01

    Eph receptors are the largest family of Receptor Tyrosine Kinases containing a single membrane-spanning segment. They are involved in a various developmental and cell-cell communication events. Although there is extensive structural information available on both the extra- and intracellular regions of Eph's in isolation, no structures are available for the entire receptor. To facilitate structural studies on functionally relevant Eph/ephrin complexes, we have developed an expression system for producing the full-length human EphA2 receptor. We successfully expressed milligram amounts of the receptor using baculovirus-based vector and insect cells. We were also able to extract the protein from the cell membranes and purify it to near homogeneity in two simple steps. The purified receptor was shown to retain its biological activity in terms of both binding to its functional ligands and being able to auto-phosphorylate the key tyrosine residues of the cytoplasmic kinase domain.

  18. High efficient expression of Lhcb2 gene from pea in E. coli and reconstitution of its expressed product with pigment in vitro

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Lhcb2 gene from pea (Pisum sativum L.) was subcloned into bacterial expression vector pET-3d, and its protein overexpressed was obtained from E. coli (BL21) containing PetpLhcb2 by site-directed mutagenesis method. Bacteria transformed with this construct yielded up to 40 percent of total protein of E. coli. Using the modified method with three subsequent cycles of freezing (1 min, -196℃) and thawing (15 min, 25℃), Lhcb2 protein purified was highly reconstituted with pigments to yield pigment-protein complexes. The reconstituted LHCB2 monomers were very similar to native LHCII monomers from spinach in partially denaturing polyacrylamide gel electrophoresis, fluorescence and absorbance spectroscopy. These results showed that Lhcb2 proteins overexpressed were reconstituted successfully with pigments and had similar organization and structure to the native LHCII monomers.

  19. High efficient expression of Lhcb2 gene from pea in E. coli and reconstitution of its expressed product with pigment in vitro

    Institute of Scientific and Technical Information of China (English)

    孙钦秒; 李良璧; 毛大璋; 匡廷云

    2000-01-01

    Lhcb2 gene from pea (Pisum sativum L.) was subcloned into bacterial expression vector pET-3d, and its protein overexpressed was obtained from E. coli (BL21) containing PetpLhcb2 by site-directed mutagenesis method. Bacteria transformed with this construct yielded up to 40 percent of total protein of E. coli. Using the modified method with three subsequent cycles of freezing (1 min, -196℃) and thawing (15 min, 25℃), Lhcb2 protein purified was highly reconstituted with pigments to yield pigment-protein complexes. The reconstituted LHCB2 monomers were very similar to native LHCll monomers from spinach in partially denaturing polyacrylamide gel electrophoresis, fluorescence and absorbance spectroscopy. These results showed that Lhcb2 proteins overexpressed were reconstituted successfully with pigments and had similar organization and structure to the native LHCII monomers.

  20. The hESC line Envy expresses high levels of GFP in all differentiated progeny.

    Science.gov (United States)

    Costa, Magdaline; Dottori, Mirella; Ng, Elizabeth; Hawes, Susan M; Sourris, Koula; Jamshidi, Pegah; Pera, Martin F; Elefanty, Andrew G; Stanley, Edouard G

    2005-04-01

    Human embryonic stem cells (hESCs) have been advanced as a potential source of cells for use in cell replacement therapies. The ability to identify hESCs and their differentiated progeny readily in transplantation experiments will facilitate the analysis of hESC potential and function in vivo. We have generated a hESC line designated 'Envy', in which robust levels of green fluorescent protein (GFP) are expressed in stem cells and all differentiated progeny.

  1. Development and application of a high-throughput cell-free expression system

    OpenAIRE

    Kai, Lei

    2012-01-01

    At the post genomics age, the study of proteomics attracts more and more attention and help to decipher the hidden information inside genes. However, the study of proteomics required large amount or large number of proteins to be synthesized. During the past several decades, heterologous overexpression of recombinant protein from bacteria, yeast, insect cells and mammalian cells was developed. Especially the E. coli system, which is the most studied expression system, was used to obtain diffe...

  2. Simple Analytic Expression with High Precision for the Barker-Henderson Diameter

    Institute of Scientific and Technical Information of China (English)

    孙久勋

    2003-01-01

    A fitting procedure is proposed to establish two analytic approximate expressions for the complicated Barker-Henderson (BH) diameter with Lennard-Jones potential in two temperature ranges. Considering that the differentiation is a process enlarging the errors and the integration decreasing the errors and that the derivative of BH diameter is important in the calculation of internal energy, we propose to fit the derivative directly, instead of usually fitting the original function and subsequently deriving its derivative. The simplicity and precision of two expressions developed are superior to the extensively used expressions in literature. The one with following form only has an average fitting 0.0063% in the reduced temperature range (0.4 ≤ kT/ε≤ 15), and can be extrapolated to a wider temperature range (0.4 ≤ kT/ε≤ 50) with an average error 0.13%, which is d/σ = 1.1755 + 0.02878 lnτ - 0.2072τ1/4 + 0.00463τ3/4.

  3. Automation of gene assignments to metabolic pathways using high-throughput expression data

    Directory of Open Access Journals (Sweden)

    Yona Golan

    2005-08-01

    Full Text Available Abstract Background Accurate assignment of genes to pathways is essential in order to understand the functional role of genes and to map the existing pathways in a given genome. Existing algorithms predict pathways by extrapolating experimental data in one organism to other organisms for which this data is not available. However, current systems classify all genes that belong to a specific EC family to all the pathways that contain the corresponding enzymatic reaction, and thus introduce ambiguity. Results Here we describe an algorithm for assignment of genes to cellular pathways that addresses this problem by selectively assigning specific genes to pathways. Our algorithm uses the set of experimentally elucidated metabolic pathways from MetaCyc, together with statistical models of enzyme families and expression data to assign genes to enzyme families and pathways by optimizing correlated co-expression, while minimizing conflicts due to shared assignments among pathways. Our algorithm also identifies alternative ("backup" genes and addresses the multi-domain nature of proteins. We apply our model to assign genes to pathways in the Yeast genome and compare the results for genes that were assigned experimentally. Our assignments are consistent with the experimentally verified assignments and reflect characteristic properties of cellular pathways. Conclusion We present an algorithm for automatic assignment of genes to metabolic pathways. The algorithm utilizes expression data and reduces the ambiguity that characterizes assignments that are based only on EC numbers.

  4. Colorectal cancers with aneuploids show high CD133 expression and poor prognosis

    Institute of Scientific and Technical Information of China (English)

    Dongdong Yu; Yonghong Zhang; You Zou; Ming Tian; Deding Tao; Junbo Hu; Jianping Gong

    2010-01-01

    Objective:The aim of the study was to investigate the relationship of DNA ploidy status in colorectal cancers with patients' prognosis and also the relationship of DNA ploidy status with expression of the colorectal cancer stem cell marker CD133.Methods:The DNA ploidy status and CD133 expression in colorectal cancers were detected by flow cytometry.The clinicopathological characteristics and progression-free survival analysis of patients was evaluated based on the clinical data.Results:DNA ploidy pattern did not correlated with gender,age,lesion diameter,histological type,depth of tumor invasion,lymphatic invasion and Dukes stage.Only primary lesion cite showed significant correlation with DNA ploidy pattern,more aneuploids were observed in colonic cancer than rectal cancer,P < 0.05.The 2-year progression-free survival rate and total progression-free time in patients with aneuploids were lower than that with diploids,P < 0.05.Tumors contained aneuploids showed higher expression of CD133 than tumors of only diploids,P < 0.05.Conclusion:Tumor DNA ploidy status is a significant prognostic factor in patients with colorectal cancer and also associated with the existence of CD133 positive colorectal cancer stem cells.

  5. PPARγ expression by rambutan peel extract in obesity rat model-induced high-calorie diet

    Institute of Scientific and Technical Information of China (English)

    Sri Rahayu Lestari; Muhammad Sasmito Djati; Achmad Rudijanto; Fatchiyah Fatchiyah

    2015-01-01

    Objective:To monitor the physiological characteristics and genes expression of obesity rat model after rambutan peel extract (RPE) treatment. Methods: Twenty-four 12-week-old male rats were divided into 4 groups: normal, obesity, obesity treated with ellagic acid (O-EA) and obesity treated withRPE30 (O-RPE30). Physiological characteristics were monitored by measuring body weight, calorie intake, size of adipocyte and level of triglyceride. Peroxisome proliferator activated receptorgamma (PPARγ), CCAAT/enhancer-binding proteinsαand fatty acid binding protein 4 (FABP4) expression were observed using immunohistochemistry, western blotting and quantitativeRT-PCR methods. Results: Body weight gain ofO-EA andO-RPE30 rats were lower than obesity group and size of adipocyte cells were smaller than obesity group (P Conclusions:RPE have anti-obesity activity by inhibiting body weight gain, declining size of adipocyte, decreasing triglyceride,PPARγ expression and mRNA level ofFABP4 gene on obesity rat model.

  6. DNA methylation in small cell lung cancer defines distinct disease subtypes and correlates with high expression of EZH2

    Science.gov (United States)

    Poirier, John T.; Gardner, Eric E.; Connis, Nick; Moreira, Andre L.; de Stanchina, Elisa; Hann, Christine L.; Rudin, Charles M.

    2015-01-01

    Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis, rapid development of resistance to chemotherapy, and genetic instability. This study profiles DNA methylation in SCLC, patient-derived xenografts (PDXs) and cell lines at single nucleotide resolution. DNA methylation patterns of primary samples are distinct from those of cell lines, while PDXs maintain a pattern closely consistent with primary samples. Clustering of DNA methylation and gene expression of primary SCLC revealed distinct disease subtypes among histologically indistinguishable primary patient samples with similar genetic alterations. SCLC is notable for dense clustering of high-level methylation in discrete promoter CpG islands, in a pattern clearly distinct from other lung cancers and strongly correlated with high expression of the E2F target and histone methyltransferase gene EZH2. Pharmacologic inhibition of EZH2 in a SCLC PDX markedly inhibited tumor growth. PMID:25746006

  7. DNA methylation in small cell lung cancer defines distinct disease subtypes and correlates with high expression of EZH2.

    Science.gov (United States)

    Poirier, J T; Gardner, E E; Connis, N; Moreira, A L; de Stanchina, E; Hann, C L; Rudin, C M

    2015-11-26

    Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis, rapid development of resistance to chemotherapy and genetic instability. This study profiles DNA methylation in SCLC, patient-derived xenografts (PDX) and cell lines at single-nucleotide resolution. DNA methylation patterns of primary samples are distinct from those of cell lines, whereas PDX maintain a pattern closely consistent with primary samples. Clustering of DNA methylation and gene expression of primary SCLC revealed distinct disease subtypes among histologically indistinguishable primary patient samples with similar genetic alterations. SCLC is notable for dense clustering of high-level methylation in discrete promoter CpG islands, in a pattern clearly distinct from other lung cancers and strongly correlated with high expression of the E2F target and histone methyltransferase gene EZH2. Pharmacologic inhibition of EZH2 in a SCLC PDX markedly inhibited tumor growth.

  8. Rasch-based high-dimensionality data reduction and class prediction with applications to microarray gene expression data

    CERN Document Server

    Kastrin, Andrej

    2010-01-01

    Class prediction is an important application of microarray gene expression data analysis. The high-dimensionality of microarray data, where number of genes (variables) is very large compared to the number of samples (obser- vations), makes the application of many prediction techniques (e.g., logistic regression, discriminant analysis) difficult. An efficient way to solve this prob- lem is by using dimension reduction statistical techniques. Increasingly used in psychology-related applications, Rasch model (RM) provides an appealing framework for handling high-dimensional microarray data. In this paper, we study the potential of RM-based modeling in dimensionality reduction with binarized microarray gene expression data and investigate its prediction ac- curacy in the context of class prediction using linear discriminant analysis. Two different publicly available microarray data sets are used to illustrate a general framework of the approach. Performance of the proposed method is assessed by re-randomization s...

  9. Detailed insight into hordeins expression in high protein cultivars as a first stage to improve feed quality

    DEFF Research Database (Denmark)

    Kaczmarczyk, Agnieszka Ewa; Aaslo, Per; Vincze, Éva

    2011-01-01

    in all samples were analyzed using UPLC method. It was noticed that there are big differences in quantity and quality of storage proteins transcripts in consecutive developmental phases and among selected cultivars. The data about mRNA expression, amino acids and total protein concentrations were plotted...... novel cereal cultivars with high protein concentration and better amino acid composition. Six barley cultivars with high protein content (Netto, PR3440, Kontiki, Fairytale, PR3528 and IC364) and Golden Promise as a control were selected for analysis of hordeins expression at four stages of grain...... development. All the known hordein coding sequences collected from commonly available databases (NCBI, HarvEST) were classified into groups and subgroups (B1, B3, C, D and Γ). Specific primers were designed with Primique and AlleleID softwares and checked with qRT-PCR first on the control cultivar Golden...

  10. Escherichia coli–expressed near full length HIV-1 envelope glycoprotein is a highly sensitive and specific diagnostic antigen

    Directory of Open Access Journals (Sweden)

    Talha Sheikh M

    2012-11-01

    Full Text Available Abstract Background The Human Immunodeficiency Virus type 1 (HIV-1 envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies. Methods A deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni(II-affinity chromatography. Biotinylated and europium(III chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n=131, that included an in-house panel and four commercially procured panels. Results In-frame deletion of three hydrophobic regions, spanning amino acid residues 1–43, 519–538 and 676–706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C, and human T cell leukemia viruses. Conclusions This study has demonstrated the expression of r-gp160 variant in E. coli, by deletion

  11. High expression of arachidonate 15-lipoxygenase and proinflammatory markers in human ischemic heart tissue

    Energy Technology Data Exchange (ETDEWEB)

    Magnusson, Lisa U.; Lundqvist, Annika [Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg Laboratory, Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg (Sweden); Asp, Julia [Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg (Sweden); Synnergren, Jane [Systems Biology Research Center, School of Life Sciences, University of Skoevde, Skoevde (Sweden); Johansson, Cecilia Thalen [Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg Laboratory, Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg (Sweden); Palmqvist, Lars [Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg (Sweden); Jeppsson, Anders [Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg (Sweden); Hulten, Lillemor Mattsson, E-mail: Lillemor.Mattsson@wlab.gu.se [Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg Laboratory, Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Sahlgrenska University Hospital, Gothenburg (Sweden)

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer We found a 17-fold upregulation of ALOX15 in the ischemic heart. Black-Right-Pointing-Pointer Incubation of human muscle cells in hypoxia showed a 22-fold upregulation of ALOX15. Black-Right-Pointing-Pointer We observed increased levels of proinflammatory markers in ischemic heart tissue. Black-Right-Pointing-Pointer Suggesting a link between ischemia and inflammation in ischemic heart biopsies. -- Abstract: A common feature of the ischemic heart and atherosclerotic plaques is the presence of hypoxia (insufficient levels of oxygen in the tissue). Hypoxia has pronounced effects on almost every aspect of cell physiology, and the nuclear transcription factor hypoxia inducible factor-1{alpha} (HIF-1{alpha}) regulates adaptive responses to low concentrations of oxygen in mammalian cells. In our recent work, we observed that hypoxia increases the proinflammatory enzyme arachidonate 15-lipoxygenase (ALOX15B) in human carotid plaques. ALOX15 has recently been shown to be present in the human myocardium, but the effect of ischemia on its expression has not been investigated. Here we test the hypothesis that ischemia of the heart leads to increased expression