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Sample records for a3ar expression level

  1. Pharmacological and Therapeutic Effects of A3 Adenosine Receptor (A3AR) Agonists

    PubMed Central

    Fishman, Pnina; Bar-Yehuda, Sara; Liang, Bruce T.; Jacobson, Kenneth A.

    2011-01-01

    The Gi-coupled A3 adenosine receptor (A3AR) mediates anti-inflammatory, anticancer and anti-ischemic protective effects. The receptor is overexpressed in inflammatory and cancer cells, while low expression is found in normal cells, rendering the A3AR as a potential therapeutic target. Highly selective A3AR agonists have been synthesized and molecular recognition in the binding site has been characterized. The present review summarizes preclinical and clinical human studies demonstrating that A3AR agonists induce specific anti-inflammatory and anticancer effects via a molecular mechanism that entails modulation of the Wnt and the NF-κB signal transduction pathways. Currently, A3AR agonists are being developed for the treatment of inflammatory diseases including rheumatoid arthritis and psoriasis; ophthalmic diseases such as dry eye syndrome and glaucoma; liver diseases such as hepatocellular carcinoma and hepatitis. PMID:22033198

  2. The A3 adenosine receptor (A3AR): therapeutic target and predictive biological marker in rheumatoid arthritis.

    PubMed

    Fishman, Pnina; Cohen, Shira

    2016-09-01

    The Gi protein-associated A3 adenosine receptor (A3AR) is over-expressed in inflammatory cells, and this high expression is also reflected in the peripheral blood mononuclear cells of patients with autoimmune inflammatory diseases such as rheumatoid arthritis, psoriasis, and Crohn's disease. CF101, a selective agonist with high affinity to the A3AR, is known to induce robust anti-inflammatory effect in experimental animal models of adjuvant-, collagen-, and tropomyosin-induced arthritis. The effect is mediated via a definitive molecular mechanism entailing deregulation of the nuclear factor-κB (NF-κB) and the Wnt signal transduction pathways resulting in apoptosis of inflammatory cells. CF101 was found to be safe and well tolerated in all preclinical, phase I, and phase II human clinical studies. In two phase II clinical studies where CF101 was administered to rheumatoid arthritis (RA) patients as a stand-alone drug, a significant anti-rheumatic effect and a direct significant correlation were found between receptor expression at baseline and patients' response to the drug, suggesting that A3AR may be utilized as a predictive biomarker. The A3AR is a promising therapeutic target in rheumatoid arthritis and can be used also as a biological marker to predict patients' response to CF101. This is a unique type of a personalized medicine approach which may pave the way for a safe and efficacious treatment for this patient population. PMID:26886128

  3. Engagement of the GABA to KCC2 signaling pathway contributes to the analgesic effects of A3AR agonists in neuropathic pain.

    PubMed

    Ford, Amanda; Castonguay, Annie; Cottet, Martin; Little, Joshua W; Chen, Zhoumou; Symons-Liguori, Ashley M; Doyle, Timothy; Egan, Terrance M; Vanderah, Todd W; De Konnick, Yves; Tosh, Dilip K; Jacobson, Kenneth A; Salvemini, Daniela

    2015-04-15

    More than 1.5 billion people worldwide suffer from chronic pain, yet current treatment strategies often lack efficacy or have deleterious side effects in patients. Adenosine is an inhibitory neuromodulator that was previously thought to mediate antinociception through the A1 and A2A receptor subtypes. We have since demonstrated that A3AR agonists have potent analgesic actions in preclinical rodent models of neuropathic pain and that A3AR analgesia is independent of adenosine A1 or A2A unwanted effects. Herein, we explored the contribution of the GABA inhibitory system to A3AR-mediated analgesia using well-characterized mouse and rat models of chronic constriction injury (CCI)-induced neuropathic pain. The deregulation of GABA signaling in pathophysiological pain states is well established: GABA signaling can be hampered by a reduction in extracellular GABA synthesis by GAD65 and enhanced extracellular GABA reuptake via the GABA transporter, GAT-1. In neuropathic pain, GABAAR-mediated signaling can be further disrupted by the loss of the KCC2 chloride anion gradient. Here, we demonstrate that A3AR agonists (IB-MECA and MRS5698) reverse neuropathic pain via a spinal mechanism of action that modulates GABA activity. Spinal administration of the GABAA antagonist, bicuculline, disrupted A3AR-mediated analgesia. Furthermore, A3AR-mediated analgesia was associated with reductions in CCI-related GAD65 and GAT-1 serine dephosphorylation as well as an enhancement of KCC2 serine phosphorylation and activity. Our results suggest that A3AR-mediated reversal of neuropathic pain increases modulation of GABA inhibitory neurotransmission both directly and indirectly through protection of KCC2 function, underscoring the unique utility of A3AR agonists in chronic pain. PMID:25878279

  4. The anti-inflammatory target A(3) adenosine receptor is over-expressed in rheumatoid arthritis, psoriasis and Crohn's disease.

    PubMed

    Ochaion, A; Bar-Yehuda, S; Cohen, S; Barer, F; Patoka, R; Amital, H; Reitblat, T; Reitblat, A; Ophir, J; Konfino, I; Chowers, Y; Ben-Horin, S; Fishman, P

    2009-01-01

    The Gi protein associated A(3) adenosine receptor (A(3)AR) was recently defined as a novel anti-inflammatory target. The aim of this study was to look at A(3)AR expression levels in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory diseases and to explore transcription factors involved receptor expression. Over-expression of A(3)AR was found in PBMCs derived from patients with rheumatoid arthritis (RA), psoriasis and Crohn's disease compared with PBMCs from healthy subjects. Bioinformatics analysis demonstrated the presence of DNA binding sites for nuclear factor-kappaB (NF-kappaB) and cyclic AMP-responsive element binding protein (CREB) in the A(3)AR gene promoter. Up-regulation of NF-kappaB and CREB was found in the PBMCs from patients with RA, psoriasis and Crohn's disease. The PI3K-PKB/Akt signaling pathway, known to regulate both the NF-kappaB and CREB, was also up-regulated in the patients' PBMCs. Taken together, NF-kappaB and CREB are involved with the over-expression of A(3)AR in patients with autoimmune inflammatory diseases. The receptor may be considered as a specific target to combat inflammation. PMID:19426966

  5. Sexual differences of imprinted genes' expression levels.

    PubMed

    Faisal, Mohammad; Kim, Hana; Kim, Joomyeong

    2014-01-01

    In mammals, genomic imprinting has evolved as a dosage-controlling mechanism for a subset of genes that play critical roles in their unusual reproduction scheme involving viviparity and placentation. As such, many imprinted genes are highly expressed in sex-specific reproductive organs. In the current study, we sought to test whether imprinted genes are differentially expressed between the two sexes. According to the results, the expression levels of the following genes differ between the two sexes of mice: Peg3, Zim1, Igf2, H19 and Zac1. The expression levels of these imprinted genes are usually greater in males than in females. This bias is most obvious in the developing brains of 14.5-dpc embryos, but also detected in the brains of postnatal-stage mice. However, this sexual bias is not obvious in 10.5-dpc embryos, a developmental stage before the sexual differentiation. Thus, the sexual bias observed in the imprinted genes is most likely attributable by gonadal hormones rather than by sex chromosome complement. Overall, the results indicate that several imprinted genes are sexually different in terms of their expression levels, and further suggest that the transcriptional regulation of these imprinted genes may be influenced by unknown mechanisms associated with sexual differentiation. PMID:24125951

  6. Population-level control of gene expression

    NASA Astrophysics Data System (ADS)

    Nevozhay, Dmitry; Adams, Rhys; van Itallie, Elizabeth; Bennett, Matthew; Balazsi, Gabor

    2011-03-01

    Gene expression is the process that translates genetic information into proteins, that determine the way cells live, function and even die. It was demonstrated that cells with identical genomes exposed to the same environment can differ in their protein composition and therefore phenotypes. Protein levels can vary between cells due to the stochastic nature of intracellular biochemical events, indicating that the genotype-phenotype connection is not deterministic at the cellular level. We asked whether genomes could encode isogenic cell populations more reliably than single cells. To address this question, we built two gene circuits to control three cell population-level characteristics: gene expression mean, coefficient of variation and non-genetic memory of previous expression states. Indeed, we found that these population-level characteristics were more predictable than the gene expression of single cells in a well-controlled environment. This research was supported by the NIH Director's New Innovator Award 1DP2 OD006481-01 and Welch Foundation Grant C-1729.

  7. A promoter-level mammalian expression atlas

    PubMed Central

    2015-01-01

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research. PMID:24670764

  8. The Level of Expressed Emotion Scale: A Useful Measure of Expressed Emotion in Adolescents?

    ERIC Educational Resources Information Center

    Nelis, Sharon M.; Rae, Gordon; Liddell, Christine

    2011-01-01

    Research has suggested that self-report measures of expressed emotion (EE) may be employed as a proxy measure of environmental stress in the home. The appropriateness of the Level of Expressed Emotion scale as a measure of perceived expressed emotion was examined in a sample of adolescents. Participants were 239 male and 422 female adolescents…

  9. How to achieve high-level expression of microbial enzymes

    PubMed Central

    Liu, Long; Yang, Haiquan; Shin, Hyun-dong; Chen, Rachel R.; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-01-01

    Microbial enzymes have been used in a large number of fields, such as chemical, agricultural and biopharmaceutical industries. The enzyme production rate and yield are the main factors to consider when choosing the appropriate expression system for the production of recombinant proteins. Recombinant enzymes have been expressed in bacteria (e.g., Escherichia coli, Bacillus and lactic acid bacteria), filamentous fungi (e.g., Aspergillus) and yeasts (e.g., Pichia pastoris). The favorable and very advantageous characteristics of these species have resulted in an increasing number of biotechnological applications. Bacterial hosts (e.g., E. coli) can be used to quickly and easily overexpress recombinant enzymes; however, bacterial systems cannot express very large proteins and proteins that require post-translational modifications. The main bacterial expression hosts, with the exception of lactic acid bacteria and filamentous fungi, can produce several toxins which are not compatible with the expression of recombinant enzymes in food and drugs. However, due to the multiplicity of the physiological impacts arising from high-level expression of genes encoding the enzymes and expression hosts, the goal of overproduction can hardly be achieved, and therefore, the yield of recombinant enzymes is limited. In this review, the recent strategies used for the high-level expression of microbial enzymes in the hosts mentioned above are summarized and the prospects are also discussed. We hope this review will contribute to the development of the enzyme-related research field. PMID:23686280

  10. Serum adropin level and ENHO gene expression in systemic sclerosis.

    PubMed

    Yolbas, Servet; Kara, Murat; Yilmaz, Musa; Aydin, Suleyman; Koca, Suleyman Serdar

    2016-06-01

    Adropin, a secreted protein, is encoded by the energy homeostasis associated (ENHO) gene. It has been implicated in the several physiological and pathological processes such as angiogenesis and apoptosis. Therefore, the aim of present study was to investigate serum adropin levels and ENHO gene expressions in systemic sclerosis (SSc) characterized by vasculopathy, inflammation, and progressive fibrosis of the skin and internal organs. The study includes 27 patients with SSc, 39 patients with Behçet's disease (BD), and 20 healthy controls (HC). Serum adropin levels and ENHO gene expressions by peripheral blood mononuclear cells were analyzed by ELISA method and by real-time PCR, respectively. The serum adropin levels were higher in the SSc and BD groups than in the HC group (p = 0.023 and p < 0.001, respectively). However, there were no significant differences among the groups in terms of ENHO gene expressions (p ANOVA = 0.149). There was no significant difference between the limited and diffuse cutaneous subtypes of SSc in terms of serum adropin level and ENHO gene expression. Moreover, serum adropin level and ENHO gene expression were not associated with the disease activity and severity indexes. ENHO gene expression was correlated with the triglyceride levels in the BD group (r = -0.426, p = 0.027). The augmented serum adropin levels may be expected in the chronic inflammatory disease and seem not to be characteristic of only SSc. However, further studies are needed to explain the precise role of adropin in SSc. PMID:27079850

  11. Calcium regulates caveolin-1 expression at the transcriptional level

    SciTech Connect

    Yang, Xiao-Yan; Huang, Cheng-Cheng; Kan, Qi-Ming; Li, Yan; Liu, Dan; Zhang, Xue-Cheng; Sato, Toshinori; Yamagata, Sadako; Yamagata, Tatsuya

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Caveolin-1 expression is regulated by calcium signaling at the transcriptional level. Black-Right-Pointing-Pointer An inhibitor of or siRNA to L-type calcium channel suppressed caveolin-1 expression. Black-Right-Pointing-Pointer Cyclosporine A or an NFAT inhibitor markedly reduced caveolin-1 expression. Black-Right-Pointing-Pointer Caveolin-1 regulation by calcium signaling is observed in several mouse cell lines. -- Abstract: Caveolin-1, an indispensable component of caveolae serving as a transformation suppressor protein, is highly expressed in poorly metastatic mouse osteosarcoma FBJ-S1 cells while highly metastatic FBJ-LL cells express low levels of caveolin-1. Calcium concentration is higher in FBJ-S1 cells than in FBJ-LL cells; therefore, we investigated the possibility that calcium signaling positively regulates caveolin-1 in mouse FBJ-S1 cells. When cells were treated with the calcium channel blocker nifedipine, cyclosporin A (a calcineurin inhibitor), or INCA-6 (a nuclear factor of activated T-cells [NFAT] inhibitor), caveolin-1 expression at the mRNA and protein levels decreased. RNA silencing of voltage-dependent L-type calcium channel subunit alpha-1C resulted in suppression of caveolin-1 expression. This novel caveolin-1 regulation pathway was also identified in mouse NIH 3T3 cells and Lewis lung carcinoma cells. These results indicate that caveolin-1 is positively regulated at the transcriptional level through a novel calcium signaling pathway mediated by L-type calcium channel/Ca{sup 2+}/calcineurin/NFAT.

  12. In plants, expression breadth and expression level distinctly and non-linearly correlate with gene structure

    PubMed Central

    2009-01-01

    Background Compactness of highly/broadly expressed genes in human has been explained as selection for efficiency, regional mutation biases or genomic design. However, highly expressed genes in flowering plants were shown to be less compact than lowly expressed ones. On the other hand, opposite facts have also been documented that pollen-expressed Arabidopsis genes tend to contain shorter introns and highly expressed moss genes are compact. This issue is important because it provides a chance to compare the selectionism and the neutralism views about genome evolution. Furthermore, this issue also helps to understand the fates of introns, from the angle of gene expression. Results In this study, I used expression data covering more tissues and employ new analytical methods to reexamine the correlations between gene expression and gene structure for two flowering plants, Arabidopsis thaliana and Oryza sativa. It is shown that, different aspects of expression pattern correlate with different parts of gene sequences in distinct ways. In detail, expression level is significantly negatively correlated with gene size, especially the size of non-coding regions, whereas expression breadth correlates with non-coding structural parameters positively and with coding region parameters negatively. Furthermore, the relationships between expression level and structural parameters seem to be non-linear, with the extremes of structural parameters possibly scale as power-laws or logrithmic functions of expression levels. Conclusion In plants, highly expressed genes are compact, especially in the non-coding regions. Broadly expressed genes tend to contain longer non-coding sequences, which may be necessary for complex regulations. In combination with previous studies about other plants and about animals, some common scenarios about the correlation between gene expression and gene structure begin to emerge. Based on the functional relationships between extreme values of structural

  13. Affective State Level Recognition in Naturalistic Facial and Vocal Expressions.

    PubMed

    Meng, Hongying; Bianchi-Berthouze, Nadia

    2014-03-01

    Naturalistic affective expressions change at a rate much slower than the typical rate at which video or audio is recorded. This increases the probability that consecutive recorded instants of expressions represent the same affective content. In this paper, we exploit such a relationship to improve the recognition performance of continuous naturalistic affective expressions. Using datasets of naturalistic affective expressions (AVEC 2011 audio and video dataset, PAINFUL video dataset) continuously labeled over time and over different dimensions, we analyze the transitions between levels of those dimensions (e.g., transitions in pain intensity level). We use an information theory approach to show that the transitions occur very slowly and hence suggest modeling them as first-order Markov models. The dimension levels are considered to be the hidden states in the Hidden Markov Model (HMM) framework. Their discrete transition and emission matrices are trained by using the labels provided with the training set. The recognition problem is converted into a best path-finding problem to obtain the best hidden states sequence in HMMs. This is a key difference from previous use of HMMs as classifiers. Modeling of the transitions between dimension levels is integrated in a multistage approach, where the first level performs a mapping between the affective expression features and a soft decision value (e.g., an affective dimension level), and further classification stages are modeled as HMMs that refine that mapping by taking into account the temporal relationships between the output decision labels. The experimental results for each of the unimodal datasets show overall performance to be significantly above that of a standard classification system that does not take into account temporal relationships. In particular, the results on the AVEC 2011 audio dataset outperform all other systems presented at the international competition. PMID:23757552

  14. NORMAL NASAL GENE EXPRESSION LEVELS USING CDNA ARRAY TECHNOLOGY

    EPA Science Inventory

    Normal Nasal Gene Expression Levels Using cDNA Array Technology.

    The nasal epithelium is a target site for chemically-induced toxicity and carcinogenicity. To detect and analyze genetic events which contribute to nasal tumor development, we first defined the gene expressi...

  15. Protein expression analyses at the single cell level.

    PubMed

    Ohno, Masae; Karagiannis, Peter; Taniguchi, Yuichi

    2014-01-01

    The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level. PMID:25197931

  16. Variability among individuals is generated at the gene expression level.

    PubMed

    Peck, Lloyd S; Thorne, Michael A S; Hoffman, Joseph I; Morley, Simon A; Clark, Melody S

    2015-07-01

    Selection acts on individuals, specifically on their differences. To understand adaptation and responses to change therefore requires knowledge of how variation is generated and distributed across traits. Variation occurs on different biological scales, from genetic through physiological to morphological, yet it is unclear which of these carries the most variability. For example, if individual variation is mainly generated by differences in gene expression, variability should decrease progressively from coding genes to morphological traits, whereas if post-translational and epigenetic effects increase variation, the opposite should occur. To test these predictions, we compared levels of variation among individuals in various measures of gene expression, physiology (including activity), and morphology in two abundant and geographically widespread Antarctic molluscs, the clam Laternula elliptica and the limpet Nacella concinna. Direct comparisons among traits as diverse as heat shock protein QPCR assays, whole transcription profiles, respiration rates, burying rate, shell length, and ash-free dry mass were made possible through the novel application of an established metric, the Wentworth Scale. In principle, this approach could be extended to analyses of populations, communities, or even entire ecosystems. We found consistently greater variation in gene expression than morphology, with physiological measures falling in between. This suggests that variability is generated at the gene expression level. These findings have important implications for refining current biological models and predictions of how biodiversity may respond to climate change. PMID:26378322

  17. Schizophrenia Gene Expression Profile Reverted to Normal Levels by Antipsychotics

    PubMed Central

    Crespo-Facorro, Benedicto; Prieto, Carlos

    2015-01-01

    Background: Despite the widespread use of antipsychotics, little is known of the molecular bases behind the action of antipsychotic drugs. A genome-wide study is needed to characterize the genes that affect the clinical response and their adverse effects. Methods: Here we show the analysis of the blood transcriptome of 22 schizophrenia patients before and after medication with atypical antipsychotics by next-generation sequencing. Results: We found that 17 genes, among the 21 495 genes analyzed, have significantly-altered expression after medication (p-value adjusted [Padj] <0.05). Six genes (ADAMTS2, CD177, CNTNAP3, ENTPD2, RFX2, and UNC45B) out of the 17 are among the 200 genes that we characterized with differential expression in a previous study between antipsychotic-naïve schizophrenia patients and controls (Sainz et al., 2013). This number of schizophrenia-altered expression genes is significantly higher than expected by chance (Chi-test, Padj 1.19E-50), suggesting that at least part of the antipsychotic beneficial effects is exerted by modulating the expression of these genes. Interestingly, all six of these genes were overexpressed in patients and reverted to control levels of expression after treatment. We also found a significant enrichment of genes related to obesity and diabetes, known adverse affects of antipsychotics. Conclusions: These results may facilitate understanding of unknown molecular mechanisms behind schizophrenia symptoms and the molecular mechanisms of antipsychotic drugs. PMID:25522406

  18. Diversity of Matriptase Expression Level and Function in Breast Cancer

    PubMed Central

    Welman, Arkadiusz; Sproul, Duncan; Mullen, Peter; Muir, Morwenna; Kinnaird, Andrew R.; Harrison, David J.; Faratian, Dana; Brunton, Valerie G.; Frame, Margaret C.

    2012-01-01

    Overexpression of matriptase has been reported in a variety of human cancers and is sufficient to trigger tumor formation in mice, but the importance of matriptase in breast cancer remains unclear. We analysed matriptase expression in 16 human breast cancer cell lines and in 107 primary breast tumors. The data revealed considerable diversity in the expression level of this protein indicating that the significance of matriptase may vary from case to case. Matriptase protein expression was correlated with HER2 expression and highest expression was seen in HER2-positive cell lines, indicating a potential role in this subgroup. Stable overexpression of matriptase in two breast cancer cell lines had different consequences. In MDA-MB-231 human breast carcinoma cells the only noted consequence of matriptase overexpression was modestly impaired growth in vivo. In contrast, overexpression of matriptase in 4T1 mouse breast carcinoma cells resulted in visible changes in morphology, actin staining and cell to cell contacts. This correlated with downregulation of the cell-cell adhesion molecule E-cadherin. These results suggest that the functions of matriptase in breast cancer are likely to be variable and cell context dependent. PMID:22514623

  19. Translation Levels Control Multi-Spanning Membrane Protein Expression

    PubMed Central

    Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.

    2012-01-01

    Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane. PMID:22563408

  20. Localizing PRL-2 expression and determining the effects of dietary Mg(2+) on expression levels.

    PubMed

    Gungabeesoon, Jeremy; Tremblay, Michel L; Uetani, Noriko

    2016-07-01

    The phosphatase of regenerating liver (PRL) is a group of protein tyrosine phosphatases that play a key role in cancer progression and metastasis. We previously showed that PRL-2 modulates intracellular Mg(2+) levels and sustains cancer phenotypes by binding to the Mg(2+) transporter CNNM3. However, the physiological functions of PRL-2 in animals remain largely unknown. To better understand which cell types are associated with PRL-2 function, we characterized its expression in mouse tissues using a PRL-2 β-galactosidase reporter mouse model. Our results demonstrated that PRL-2 was ubiquitously expressed, with the highest expression levels observed in the hippocampal pyramidal neurons, ependymal cells, cone and rod photoreceptor cells, endocardium, vascular and bronchial smooth muscle, and collecting ducts in the kidney. On the other hand, PRL-2 expression was undetectable or very low in the parenchymal cells of the liver and pancreas. Our results also indicated that PRL-2 is involved in cell-type-specific Mg(2+) homeostasis and that PRL-2 expression is potentially inversely regulated by dietary Mg(2+) levels. PMID:27015884

  1. The genetic architecture of gene expression levels in wild baboons

    PubMed Central

    Tung, Jenny; Zhou, Xiang; Alberts, Susan C; Stephens, Matthew; Gilad, Yoav

    2015-01-01

    Primate evolution has been argued to result, in part, from changes in how genes are regulated. However, we still know little about gene regulation in natural primate populations. We conducted an RNA sequencing (RNA-seq)-based study of baboons from an intensively studied wild population. We performed complementary expression quantitative trait locus (eQTL) mapping and allele-specific expression analyses, discovering substantial evidence for, and surprising power to detect, genetic effects on gene expression levels in the baboons. eQTL were most likely to be identified for lineage-specific, rapidly evolving genes; interestingly, genes with eQTL significantly overlapped between baboons and a comparable human eQTL data set. Our results suggest that genes vary in their tolerance of genetic perturbation, and that this property may be conserved across species. Further, they establish the feasibility of eQTL mapping using RNA-seq data alone, and represent an important step towards understanding the genetic architecture of gene expression in primates. DOI: http://dx.doi.org/10.7554/eLife.04729.001 PMID:25714927

  2. The Expression Level of Septin12 Is Critical for Spermiogenesis

    PubMed Central

    Lin, Ying-Hung; Lin, Yung-Ming; Wang, Ya-Yun; Yu, I-Shing; Lin, Yi-Wen; Wang, Yun-Han; Wu, Ching-Ming; Pan, Hsien-An; Chao, Shin-Chih; Yen, Pauline H.; Lin, Shu-Wha; Kuo, Pao-Lin

    2009-01-01

    Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis. PMID:19359518

  3. Comprehensive analysis of Arabidopsis expression level polymorphisms with simple inheritance.

    PubMed

    Plantegenet, Stephanie; Weber, Johann; Goldstein, Darlene R; Zeller, Georg; Nussbaumer, Cindy; Thomas, Jérôme; Weigel, Detlef; Harshman, Keith; Hardtke, Christian S

    2009-01-01

    In Arabidopsis thaliana, gene expression level polymorphisms (ELPs) between natural accessions that exhibit simple, single locus inheritance are promising quantitative trait locus (QTL) candidates to explain phenotypic variability. It is assumed that such ELPs overwhelmingly represent regulatory element polymorphisms. However, comprehensive genome-wide analyses linking expression level, regulatory sequence and gene structure variation are missing, preventing definite verification of this assumption. Here, we analyzed ELPs observed between the Eil-0 and Lc-0 accessions. Compared with non-variable controls, 5' regulatory sequence variation in the corresponding genes is indeed increased. However, approximately 42% of all the ELP genes also carry major transcription unit deletions in one parent as revealed by genome tiling arrays, representing a >4-fold enrichment over controls. Within the subset of ELPs with simple inheritance, this proportion is even higher and deletions are generally more severe. Similar results were obtained from analyses of the Bay-0 and Sha accessions, using alternative technical approaches. Collectively, our results suggest that drastic structural changes are a major cause for ELPs with simple inheritance, corroborating experimentally observed indel preponderance in cloned Arabidopsis QTL. PMID:19225455

  4. Massively Parallel Interrogation of the Effects of Gene Expression Levels on Fitness.

    PubMed

    Keren, Leeat; Hausser, Jean; Lotan-Pompan, Maya; Vainberg Slutskin, Ilya; Alisar, Hadas; Kaminski, Sivan; Weinberger, Adina; Alon, Uri; Milo, Ron; Segal, Eran

    2016-08-25

    Data of gene expression levels across individuals, cell types, and disease states is expanding, yet our understanding of how expression levels impact phenotype is limited. Here, we present a massively parallel system for assaying the effect of gene expression levels on fitness in Saccharomyces cerevisiae by systematically altering the expression level of ∼100 genes at ∼100 distinct levels spanning a 500-fold range at high resolution. We show that the relationship between expression levels and growth is gene and environment specific and provides information on the function, stoichiometry, and interactions of genes. Wild-type expression levels in some conditions are not optimal for growth, and genes whose fitness is greatly affected by small changes in expression level tend to exhibit lower cell-to-cell variability in expression. Our study addresses a fundamental gap in understanding the functional significance of gene expression regulation and offers a framework for evaluating the phenotypic effects of expression variation. PMID:27545349

  5. Multi-level Expression Design Language: Requirement level (MEDL-R) system evaluation

    NASA Technical Reports Server (NTRS)

    1980-01-01

    An evaluation of the Multi-Level Expression Design Language Requirements Level (MEDL-R) system was conducted to determine whether it would be of use in the Goddard Space Flight Center Code 580 software development environment. The evaluation is based upon a study of the MEDL-R concept of requirement languages, the functions performed by MEDL-R, and the MEDL-R language syntax. Recommendations are made for changes to MEDL-R that would make it useful in the Code 580 environment.

  6. Sex determines the expression level of one third of the actively expressed genes in bovine blastocysts.

    PubMed

    Bermejo-Alvarez, P; Rizos, D; Rath, D; Lonergan, P; Gutierrez-Adan, A

    2010-02-23

    Although genetically identical for autosomal Chrs (Chr), male and female preimplantation embryos could display sex-specific transcriptional regulation. To illustrate sex-specific differences at the mRNA level, we compared gene-expression patterns between male and female blastocysts by DNA microarray comparison of nine groups of 60 bovine in vitro-produced blastocysts of each sex. Almost one-third of the transcripts detected showed sexual dimorphism (2,921 transcripts; false-discovery rate, P < 0.05), suggesting that in the absence of hormonal influences, the sex Chrs impose an extensive transcriptional regulation upon autosomal genes. Six genes were analyzed by qPCR in in vivo-derived embryos, which displayed similar sexual dimorphism. Ontology analysis suggested a higher global transcriptional level in females and a more active protein metabolism in males. A gene homolog to an X-linked gene involved in network interactions during spliceosome assembly was found in the Y-Chr. Most of the X-linked-expressed transcripts (88.5%) were up-regulated in females, but most of them (70%) exhibited fold-changes lower than 1.6, suggesting that X-Chr inactivation is partially achieved at the blastocyst stage. Almost half of the transcripts up-regulated in female embryos exhibiting more than 1.6-fold change were present in the X-Chr and eight of them were selected to determine a putative paternal imprinting by gene expression comparison with parthenogenetic embryos. Five (BEX, CAPN6, BEX2, SRPX2, and UBE2A) exhibited a higher expression in females than in parthenotes, suggesting that they are predominantly expressed by the paternal inherited X-Chr and that imprinting may increase the transcriptional skew caused by double X-Chr dosage. PMID:20133684

  7. Sex determines the expression level of one third of the actively expressed genes in bovine blastocysts

    PubMed Central

    Bermejo-Alvarez, P.; Rizos, D.; Rath, D.; Lonergan, P.; Gutierrez-Adan, A.

    2010-01-01

    Although genetically identical for autosomal Chrs (Chr), male and female preimplantation embryos could display sex-specific transcriptional regulation. To illustrate sex-specific differences at the mRNA level, we compared gene-expression patterns between male and female blastocysts by DNA microarray comparison of nine groups of 60 bovine in vitro-produced blastocysts of each sex. Almost one-third of the transcripts detected showed sexual dimorphism (2,921 transcripts; false-discovery rate, P < 0.05), suggesting that in the absence of hormonal influences, the sex Chrs impose an extensive transcriptional regulation upon autosomal genes. Six genes were analyzed by qPCR in in vivo-derived embryos, which displayed similar sexual dimorphism. Ontology analysis suggested a higher global transcriptional level in females and a more active protein metabolism in males. A gene homolog to an X-linked gene involved in network interactions during spliceosome assembly was found in the Y-Chr. Most of the X-linked-expressed transcripts (88.5%) were up-regulated in females, but most of them (70%) exhibited fold-changes lower than 1.6, suggesting that X-Chr inactivation is partially achieved at the blastocyst stage. Almost half of the transcripts up-regulated in female embryos exhibiting more than 1.6-fold change were present in the X-Chr and eight of them were selected to determine a putative paternal imprinting by gene expression comparison with parthenogenetic embryos. Five (BEX, CAPN6, BEX2, SRPX2, and UBE2A) exhibited a higher expression in females than in parthenotes, suggesting that they are predominantly expressed by the paternal inherited X-Chr and that imprinting may increase the transcriptional skew caused by double X-Chr dosage. PMID:20133684

  8. Naringin promotes differentiation of bone marrow stem cells into osteoblasts by upregulating the expression levels of microRNA-20a and downregulating the expression levels of PPARγ.

    PubMed

    Fan, Jifeng; Li, Jie; Fan, Qinbo

    2015-09-01

    Naringin is a dihydrotestosterone flavonoid compound that significantly inhibits bone loss, improves bone density, and enhances biomechanical anti‑compression performance. Previous studies have demonstrated that naringin improves the activity levels of osteocalcin (OC) and alkaline phosphatase (ALP) in MC3T3‑E1 osteoblast precursor cells. The present study investigated the effects of naringin on osteoblastic differentiation and inhibition of adipocyte formation in bone marrow stem cells (BMSCs). The levels of osteogenesis were modulated via upregulation of the expression levels of microRNA (miR)‑20a, and downregulation of the expression levels of peroxisome proliferator‑activated receptor γ (PPARγ). The results indicated that naringin significantly enhanced BMSC proliferation in a dose‑dependent manner. In addition, naringin significantly increased the mRNA expression levels of OC, ALP, and collagen type I. Furthermore, naringin decreased the protein expression levels of PPARγ, and increased the expression levels of miR‑20a in the BMSCs. These results suggested that miR‑20a may regulate the expression of PPARγ in BMSCs. To our knowledge, this is the first study to report naringin‑induced osteogenesis via upregulation of the expression levels of miR‑20a, and downregulation of the expression levels of PPARγ. These results indicated the important role of naringin in BMSC differentiation. PMID:26126997

  9. GTP Cyclohydrolase I Expression, Protein, and Activity Determine Intracellular Tetrahydrobiopterin Levels, Independent of GTP Cyclohydrolase Feedback Regulatory Protein Expression

    PubMed Central

    Tatham, Amy L.; Crabtree, Mark J.; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J.; Channon, Keith M.

    2009-01-01

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r2 = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r2 = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression. PMID:19286659

  10. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression. PMID:19286659

  11. Prognostic correlation between MTA2 expression level and colorectal cancer

    PubMed Central

    Ding, Weijun; Hu, Wei; Yang, Haihua; Ying, Ting; Tian, Ye

    2015-01-01

    Objectives: Association of MTA2 expression with presence, development, metastasis and prognosis of colorectal cancer (CRC) was investigated. Methods: 90 CRC-related cases with follow-up information were made into tissue microarrays according to the paired principle of cancer tissues and the adjacent tissues. Subsequently, the expression of MAT2 was detected with immunohistochemical analysis and SPSS software was finally utilized to analyze the relationships between experimental data and clinical indicatives. Results: Expression of MTA2 in CRC tissues were notably higher than their adjacent tissues (P < 0.001) and showed significant positive correlation with tumor grade (r2 > 0, P < 0.01). Moreover, survival analysis indicated that MTA2 expression in cancer tissues, serving as an independent correlation factor, was significantly correlated with poor prognosis (P = 0.004). Conclusions: MTA2 is a crucial biomarker that is closely related with prognosis of CRC and also a potential molecular target for evaluating the prognosis and treatment of CRC. PMID:26261611

  12. Quantifying the Effect of DNA Packaging on Gene Expression Level

    NASA Astrophysics Data System (ADS)

    Kim, Harold

    2010-10-01

    Gene expression, the process by which the genetic code comes alive in the form of proteins, is one of the most important biological processes in living cells, and begins when transcription factors bind to specific DNA sequences in the promoter region upstream of a gene. The relationship between gene expression output and transcription factor input which is termed the gene regulation function is specific to each promoter, and predicting this gene regulation function from the locations of transcription factor binding sites is one of the challenges in biology. In eukaryotic organisms (for example, animals, plants, fungi etc), DNA is highly compacted into nucleosomes, 147-bp segments of DNA tightly wrapped around histone protein core, and therefore, the accessibility of transcription factor binding sites depends on their locations with respect to nucleosomes - sites inside nucleosomes are less accessible than those outside nucleosomes. To understand how transcription factor binding sites contribute to gene expression in a quantitative manner, we obtain gene regulation functions of promoters with various configurations of transcription factor binding sites by using fluorescent protein reporters to measure transcription factor input and gene expression output in single yeast cells. In this talk, I will show that the affinity of a transcription factor binding site inside and outside the nucleosome controls different aspects of the gene regulation function, and explain this finding based on a mass-action kinetic model that includes competition between nucleosomes and transcription factors.

  13. Insert sequence length determines transfection efficiency and gene expression levels in bicistronic mammalian expression vectors

    PubMed Central

    Payne, Andrew J; Gerdes, Bryan C; Kaja, Simon; Koulen, Peter

    2013-01-01

    Bicistronic expression vectors have been widely used for co-expression studies since the initial discovery of the internal ribosome entry site (IRES) about 25 years ago. IRES sequences allow the 5’ cap-independent initiation of translation of multiple genes on a single messenger RNA strand. Using a commercially available mammalian expression vector containing an IRES sequence with a 3’ green fluorescent protein fluorescent marker, we found that sequence length of the gene of interest expressed 5’ of the IRES site influences both expression of the 3’ fluorescent marker and overall transfection efficiency of the vector construct. Furthermore, we generated a novel construct expressing two distinct fluorescent markers and found that high expression of one gene can lower expression of the other. Observations from this study indicate that caution is warranted in the design of experiments utilizing an IRES system with a short 5’ gene of interest sequence (<300 bp), selection of single cells based on the expression profile of the 3’ optogenetic fluorescent marker, and assumptions made during data analysis. PMID:24380024

  14. Level of Aspiration: A Behavioral Expression of Self-Concept.

    ERIC Educational Resources Information Center

    Kay, Richard S.; Felker, Donald W.

    The present study was designed to investigate the role of expectations in self-concept and level of aspiration (LOA) behavior. Specifically, the focus was to investigate self-concept and LOA as covariates and to describe the nature of the relationship if, in fact, one existed. A sample of 80 third and fourth grade students was selected from three…

  15. Bisphenol a influences blastocyst implantation via regulating integrin β3 and trophinin expression levels

    PubMed Central

    Pan, Xiaoyan; Wang, Xiyan; Wang, Zhengchao; Wang, Xuenan; Dou, Zhaohua; Li, Zhixin

    2015-01-01

    Objective: This study is to investigate effects of bisphenol A (BPA) on the blastocyst implantation in endometrium. Methods: Pregnant mice were orally administered with BPA. Implantation sites were examined, and serum estrogen level was assayed with ELISA. Protein expression levels were detected with immunohistochemistry and immunofluorescence staining. Results: High doses (400 and 600 mg/kg/day) of BPA remarkably reduced the implantation sites in the pregnant mice. No significant differences were observed in the serum estrogen level across the groups. Moreover, high doses (400 and 600 mg/kg/day) of BPA significantly declined the expression level of endometrial estrogen receptor α (ERα) in the pregnant mice. In addition, high doses (400 and 600 mg/kg/day) of BPA significantly declined the expression levels of integrin β3 and trophinin in the endometrium and blastocysts. Conclusion: BPA declines ERα expression in endometrium, and inhibits adhesion protein expression in endometrium and blastocysts, causing the adhesion failure of blastocyst implantation. PMID:26884915

  16. The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome

    PubMed Central

    Hurst, Laurence D.; Ghanbarian, Avazeh T.; Forrest, Alistair R. R.; Huminiecki, Lukasz

    2015-01-01

    X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression

  17. The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

    PubMed

    Hurst, Laurence D; Ghanbarian, Avazeh T; Forrest, Alistair R R; Huminiecki, Lukasz

    2015-12-01

    X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression

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

    PubMed

    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. PMID:26892536

  19. Gene expression analyses in maize inbreds and hybrids with varying levels of heterosis

    PubMed Central

    Stupar, Robert M; Gardiner, Jack M; Oldre, Aaron G; Haun, William J; Chandler, Vicki L; Springer, Nathan M

    2008-01-01

    Background Heterosis is the superior performance of F1 hybrid progeny relative to the parental phenotypes. Maize exhibits heterosis for a wide range of traits, however the magnitude of heterosis is highly variable depending on the choice of parents and the trait(s) measured. We have used expression profiling to determine whether the level, or types, of non-additive gene expression vary in maize hybrids with different levels of genetic diversity or heterosis. Results We observed that the distributions of better parent heterosis among a series of 25 maize hybrids generally do not exhibit significant correlations between different traits. Expression profiling analyses for six of these hybrids, chosen to represent diversity in genotypes and heterosis responses, revealed a correlation between genetic diversity and transcriptional variation. The majority of differentially expressed genes in each of the six different hybrids exhibited additive expression patterns, and ~25% exhibited statistically significant non-additive expression profiles. Among the non-additive profiles, ~80% exhibited hybrid expression levels between the parental levels, ~20% exhibited hybrid expression levels at the parental levels and ~1% exhibited hybrid levels outside the parental range. Conclusion We have found that maize inbred genetic diversity is correlated with transcriptional variation. However, sampling of seedling tissues indicated that the frequencies of additive and non-additive expression patterns are very similar across a range of hybrid lines. These findings suggest that heterosis is probably not a consequence of higher levels of additive or non-additive expression, but may be related to transcriptional variation between parents. The lack of correlation between better parent heterosis levels for different traits suggests that transcriptional diversity at specific sets of genes may influence heterosis for different traits. PMID:18402703

  20. mef2 activity levels differentially affect gene expression during Drosophila muscle development

    PubMed Central

    Elgar, Stuart J.; Han, Jun; Taylor, Michael V.

    2008-01-01

    Cell differentiation is controlled by key transcription factors, and a major question is how they orchestrate cell-type-specific genetic programs. Muscle differentiation is a well studied paradigm in which the conserved Mef2 transcription factor plays a pivotal role. Recent genomic studies have identified a large number of mef2-regulated target genes with distinct temporal expression profiles during Drosophila myogenesis. However, the question remains as to how a single transcription factor can control such diverse patterns of gene expression. In this study we used a strategy combining genomics and developmental genetics to address this issue in vivo during Drosophila muscle development. We found that groups of mef2-regulated genes respond differently to changes in mef2 activity levels: some require higher levels for their expression than others. Furthermore, this differential requirement correlates with when the gene is first expressed during the muscle differentiation program. Genes that require higher levels are activated later. These results implicate mef2 in the temporal regulation of muscle gene expression, and, consistent with this, we show that changes in mef2 activity levels can alter the start of gene expression in a predictable manner. Together these results indicate that Mef2 is not an all-or-none regulator; rather, its action is more subtle, and levels of its activity are important in the differential expression of muscle genes. This suggests a route by which mef2 can orchestrate the muscle differentiation program and contribute to the stringent regulation of gene expression during myogenesis. PMID:18198273

  1. HLA-C expression levels define permissible mismatches in hematopoietic cell transplantation.

    PubMed

    Petersdorf, Effie W; Gooley, Theodore A; Malkki, Mari; Bacigalupo, Andrea P; Cesbron, Anne; Du Toit, Ernette; Ehninger, Gerhard; Egeland, Torstein; Fischer, Gottfried F; Gervais, Thibaut; Haagenson, Michael D; Horowitz, Mary M; Hsu, Katharine; Jindra, Pavel; Madrigal, Alejandro; Oudshoorn, Machteld; Ringdén, Olle; Schroeder, Marlis L; Spellman, Stephen R; Tiercy, Jean-Marie; Velardi, Andrea; Witt, Campbell S; O'Huigin, Colm; Apps, Richard; Carrington, Mary

    2014-12-18

    Life-threatening graft-versus-host disease (GVHD) limits the use of HLA-C-mismatched unrelated donors in transplantation. Clinicians lack criteria for donor selection when HLA-C-mismatched donors are a patient's only option for cure. We examined the role for HLA-C expression levels to identify permissible HLA-C mismatches. The median fluorescence intensity, a proxy of HLA-C expression, was assigned to each HLA-C allotype in 1975 patients and their HLA-C-mismatched unrelated transplant donors. The association of outcome with the level of expression of patients' and donors' HLA-C allotypes was evaluated in multivariable models. Increasing expression level of the patient's mismatched HLA-C allotype was associated with increased risks of grades III to IV acute GVHD, nonrelapse mortality, and mortality. Increasing expression level among HLA-C mismatches with residue 116 or residue 77/80 mismatching was associated with increased nonrelapse mortality. The immunogenicity of HLA-C mismatches in unrelated donor transplantation is influenced by the expression level of the patient's mismatched HLA-C allotype. HLA-C expression levels provide new information on mismatches that should be avoided and extend understanding of HLA-C-mediated immune responses in human disease. PMID:25323824

  2. HLA-C expression levels define permissible mismatches in hematopoietic cell transplantation

    PubMed Central

    Gooley, Theodore A.; Malkki, Mari; Bacigalupo, Andrea P.; Cesbron, Anne; Du Toit, Ernette; Ehninger, Gerhard; Egeland, Torstein; Fischer, Gottfried F.; Gervais, Thibaut; Haagenson, Michael D.; Horowitz, Mary M.; Hsu, Katharine; Jindra, Pavel; Madrigal, Alejandro; Oudshoorn, Machteld; Ringdén, Olle; Schroeder, Marlis L.; Spellman, Stephen R.; Tiercy, Jean-Marie; Velardi, Andrea; Witt, Campbell S.; O’Huigin, Colm; Apps, Richard; Carrington, Mary

    2014-01-01

    Life-threatening graft-versus-host disease (GVHD) limits the use of HLA-C-mismatched unrelated donors in transplantation. Clinicians lack criteria for donor selection when HLA-C-mismatched donors are a patient’s only option for cure. We examined the role for HLA-C expression levels to identify permissible HLA-C mismatches. The median fluorescence intensity, a proxy of HLA-C expression, was assigned to each HLA-C allotype in 1975 patients and their HLA-C-mismatched unrelated transplant donors. The association of outcome with the level of expression of patients’ and donors’ HLA-C allotypes was evaluated in multivariable models. Increasing expression level of the patient’s mismatched HLA-C allotype was associated with increased risks of grades III to IV acute GVHD, nonrelapse mortality, and mortality. Increasing expression level among HLA-C mismatches with residue 116 or residue 77/80 mismatching was associated with increased nonrelapse mortality. The immunogenicity of HLA-C mismatches in unrelated donor transplantation is influenced by the expression level of the patient’s mismatched HLA-C allotype. HLA-C expression levels provide new information on mismatches that should be avoided and extend understanding of HLA-C-mediated immune responses in human disease. PMID:25323824

  3. Identification of Xenologs and Their Characteristic Low Expression Levels in the Cyanobacterium Synechococcus elongatus.

    PubMed

    Álvarez-Canales, Gilberto; Arellano-Álvarez, Guadalupe; González-Domenech, Carmen M; de la Cruz, Fernando; Moya, Andrés; Delaye, Luis

    2015-06-01

    Horizontal gene transfer (HGT) is a central process in prokaryotic evolution. Once a gene is introduced into a genome by HGT, its contribution to the fitness of the recipient cell depends in part on its expression level. Here we show that in Synechococcus elongatus PCC 7942, xenologs derived from non-cyanobacterial sources exhibited lower expression levels than native genes in the genome. In accord with our observation, xenolog codon adaptation indexes also displayed relatively low expression values. These results are in agreement with previous reports that suggested the relative neutrality of most xenologs. However, we also demonstrated that some of the xenologs detected participated in cellular functions, including iron starvation acclimation and nitrate reduction, which corroborate the role of HGT in bacterial adaptation. For example, the expression levels of some of the xenologs detected are known to increase under iron-limiting conditions. We interpreted the overall pattern as an indication that there is a selection pressure against high expression levels of xenologs. However, when a xenolog protein product confers a selective advantage, natural selection can further modulate its expression level to meet the requirements of the recipient cell. In addition, we show that ORFans did not exhibit significantly lower expression levels than native genes in the genome, which suggested an origin other than xenology. PMID:26040248

  4. Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

    PubMed

    Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

    2015-11-20

    The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions. PMID:26478262

  5. Effects of elevated peroxidase levels and corn earworm feeding on gene expression in tomato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato gene arrays were used to investigate how high levels of transgenic peroxidase expression and feeding by the corn earworm, Helicoverpa zea, affected expression of defensive and other genes. High peroxidase activity significantly upregulated proteinase inhibitors and a few other defensive gene...

  6. Predicting Gene Expression Level from Relative Codon Usage Bias: An Application to Escherichia coli Genome

    PubMed Central

    Roymondal, Uttam; Das, Shibsankar; Sahoo, Satyabrata

    2009-01-01

    We present an expression measure of a gene, devised to predict the level of gene expression from relative codon bias (RCB). There are a number of measures currently in use that quantify codon usage in genes. Based on the hypothesis that gene expressivity and codon composition is strongly correlated, RCB has been defined to provide an intuitively meaningful measure of an extent of the codon preference in a gene. We outline a simple approach to assess the strength of RCB (RCBS) in genes as a guide to their likely expression levels and illustrate this with an analysis of Escherichia coli (E. coli) genome. Our efforts to quantitatively predict gene expression levels in E. coli met with a high level of success. Surprisingly, we observe a strong correlation between RCBS and protein length indicating natural selection in favour of the shorter genes to be expressed at higher level. The agreement of our result with high protein abundances, microarray data and radioactive data demonstrates that the genomic expression profile available in our method can be applied in a meaningful way to the study of cell physiology and also for more detailed studies of particular genes of interest. PMID:19131380

  7. Optimality and evolutionary tuning of the expression level of a protein.

    PubMed

    Dekel, Erez; Alon, Uri

    2005-07-28

    Different proteins have different expression levels. It is unclear to what extent these expression levels are optimized to their environment. Evolutionary theories suggest that protein expression levels maximize fitness, but the fitness as a function of protein level has seldom been directly measured. To address this, we studied the lac system of Escherichia coli, which allows the cell to use the sugar lactose for growth. We experimentally measured the growth burden due to production and maintenance of the Lac proteins (cost), as well as the growth advantage (benefit) conferred by the Lac proteins when lactose is present. The fitness function, given by the difference between the benefit and the cost, predicts that for each lactose environment there exists an optimal Lac expression level that maximizes growth rate. We then performed serial dilution evolution experiments at different lactose concentrations. In a few hundred generations, cells evolved to reach the predicted optimal expression levels. Thus, protein expression from the lac operon seems to be a solution of a cost-benefit optimization problem, and can be rapidly tuned by evolution to function optimally in new environments. PMID:16049495

  8. Correlations Between Gene Expression and Mercury Levels in Blood of Boys With and Without Autism

    PubMed Central

    Green, Peter G.; Tian, Yingfang; Hertz-Picciotto, Irva; Pessah, Isaac N.; Hansen, Robin; Yang, Xiaowei; Teng, Jennifer; Gregg, Jeffrey P.; Ashwood, Paul; Van de Water, Judy; Sharp, Frank R.

    2009-01-01

    Gene expression in blood was correlated with mercury levels in blood of 2- to 5-year-old boys with autism (AU) compared to age-matched typically developing (TD) control boys. This was done to address the possibility that the two groups might metabolize toxicants, such as mercury, differently. RNA was isolated from blood and gene expression assessed on whole genome Affymetrix Human U133 expression microarrays. Mercury levels were measured using an inductively coupled plasma mass spectrometer. Analysis of covariance (ANCOVA) was performed and partial correlations between gene expression and mercury levels were calculated, after correcting for age and batch effects. To reduce false positives, only genes shared by the ANCOVA models were analyzed. Of the 26 genes that correlated with mercury levels in both AU and TD boys, 11 were significantly different between the groups (P(Diagnosis*Mercury) ≤ 0.05). The expression of a large number of genes (n = 316) correlated with mercury levels in TD but not in AU boys (P ≤ 0.05), the most represented biological functions being cell death and cell morphology. Expression of 189 genes correlated with mercury levels in AU but not in TD boys (P ≤ 0.05), the most represented biological functions being cell morphology, amino acid metabolism, and antigen presentation. These data and those in our companion study on correlation of gene expression and lead levels show that AU and TD children display different correlations between transcript levels and low levels of mercury and lead. These findings might suggest different genetic transcriptional programs associated with mercury in AU compared to TD children. Electronic supplementary material The online version of this article (doi:10.1007/s12640-009-9137-7) contains supplementary material, which is available to authorized users. PMID:19937285

  9. High-level expression of the bacterial opd gene in Drosophila melanogaster: improved inducible insecticide resistance.

    PubMed

    Benedict, M Q; Scott, J A; Cockburn, A F

    1994-11-01

    The bacterial parathion hydrolase gene (opd) was expressed in transformed D. melanogaster under the control of an hsp70 promoter. Transformed lines carrying chimaeric genes designed for either cytoplasmic or secretory expression exhibited high- or low-level heat-shock-inducible transient resistance to paraoxon respectively. Greatest levels of resistance occurred approximately 12-16 h after heat shock and well after periods of maximal transcription. Insecticide resistance conferred by the cytoplasmic form of opd is expressed as a semidominant trait. PMID:7704308

  10. Dual transcriptional-translational cascade permits cellular level tuneable expression control

    PubMed Central

    Morra, Rosa; Shankar, Jayendra; Robinson, Christopher J.; Halliwell, Samantha; Butler, Lisa; Upton, Mathew; Hay, Sam; Micklefield, Jason; Dixon, Neil

    2016-01-01

    The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems. PMID:26405200

  11. Development of Multigene Expression Signature Maps at the Protein Level from Digitized Immunohistochemistry Slides

    PubMed Central

    Metzger, Gregory J.; Dankbar, Stephen C.; Henriksen, Jonathan; Rizzardi, Anthony E.; Rosener, Nikolaus K.; Schmechel, Stephen C.

    2012-01-01

    Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC) is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps) of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions. PMID:22438942

  12. [Expression of erythropoietin receptor in leukemia cells and relation of erythropoietin level with leukemic anemia].

    PubMed

    Feng, Mei; Li, Yu-Cui

    2008-12-01

    This study was purposed to investigate the expression of erythropoietin receptor (EPOR) in leukemic cells and the relationship of serum erythropoietin level with anemia in acute leukemia patients, so as to provide a new theoretical basis for the cytokine therapy in acute leukemia with anemia. The EPOR in 30 AL patients was detected by using reverse transcription polymerase chain reaction (RT-PCR), the level of serum erythropoietin was detected by chemiluminescence analysis, the hemoglobin level was assayed by automatic blood counting instrument. The results indicated that EPOR was expressed in 18 out of 30 AL patients, the expression rate of EPOR in AL patients was 60%, however, but the EPOR expression rate in AML was 61.9% (13/21) and 55.6% (5/9) in ALL, the EPOR expression rate was no significant difference between AML and ALL. The EPOR expression rate was significantly lower than that in control group (86.7%) (p<0.05). The relative level of EPOR expression in AML was higher than that in ALL (p<0.05), the average level of EPOR expression in AL was significantly lower than that in control group (p<0.01). The level of sEPO in 30 AL patients was significantly higher than that in control group (p<0.01), and there was negative correlation between the levels of sEPO and Hb (p<0.01). It is concluded that the EPOR is expressed in cells of AL, but the expressive level is low. The EPOR expression rate shows no significant difference between AML and ALL. The mechanism of negative feedback to anemia in acute leukemia is intact. Anemia of acute leukemia is not completely associated with inadequate erythropoietin production and relates to hemopoiesis defect that considered as the main reason. Recombinant human erythropoietin is widely used in treatment of anemia caused by acute leukemia. Whether the treatment with rh-EPO for acute leukemia with anemia will enhance the proliferation of leukemia cells, this problem should be explored further. PMID:19099624

  13. Correlated expression levels of endothelin receptor B and Plexin C1 in melanoma

    PubMed Central

    Kumasaka, Mayuko Y; Yajima, Ichiro; Iida, Machiko; Takahashi, Hiro; Inoue, Yuji; Fukushima, Satoshi; Ihn, Hironobu; Takeda, Kozue; Naito, Yuji; Yoshikawa, Toshikazu; Kato, Masashi

    2015-01-01

    Discussion concerning the effect of endothelin receptor B (Ednrb) on melanoma continues because Ednrb has been reported to have both tumor promoting and suppressive effects for melanoma. In order to examine Ednrb-related signaling in melanomagenesis, DNA microarray analysis for a melanoma from a RFP/RET-transgenic mouse (RET-mouse) and a melanoma from an Ednrb-heterozygously deleted RET-mouse [Ednrb(+/-);RET-mouse], in both of which melanoma spontaneously develops, was performed in this study. We found that the expression level of Plexin C1 (PlxnC1), a suppressor for melanoma, in a melanoma from an Ednrb(+/-);RET-mouse was drastically decreased compared to that in a melanoma from a RET-mouse. Therefore, we further examined the correlation between Ednrb and PlxnC1 expression levels in melanomas. PlxnC1 transcript expression levels in melanomas from Ednrb(+/-);RET-mice were lower than those in melanomas from RET-mice. A strong correlation between Ednrb and PlxnC1 transcript expression levels (R = 0.78, p < 0.01) was also found in melanomas from both RET-mice and Ednrb(+/-);RET-mice. Correspondingly, there was a significant correlation between transcript (R = 0.80; p < 0.01) and protein (R = 0.60; p < 0.01) expression levels of EDNRB and PLXNC1 in human primary melanomas. Together with our results showing that the expression level of PLXNC1 transcript was reduced in EDNRB-depleted human melanoma cells, our results showing positively correlated expression levels of Ednrb/EDNRB and PlxnC1/PLXNC1 in melanoma suggest that PlxnC1/PLXNC1 is involved in the Ednrb/EDNRB-mediated suppressive effect on melanoma. PMID:26045990

  14. Impact of STAT/SOCS mRNA Expression Levels after Major Injury

    PubMed Central

    Brumann, M.; Matz, M.; Kusmenkov, T.; Stegmaier, J.; Biberthaler, P.; Kanz, K.-G.; Mutschler, W.; Bogner, V.

    2014-01-01

    Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0 h–72 h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6 h after trauma. SOCS 1 levels significantly decreased 6 h–72 h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6 h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6 h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance. PMID:24648661

  15. The Genomic Landscape of Position Effects on Protein Expression Level and Noise in Yeast.

    PubMed

    Chen, Xiaoshu; Zhang, Jianzhi

    2016-05-25

    Position effect, the influence of the chromosomal location of a gene on its activity, is a fundamental property of the genome. By placing a GFP gene cassette at 482 different locations across all chromosomes in budding yeast, we quantified the position effects on protein expression level and noise at the genomic scale. The position effects are significant, altering the mean protein expression level by up to 15 times and expression noise by up to 20 times. DNA replication timing, 3D chromosomal conformation, and several histone modifications are major covariates of position effects. Essential genes are enriched in genomic regions with inherently low expression noise, supporting the hypothesis that chromosomal clustering of essential genes results from selection against their expressional stochasticity. Position effects exhibit significant interactions with promoters. Together, our results suggest that position effects have shaped the evolution of chromosome organization and should inform future genome engineering efforts. PMID:27185547

  16. A Theoretical Lower Bound for Selection on the Expression Levels of Proteins

    PubMed Central

    Price, Morgan N.; Arkin, Adam P.

    2016-01-01

    We use simple models of the costs and benefits of microbial gene expression to show that changing a protein’s expression away from its optimum by 2-fold should reduce fitness by at least 0.2·P, where P is the fraction the cell’s protein that the gene accounts for. As microbial genes are usually expressed at above 5 parts per million, and effective population sizes are likely to be above 106, this implies that 2-fold changes to gene expression levels are under strong selection, as Ne·s≫1, where Ne is the effective population size and s is the selection coefficient. Thus, most gene duplications should be selected against. On the other hand, we predict that for most genes, small changes in the expression will be effectively neutral. PMID:27289091

  17. A Theoretical Lower Bound for Selection on the Expression Levels of Proteins.

    PubMed

    Price, Morgan N; Arkin, Adam P

    2016-01-01

    We use simple models of the costs and benefits of microbial gene expression to show that changing a protein's expression away from its optimum by 2-fold should reduce fitness by at least [Formula: see text], where P is the fraction the cell's protein that the gene accounts for. As microbial genes are usually expressed at above 5 parts per million, and effective population sizes are likely to be above 10(6), this implies that 2-fold changes to gene expression levels are under strong selection, as [Formula: see text], where Ne is the effective population size and s is the selection coefficient. Thus, most gene duplications should be selected against. On the other hand, we predict that for most genes, small changes in the expression will be effectively neutral. PMID:27289091

  18. Dscam Expression Levels Determine Presynaptic Arbor Sizes in Drosophila Sensory Neurons

    PubMed Central

    Kim, Jung Hwan; Wang, Xin; Coolon, Rosemary; Ye, Bing

    2013-01-01

    SUMMARY Expression of the Down syndrome cell-adhesion molecule (Dscam) is increased in the brains of patients with several neurological disorders. Although Dscam is critically involved in many aspects of neuronal development, little is known about either the mechanism that regulates its expression or the functional consequences of dysregulated Dscam expression. Here, we show that Dscam expression levels serve as an instructive code for the size control of presynaptic arbor. Two convergent pathways, involving dual leucine zipper kinase (DLK) and fragile X mental retardation protein (FMRP), control Dscam expression through protein translation. Defects in this regulation of Dscam translation lead to exuberant presynaptic arbor growth in Drosophila somatosensory neurons. Our findings demonstrate a previously unknown aspect of Dscam function and provide insights into how dysregulated Dscam may contribute to the pathogenesis of neurological disorders. PMID:23764288

  19. The human phenolsulphotransferase polymorphism is determined by the level of expression of the enzyme protein.

    PubMed Central

    Jones, A L; Roberts, R C; Coughtrie, M W

    1993-01-01

    We have examined the expression of platelet phenolsulphotransferase (PST) in 60 individuals. Using an antibody which recognizes both forms of PST present in man (P-PST and M-PST), we determined that the polymorphism of platelet P-PST activity is determined by the level of expression of the enzyme protein. The implications for susceptibility to adverse drug reactions and chemical carcinogenesis are discussed. Images Figure 1 Figure 2 Figure 3 PMID:8257413

  20. An alternative method of enhancing the expression level of heterologous protein in Escherichia coli.

    PubMed

    Yin, Jun; Tian, Hong; Bao, Lichen; Dai, Xin; Gao, Xiangdong; Yao, Wenbing

    2014-12-12

    Though numerous strategy options are available for achieving high expression levels of genes in Escherichia coli, not every gene can be efficiently expressed in this organism. By investigating the relationship between the mRNA secondary structure of translational initiation region (TIR) and gene expression in E.coli, we establish a simple method to design sequences of appropriate TIR (from -35 to +36) that meet a specific expression level as we need. Using this method, overexpression of native human humor necrosis factor α and extracellular domain of Her2/neu protein (aa 23-146) in E. coli were achieved. Differences in expression appeared was mainly related to the efficiency of translation initiation and the stability of mRNA secondary structure, because the intracellular mRNA levels analyzed by real-time RT-PCR were quite similar. Our approach can overcome the steric hindrance of translation startup, and therefore promote translation smoothly to acquire high expression of exogenous protein. PMID:25449272

  1. Effects of dietary roughage levels on the expression of adipogenic transcription factors in Wagyu steers.

    PubMed

    Yamada, T; Kawakami, S-I; Nakanishi, N

    2009-12-01

    We hypothesized that dietary roughage level would alter the expression levels of adipogenic transcription factors in adipose tissue of Japanese black (Wagyu) steers. Steers were fed whole crop rice silage at three levels: (1) high-roughage feeding group, fed 8kg silage and 5kg concentrate (HR); (2) middle roughage feeding group, fed 5kg silage and 6kg concentrate (MR); and (3) low roughage feeding group, fed 2kg silage and 7kg concentrate (LR) from 22 to 30months of age. In subcutaneous adipose tissue, there were no significant differences in the expression of the adipogenic transcription factors and adipocyte size among feeding groups. In mesenteric adipose tissue, the expression of C/EBPα in the LR and MR groups was significantly higher than that in the HR group. Adipocyte size in the mesenteric adipose tissue of the LR group was significantly larger than that of the HR group. In intermuscular adipose tissue, the expression of C/EBPβ-LAP in the LR group was significantly higher than that in the HR group, and the expression of C/EBPβ-LIP in the LR and MR groups was significantly higher than that in the HR group. Adipocyte size in the intermuscular adipose tissue of the LR and MR groups was significantly smaller than that of the HR group. These results suggest that dietary roughage revel affects the adipose tissue depot-specific differences in C/EBP family expression pattern and adipocyte cellularity in Wagyu steers. PMID:20416623

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

    PubMed Central

    Gao, Meili; Li, Yongfei; Xue, Xiaochang; Wang, Xianfeng; Long, Jiangang

    2012-01-01

    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. PMID:23093835

  3. Expression of fas protein on CD4+T cells irradiated by low level He-Ne

    NASA Astrophysics Data System (ADS)

    Nie, Fan; Zhu, Jing; Zhang, Hui-Guo

    2005-07-01

    Objective: To investigate the influence on the Expression of Fas protein on CD4+ T cells irradiated by low level He-Ne laser in the cases of psoriasis. Methods:the expression of CD4+ T Fas protein was determined in the casee of psoriasis(n=5) pre and post-low level laser irradiation(30 min、60min and 120min)by flow cytometry as compared withthe control(n=5). Results:In the cases of psoriasis,the expression of CD4+T FAS protein 21.4+/-3.1% was increased significantly than that of control group 16.8+/-2.1% pre-irradiation, p<0.05in the control,there is no difference between pre and post- irradiation,p>0.05in the cases , the expression of CD4+T Fas protein wae positively corelated to the irradiation times, when the energy density arrived to 22.92J/cm2(60 minutes)and 45.84J/cm2(120minutes), the expression of CD4+ T Fas protein was increased significantly as compared with pre-irradiation,p<0.05.Conclusion: The expression of CD4+T Fas protein may be increased by low level He-Ne laser irradiation ,the uncontrolled status of apoptosis could be corrected.

  4. Protein pheromone expression levels predict and respond to the formation of social dominance networks

    PubMed Central

    Nelson, Adam C.; Cunningham, Christopher B.; Ruff, James S.; Potts, Wayne K.

    2015-01-01

    Communication signals are key regulators of social networks, and are thought to be under selective pressure to honestly reflect social status, including dominance status. The odors of dominants and nondominants differentially influence behavior, and identification of the specific pheromones associated with, and predictive of, dominance status is essential for understanding the mechanisms of network formation and maintenance. In mice, major urinary proteins (MUPs) are excreted in extraordinary large quantities and expression level has been hypothesized to provide an honest signal of dominance status. Here, we evaluate whether MUPs are associated with dominance in wild-derived mice by analyzing expression levels before, during, and after competition for reproductive resources over three days. During competition, dominant males have 24% greater urinary MUP expression than nondominants. The MUP darcin, a pheromone that stimulates female attraction, is predictive of dominance status: dominant males have higher darcin expression before competition. Dominants also have a higher ratio of darcin to other MUPs before and during competition. These differences appear transient, because there are no differences in MUPs or darcin after competition. We also find MUP expression is affected by sire dominance status: socially naive sons of dominant males have lower MUP expression, but this apparent repression is released during competition. A requisite condition for the evolution of communication signals is honesty, and we provide novel insight into pheromones and social networks by showing that MUP and darcin expression is a reliable signal of dominance status, a primary determinant of male fitness in many species. PMID:25867293

  5. Expression of DNA repair genes in burned skin exposed to low-level red laser.

    PubMed

    Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

    2014-11-01

    Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin. PMID:24930134

  6. Interactions between copy number and expression level of genes involved in fluconazole resistance in Candida glabrata

    PubMed Central

    Abbes, Salma; Mary, Charles; Sellami, Hayet; Michel-Nguyen, Annie; Ayadi, Ali; Ranque, Stéphane

    2013-01-01

    Objectives: This study aimed to elucidate the relative involvement of drug resistance gene copy number and overexpression in fluconazole resistance in clinical C. glabrata isolates using a population-based approach. Methods: Fluconazole resistance levels were quantified using the minimal inhibitory concentration (MIC) via Etest method. Both gene expression levels and gene copy number of CgCDR1, CgPDH1, CgERG11, and CgSNQ2 were assessed via quantitative real-time PCR. The influence of the main effects and first-level interactions of both the expression level and copy number of these genes on fluconazole resistance levels were analyzed using a multivariate statistical model. Results: Forty-three C. glabrata isolates were collected from 30 patients during in a hospital survey. In the multivariate analysis, C. glabrata fluconazole MICs were independently increased by CgSNQ2 overexpression (p < 10−4) and the interaction between CgPDH1 gene copy number and CgPDH1 expression level (p = 0.038). In contrast, both CgPDH1 overexpression (p = 0.049) and the interaction between CgSNQ2 and CgERG11 expression (p = 0.003) led to a significant decrease in fluconazole MICs. Conclusion: Fluconazole resistance in C. glabrata involves complex interactions between drug resistance gene expression and/or copy number. The population-based multivariate analysis highlighted the involvement of the CgSNQ2 gene in fluconazole resistance and the complex effect of the other genes such as PDH1 for which overexpression was associated with reduced fluconazole resistance levels, while the interaction between PDH1 overexpression and copy number was associated with increased resistance levels. PMID:24273749

  7. Restored expression levels of TET1 decrease the proliferation and migration of renal carcinoma cells

    PubMed Central

    FAN, MIN; HE, XIAOZHOU; XU, XIANLIN

    2015-01-01

    Renal carcinoma is the most common type of kidney cancer in adults and is responsible for ~90–95% of the cases of kidney cancer. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is a member of the TET family of enzymes, and is expressed at low levels in multiple malignancies. In the present study, a series of experiments were designed and performed to investigate whether the expression of TET1 is clinically correlated with clinical outcomes in renal carcinoma, and to examine the associations between TET1 expression level and the proliferation and migration in renal carcinoma cells. As a result, TET1 was observed to exhibit markedly low expression levels in 54 tumor tissue samples from 54 patients with renal carcinoma. Furthermore, statistical analysis revealed a clinical correlation between low expression levels of TET1 and the prognosis of patients with renal carcinoma. When TET1 was overexpressed in renal carcinoma cells, the viability and invasive abilities of the cells were decreased, and the rate of apoptosis was increased. In conclusion, the results demonstrated that TET1 is involved in tumor inhibition in renal carcinoma by promoting cell apoptosis and inhibiting cell proliferation and invasion, which may be exploited as a novel therapeutic target in the treatment of renal carcinoma. PMID:26165803

  8. Low-level lasers affect uncoupling protein gene expression in skin and skeletal muscle tissues

    NASA Astrophysics Data System (ADS)

    Canuto, K. S.; Sergio, L. P. S.; Paoli, F.; Mencalha, A. L.; Fonseca, A. S.

    2016-03-01

    Wavelength, frequency, power, fluence, and emission mode determine the photophysical, photochemical, and photobiological responses of biological tissues to low-level lasers. Free radicals are involved in these responses acting as second messengers in intracellular signaling processes. Irradiated cells present defenses against these chemical species to avoid unwanted effects, such as uncoupling proteins (UCPs), which are part of protective mechanisms and minimize the effects of free radical generation in mitochondria. In this work UCP2 and UCP3 mRNA gene relative expression in the skin and skeletal muscle tissues of Wistar rats exposed to low-level red and infrared lasers was evaluated. Samples of the skin and skeletal muscle tissue of Wistar rats exposed to low-level red and infrared lasers were withdrawn for total RNA extraction, cDNA synthesis, and the evaluation of gene expression by quantitative polymerase chain reaction. UCP2 and UCP3 mRNA expression was differently altered in skin and skeletal muscle tissues exposed to lasers in a wavelength-dependent effect, with the UCP3 mRNA expression dose-dependent. Alteration on UCP gene expression could be part of the biostimulation effect and is necessary to make cells exposed to red and infrared low-level lasers more resistant or capable of adapting in damaged tissues or diseases.

  9. Effects of phased joint intervention on Rho/ROCK expression levels in patients with portal hypertension

    PubMed Central

    Shi, Min; Wei, Jue; Meng, Wen-Ying; Wang, Na; Wang, Ting; Wang, Yu-Gang

    2016-01-01

    The current study investigated the effects of phased joint intervention on clinical efficacy and Rho/Rho-associated coil protein kinase (ROCK) expression in patients with portal hypertension complicated by esophageal variceal bleeding (EVB) and hypersplenism. Patients with portal hypertension (n=53) caused by liver cirrhosis complicated by EVB and hypersplenism treated with phased joint intervention were assessed, and portal hemodynamics, blood, liver function, complications, and rebleeding incidence were analyzed. Reverse transcription-quantitative polymerase chain reaction was used to measure Rho, ROCK1 and ROCK2 mRNA expression levels in peripheral blood mononuclear cells prior to and following phased joint intervention, and western blotting was employed to determine the protein expression levels of Rho, ROCK1, ROCK2, phosphorylated (p) myosin phosphatase target subunit 1 (MYPT1) and total-MYPT1. All patients underwent an emergency assessment of hemostasis with a 100% success rate. Varicose veins were alleviated, and portal hemodynamics and liver function improved following intervention. Furthermore, preoperative and postoperative expression levels of Rho, ROCK1 and ROCK2 mRNA were higher compared with the control group. Notably, the mRNA expression levels of Rho, ROCK1 and ROCK2 in the postoperative group were significantly lower when compared with the preoperative group. Protein expression levels of Rho, ROCK1, ROCK2 and pMYPT1 in the postoperative group were lower, as compared with the preoperative group. Concentration levels of transforming growth factor-β1, connective tissue growth factor and platelet-derived growth factor in peripheral blood were significantly reduced following phased joint intervention. Therefore, the present findings demonstrated that phased joint intervention is able to effectively treat EVB and hypersplenism, and improve liver function. The efficacy of phased joint intervention may be associated with its role in the regulation of the

  10. Different 3' end regions strongly influence the level of gene expression in plant cells.

    PubMed Central

    Ingelbrecht, I L; Herman, L M; Dekeyser, R A; Van Montagu, M C; Depicker, A G

    1989-01-01

    We have investigated the functional role of a 3' end region on the expression of a reporter gene in plant cells. In stably transformed plants, expression of the reporter gene without a plant gene 3' end is variable and depends on the fortuitous presence of polyadenylation signals in the downstream sequences. When the reporter gene is flanked by pBR322 DNA, 3'-processing and polyadenylation occurs at (a) cryptic site(s) within these vector sequences. Using a transient gene expression system, we present a deletion analysis of the 3' end of the octopine synthase gene showing that the most proximal polyadenylation signal per se is not sufficient to ensure expression but that a downstream (G)T-rich sequence is also required. Optimal expression of the fusion gene requires more than 98 base pairs and at most 142 base pairs downstream from the most distal polyadenylation site. We analyzed the expression of chimeric genes with 3' end sequences originating from different plant genes. In the transient expression assay, all constructs direct similar neomycin phosphotransferase II activities. However, in stably transformed tissue, the gene constructs displayed characteristic expression levels which varied as much as 60-fold. This result suggests a role for 3' end sequences in post-transcriptional processes such as efficiency of 3'-processing and/or mRNA stability. PMID:2562510

  11. Relationship between TLR4 and MCP2 expression levels and habitual abortion.

    PubMed

    Li, X P; Song, L N; Tian, L P; Zhang, Y S

    2016-01-01

    Habitual abortion is associated with the altered expression of multiple genes. This study was carried out to investigate the relationship between expression of Toll-like receptor 4 (TLR4) and monocyte chemotactic protein 2 (MCP2 or CCL8) and habitual abortion. This was done by detecting and comparing their relative expression in peripheral blood and placental villi of patients and healthy fertile women. Based on our previous research, 85 subjects with habitual abortion (study group) and 40 healthy fertile women (control group), who were admitted to our hospital between June 2013 and December 2014, were enrolled in this study. After these subjects signed written informed consent, peripheral blood samples and villous tissues were collected, from which the total RNA was extracted. The expression of TLR4 and MCP2 was detected with quantitative reverse transcription-polymerase chain reaction, using GAPDH as a reference control. The expression of TLR4 and MCP2 in the peripheral blood and villous tissues of the study group was significantly higher than that of the control group (P < 0.05). A positive correlation was also observed between the changes in expression levels of TLR4 and MCP2. In conclusion, TLR4 and MCP2 expression correlated with the occurrence of habitual abortion. Detecting expression changes in TLR4 and MCP2 in the peripheral blood is a feasible method for predicting the occurrence of abortion in women of child-bearing age. PMID:27173235

  12. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  13. TSLP Expression and High Serum TSLP Level Indicate a Poor Prognosis in Gastric Cancer Patients

    PubMed Central

    Watanabe, Joji; Saito, Hiroaki; Miyatani, Kozo; Ikeguchi, Masahide; Umekita, Yoshihisa

    2015-01-01

    Background Thymic stromal lymphopoietin (TSLP) plays an important role in promoting tumor survival, by manipulating the immune response and angiogenesis. However, the clinical significance of TSLP in gastric cancer is unclear. Methods Immunohistochemistry was used to investigate TSLP expression in non-cancerous gastric mucosa and gastric cancer tissue from patients with gastric cancer. Serum TSLP levels were measured using an enzyme-linked immunosorbent assay. Results Tumors with TSLP expression were significantly larger than those without TSLP expression. TSLP expression was observed more frequently in advanced (T2/T3/T4) than in early (T1) gastric cancer and in stage 3/4 than in stage 1/2. Lymph node metastasis, liver metastasis, positive peritoneal lavage cytology, lymphatic invasion, and vascular invasion occurred significantly more often in TSLP-expressing than in non-expressing tumors. The prognosis of patients with TSLP-positive tumors was significantly worse than that of patients with TSLP-negative tumors. Patients with high serum TSLP concentrations also had a significantly worse prognosis than those with low concentrations. Multivariate analysis identified serum TSLP level as an independent prognostic indicator. Conclusion TSLP is closely related to the progression of gastric cancer and may predict survival in these patients. PMID:26538800

  14. Analysis of gene expression patterns and levels in maize hybrids and their parents.

    PubMed

    Nie, H S; Li, S P; Shan, X H; Wu, Y; Su, S Z; Liu, H K; Han, J Y; Yuan, Y P

    2015-01-01

    Heterosis has greatly contributed to conventional plant breeding and is widely used to increase crop plant productivity. However, although some studies have explored the mechanisms of heterosis at the genomic and transcriptome level, these mechanisms still remain unclear. The growth and development of maize seedlings and immature embryos have an important impact on subsequent production. This study investigated differentially expressed genes (DEGs) between parents and reciprocal hybrids in the seedling leaves, roots, and immature embryo 15 days after pollination using amplified fragment length polymorphism (AFLP)-based transcript profiling (cDNA-AFLP). We isolated 180, 170, and 108 genes from the leaves, roots, and immature embryos, respectively, that were differentially expressed between hybrids and parents. Sequencing and functional analysis revealed that 107 transcript-derived fragments in the roots and leaves and 90 in the immature embryos were involved in known functions, whereas many DEGs had roles in plant growth and development, photosynthesis, signal transduction, and seed germination. Quantitative reverse-transcription polymerase chain reaction analysis of relative expression levels between reciprocal hybrids and both parental genotypes of selected genes produced results that were consistent with cDNA-AFLP. We validated the expression patterns of 15 selected genes related to heterosis formation and revealed that most showed non-additive expression in one or both hybrids, including dominant, underdominant, and overdominant expression. This indicates that gene-regulatory interactions among parental alleles play an important role in heterosis during the early developmental stages of maize. PMID:26634505

  15. High level expression of kringle 5 fragment of plasminogen in Pichia pastoris.

    PubMed

    Zhou, Yufei; Zheng, Quan; Gao, Jin; Gu, Jun

    2005-02-01

    Angiogensis can be blocked by inhibitors such as endostatin and angiostatin. The kringle 5 fragment of plasminogen also has a potent inhibitory effect on endothelial cell proliferation and leads to the inhibition of angiogenesis. It has promise in anti-angiogenic therapy due to its small size and potent inhibitory effect. Preparation of kringle 5 has been achieved through the proteolysis of native plasminogen and recombinant DNA technology. Bacterially expressed recombinant kringle 5 is mainly insoluble and expressed at low level. The refolding yield is also low. To produce recombinant human kringle 5 in a large quantity, we have genetically modified a strain of Pichia pastoris. On methanol induction, this strain expressed and secreted biologically active, recombinant kringle 5. The expression level of the engineered strain in culture reached more than 300 mgl(-1). Purification was easily achieved by precipitation, hydrophobic and DEAE ion exchange chromatography. The recovery of recombinant kringle 5 was about 50% after purification. Yeast-expressed kringle 5 has a higher activity in anti-endothelial proliferation than bacterially expressed kringle 5. PMID:15717125

  16. Elevated gene expression levels distinguish human from non-human primate brains

    PubMed Central

    Cáceres, Mario; Lachuer, Joel; Zapala, Matthew A.; Redmond, John C.; Kudo, Lili; Geschwind, Daniel H.; Lockhart, David J.; Preuss, Todd M.; Barlow, Carrolee

    2003-01-01

    Little is known about how the human brain differs from that of our closest relatives. To investigate the genetic basis of human specializations in brain organization and cognition, we compared gene expression profiles for the cerebral cortex of humans, chimpanzees, and rhesus macaques by using several independent techniques. We identified 169 genes that exhibited expression differences between human and chimpanzee cortex, and 91 were ascribed to the human lineage by using macaques as an outgroup. Surprisingly, most differences between the brains of humans and non-human primates involved up-regulation, with ≈90% of the genes being more highly expressed in humans. By contrast, in the comparison of human and chimpanzee heart and liver, the numbers of up- and down-regulated genes were nearly identical. Our results indicate that the human brain displays a distinctive pattern of gene expression relative to non-human primates, with higher expression levels for many genes belonging to a wide variety of functional classes. The increased expression of these genes could provide the basis for extensive modifications of cerebral physiology and function in humans and suggests that the human brain is characterized by elevated levels of neuronal activity. PMID:14557539

  17. Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells.

    PubMed

    Kwaks, T H J; Sewalt, R G A B; van Blokland, R; Siersma, T J; Kasiem, M; Kelder, A; Otte, A P

    2005-01-12

    Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently. PMID:15607223

  18. Levels of Lycopene β-Cyclase 1 Modulate Carotenoid Gene Expression and Accumulation in Daucus carota

    PubMed Central

    Moreno, Juan Camilo; Pizarro, Lorena; Fuentes, Paulina; Handford, Michael; Cifuentes, Victor; Stange, Claudia

    2013-01-01

    Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and β-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and β-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots. PMID:23555569

  19. Association between plasma selenium level and NRF2 target genes expression in humans.

    PubMed

    Reszka, Edyta; Wieczorek, Edyta; Jablonska, Ewa; Janasik, Beata; Fendler, Wojciech; Wasowicz, Wojciech

    2015-04-01

    Animal studies in rodent and in vitro studies indicate compensatory role of nuclear factor (erythroid-derived 2)-like (Nrf2) and Nrf2-regulated antioxidant and phase II biotransformation enzymes for the dietary selenium (Se) deficiency or for the loss of selenoproteins. To explore associations between plasma Se level and NRF2-regulated cytoprotective genes expression, an observational study was conducted in a population of 96 healthy non-smoking men living in Central Poland aged 18-83 years with relatively low plasma Se level. NRF2, KEAP2, CAT, EPHX1, GCLC, GCLM, GPX2, GSR, GSTA1, GSTM1, GSTP1, GSTT1, HMOX1, NQO1, PRDX1, SOD1, SOD2, TXNRD1 transcript levels in peripheral blood leukocytes and polymorphism of NRF2-617C/A (rs6721961) in blood genomic DNA were determined by means of quantitative real-time PCR. Mean plasma Se level was found to be 51.10±15.25μg/L (range 23.86-96.18μg/L). NRF2 mRNA level was positively correlated with expression of investigated NRF2-target genes. The multivariate linear regression adjusting for selenium status showed that plasma Se level was significantly inversely associated only with expression of GSTP1 (β-coef.=-0.270, p=0.009), PRDXR1 (β-coef.=-0.245, p=0.017) and SOD2 with an inverse trend toward significance (β-coef.=-0.186, p=0.074), but without an effect of NRF2 gene variants. NRF2 expression was inversely associated with age (r=-0.23, p=0.03) and body mass index (r=-0.29, p<0.001). The findings may suggest a possible link between plasma Se level and cytoprotective response at gene level in humans. PMID:25524402

  20. The RNA-binding protein gene, hermes, is expressed at high levels in the developing heart.

    PubMed

    Gerber, W V; Yatskievych, T A; Antin, P B; Correia, K M; Conlon, R A; Krieg, P A

    1999-01-01

    In a screen for novel sequences expressed during embryonic heart development we have isolated a gene which encodes a putative RNA-binding protein. This protein is a member of one of the largest families of RNA-binding proteins, the RRM (RNA Recognition Motif) family. The gene has been named hermes (for HEart, RRM Expressed Sequence). The hermes protein is 197-amino acids long and contains a single RRM domain. In situ hybridization analysis indicates that hermes is expressed at highest levels in the myocardium of the heart and to a lesser extent in the ganglion layer of the retina, the pronephros and epiphysis. Expression of hermes in each of these tissues begins at approximately the time of differentiation and is maintained throughout development. Analysis of the RNA expression of the hermes orthologues from chicken and mouse reveals that, like Xenopus, the most prominent tissue of expression is the developing heart. The sequence and expression pattern of hermes suggests a role in post-transcriptional regulation of heart development. PMID:10096065

  1. Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding.

    PubMed

    Chappell, Paul; Meziane, El Kahina; Harrison, Michael; Magiera, Łukasz; Hermann, Clemens; Mears, Laura; Wrobel, Antony G; Durant, Charlotte; Nielsen, Lise Lotte; Buus, Søren; Ternette, Nicola; Mwangi, William; Butter, Colin; Nair, Venugopal; Ahyee, Trudy; Duggleby, Richard; Madrigal, Alejandro; Roversi, Pietro; Lea, Susan M; Kaufman, Jim

    2015-01-01

    Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. PMID:25860507

  2. Gene expression in diplosporous and sexual Eragrostis curvula genotypes with differing ploidy levels.

    PubMed

    Cervigni, Gerardo D L; Paniego, Norma; Pessino, Silvina; Selva, Juan P; Díaz, Marina; Spangenberg, Germán; Echenique, Viviana

    2008-05-01

    The molecular nature of gene expression during the initiation and progress of diplosporous apomixis is still unknown. Moreover, the basis of the close correlation between diplospory and polyploidy is not clarified yet. A comparative expression analysis was performed based on expressed sequence tags (ESTs) sequencing and differential display in an Eragrostis curvula diplosporous tetraploid genotype (T, 4x apo), a sexual diploid derivative obtained from tissue culture (D, 2x sex) and an artificial sexual tetraploid obtained from the diploid seeds after colchicine treatment (C, 4x sex). From a total of 8,884 unigenes sequenced from inflorescence-derived libraries, 112 (1.26%) showed significant differential expression in individuals with different ploidy level and/or variable reproductive mode. Independent comparisons between plants with different reproductive mode (same ploidy) or different ploidy level (same reproductive mode) allowed the identification of genes modulated in response to diplosporous development or polyploidization, respectively. Surprisingly, a group of genes (Group 3) were differentially expressed or silenced only in the 4x sex plant, presenting similar levels of expression in the 4x apo and the 2x sex genotypes. A group of randomly selected differential genes was validated by QR-PCR. Differential display analysis showed that in general the 4x apo and 4x sex expression profiles were more related and different from the 2x sex one, but confirmed the existence of Group 3-type genes, in both inflorescences and leaves. The possible biological significance for the occurrence of this particular group of genes is discussed. In silico mapping onto the rice genome was used to identify candidates mapping to the region syntenic to the diplospory locus. PMID:18311543

  3. Correlation between radiation dose and p53 protein expression levels in human lymphocytes.

    PubMed

    Cavalcanti, Mariana B; Fernandes, Thiago S; Silva, Edvane B; Amaral, Ademir

    2015-09-01

    The aim of this research was to evaluate the relationship between p53 protein levels and absorbed doses from in vitro irradiated human lymphocytes. For this, samples of blood from 23 donors were irradiated with 0.5; 1; 2; and 4 Gy from a Cobalt-60 source, and the percentages of lymphocytes expressing p53 were scored using Flow Cytometry. The subjects were divided into 3 groups, in accordance with the p53 levels expressed per radiation dose: low (Group I), high (Group II), and excessive levels (Group III). For all groups, the analyses showed that the p53 expression levels increase with the absorbed dose. Particularly for groups I and II, the correlation between this protein expression and the dose follows the linear-quadratic model, such as for radioinduced chromosomal aberrations. In conclusion, our findings indicate possible applications of this approach in evaluating individual radiosensitivity prior to radiotherapeutical procedures as well as in medical surveillance of occupationally exposed workers. Furthermore, due to the rapidity of flow-cytometric analyses, the methodology here employed would play an important role in emergency responses to a large-scale radiation incident where many people may have been exposed. PMID:26312422

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

    PubMed

    Shirk, Paul D; Furlong, Richard B

    2016-01-01

    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 that are involved in integration of the densovirus in insect cell chromosomes and a promoter/enhancer system that results in high levels of expression. The plasmid also contains two markers that permit selection of transformed insect cells by antibiotic resistance or by cell-sorting for fluorescent protein expression. Transformation of Bombyx mori Bm5 or Spodoptera frugiperda Sf9 cultured cells with the pDP9 vectors results in the integration of the pDP9 plasmid into genomic DNA of Bm5 and Sf9 cells. pDP9 contains a multiple cloning site (MCS) 3' of the densoviral P9 promoter and insertion of a protein coding sequence within the MCS results in high level expression by pDP9 transformed cells. P9 driven transcription in the pDP9 transformed Sf9 cells produced foreign gene transcript levels that were 30 fold higher than actin 3 driven transgenes and equivalent to hr5IE1 driven transgenes. The pDP9 vector transformation results in the efficient selection of clones for assessment of promoter activity. PMID:26794473

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Elevated interleukin-6 expression levels are associated with intervertebral disc degeneration

    PubMed Central

    DENG, XIAO; ZHAO, FENG; KANG, BAOLIN; ZHANG, XIN

    2016-01-01

    The present study aimed to investigate whether serum interleukin-6 (IL-6) expression levels were associated with the onset and progression of intervertebral disc degeneration (IDD). A comprehensive meta-analysis of the scientific literature from numerous electronic databases was performed, in order to obtain published studies associated with the topic of interest. Relevant case-control studies that had previously assessed a correlation between IL-6 expression levels and IDD were identified using predetermined inclusion and exclusion criteria. The STATA version 12.0 software was used for statistical analysis of the extracted data. A total of 112 studies were initially retrieved, with eight studies meeting the inclusion criteria. These contained a total of 392 subjects, of which 263 were patients with IDD and 129 were healthy controls. A meta-analysis of the eight studies demonstrated that serum IL-6 protein expression levels may be associated with IDD, and this was irrespective of IDD subtype (bulging, protrusion, or sequestration). Notably, serum expression levels of the IL-6 protein were upregulated in intervertebral disc (IVD) protrusion tissue, as compared with normal IVD tissue; thus suggesting that IL-6 may have an important role in the pathophysiological process of IDD. PMID:27073460

  7. High level expression of organophosphorus hydrolase in Pichia pastoris by multicopy ophcM assembly.

    PubMed

    Shen, Wei; Shu, Min; Ma, Lixin; Ni, Hong; Yan, Hong

    2016-03-01

    The residues of organophosphorus pesticides bring serious impact on the environmental safety and people's health. Biodegradation of organophosphorus pesticides is recognized as an ideal method. An organophosphorus hydrolase (OPHCM) from Pseudomonas pseudoalcaligenes was synthesized and expressed in Pichia pastoris. The yield reached approximately 470 mg/l after a 6-d induction in shake flasks. To improve the enzyme production, we describe a novel approach to express OPHCM efficiently with a biobrick assembly method in vitro. Four recombinant plasmids containing 1-4 copies of ophcM-expressing cassettes were constructed and transformed into P. pastoris. Increasing the copy number of ophcM gene enhanced the expression level of OPHCM. The maximum yield and specific activity in P. pastoris harboring two-copy tandem ophcM-expressing cassettes reached 610 mg/l after a 6-d induction in shake flasks and 7.8 g/l in high-density fermentation with specific activity of 13.7 U/mg. The optimum pH and temperature of the recombinant OPHCM activity were 11.0 and 50 °C, respectively. In addition, the enzyme activity of recombinant OPHCM enhanced 57.6% and 30.1% in the presence of 1 mM Cd(2+) and 5% glycerol, respectively. The high expression and good properties of recombinant OPHCM provide an effective solution to solve the pollution of organophosphorus pesticides in the environment. Moreover, the approach for generating multicopy gene expressing vectors here will benefit the study for enhancing the expression level of genes of interest. PMID:26611611

  8. High Levels of MeCP2 Depress MHC Class I Expression in Neuronal Cells

    PubMed Central

    Miralvès, Julie; Magdeleine, Eddy; Kaddoum, Lara; Brun, Hélène; Peries, Sophie; Joly, Etienne

    2007-01-01

    Background The expression of MHC class I genes is repressed in mature neurons. The molecular basis of this regulation is poorly understood, but the genes are particularly rich in CpG islands. MeCP2 is a transcriptional repressor that binds to methylated CpG dinucleotides; mutations in this protein also cause the neurodevelopmental disease called Rett syndrome. Because MHC class I molecules play a role in neuronal connectivity, we hypothesised that MeCP2 might repress MHC class I expression in the CNS and that this might play a role in the pathology of Rett syndrome. Methodology We show here that transiently transfected cells expressing high levels of MeCP2 specifically downregulate cell-surface expression of MHC class I molecules in the neuronal cell line N2A and they prevent the induction of MHC class I expression in response to interferon in these cells, supporting our first hypothesis. Surprisingly, however, overexpression of the mutated forms of MeCP2 that cause Rett syndrome had a similar effect on MHC class I expression as the wild-type protein. Immunohistological analyses of brain slices from MECP2 knockout mice (the MeCP2tm1.1Bird strain) demonstrated a small but reproducible increase in MHC class I when compared to their wild type littermates, but we found no difference in MHC class I expression in primary cultures of mixed glial cells (mainly neurons and astrocytes) from the knockout and wild-type mice. Conclusion These data suggest that high levels of MeCP2, such as those found in mature neurons, may contribute to the repression of MHC expression, but we find no evidence that MeCP2 regulation of MHC class I is important for the pathogenesis of Rett syndrome. PMID:18159237

  9. PAQR3 gene expression and its methylation level in colorectal cancer tissues

    PubMed Central

    Li, Ri-Heng; Zhang, Ai-Min; Li, Shuang; Li, Tian-Yang; Wang, Lian-Jing; Zhang, Hao-Ran; Shi, Jian-Wei; Liu, Xiao-Rui; Chen, Yuan; Chen, Ya-Chao; Wei, Teng-Yao; Gao, Ying; Li, Wei; Tang, Hong-Ying; Tang, Mei-Yu

    2016-01-01

    The aim of the present study was to investigate the PAQR3 gene expression and its methylation level in colorectal cancer tissues, as well as the association with colorectal cancer clinical data. In total, 54 cases of colorectal cancer tissue samples and normal adjacent tissue samples were collected between June, 2013 and July, 2014. RT-PCR and western blot analysis were used to detect the mRNA and protein levels of PAQR3 in colorectal samples, respectively. MSP was used to detect the methylation level of PAQR3 gene in colorectal samples, which was compared with colorectal data. The results showed that a decreased expression level of PAQR3 mRNA in colorectal cancer tissues and the expression reduction rate was 57.4% (31/54). Similarly, the expression level of PAQR3 protein was reduced in cancer tissues, and the reduction rate was 46.3% (25/54), while the protein expression reduction rate in cancer adjacent tissue was 5.6% (3/54), and the difference was statistically significant (P<0.05). Furthermore, the methylation rates of PAQR3 in cancer tissues and cancer adjacent tissues were 33.3% (18/54) and 5.6% (3/54), respectively. In addition, PAQR3 mRNA and protein levels in colorectal cancer tissues were associated with the differentiation degree, lymphatic metastasis and tumor infiltration depth. The methylation level of PAQR3 was associated with age, differentiated degree, lymphatic metastasis and tumor infiltration depth. In conclusion, the expression of PAQR3 mRNA and protein in colorectal cancer was reduced and methylation of PAQR3 occurred. Although the PAQR3 mRNA and protein levels were not associated with gender, age or the location of tumor, there was an association with differentiation degree, lymphatic metastasis and tumor infiltration depth. In addition, the methylation level of PAQR3 was not correlated with gender or tumor location, but was correlated with age, differentiation degree, lymphatic metastasis and tumor infiltration depth. PMID:27588124

  10. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes

    PubMed Central

    Soltani, Mohammad; Vargas-Garcia, Cesar A.; Antunes, Duarte; Singh, Abhyudai

    2016-01-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  11. Rabies Virus Vector Transgene Expression Level and Cytotoxicity Improvement Induced by Deletion of Glycoprotein Gene

    PubMed Central

    Ohara, Shinya; Sato, Sho; Oyama, Kei; Tsutsui, Ken-Ichiro; Iijima, Toshio

    2013-01-01

    The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level. PMID:24244660

  12. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes.

    PubMed

    Soltani, Mohammad; Vargas-Garcia, Cesar A; Antunes, Duarte; Singh, Abhyudai

    2016-08-01

    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells. PMID:27536771

  13. Effect of temperature on oxidative stress, antioxidant levels and uncoupling protein expression in striped hamsters.

    PubMed

    Zhou, Si-Si; Cao, Li-Li; Xu, Wei-Dong; Cao, Jing; Zhao, Zhi-Jun

    2015-11-01

    According to the rate of living-free radical hypothesis, higher metabolic rates should increase reactive oxygen species (ROS) production. However, the "uncoupling to survive" hypothesis postulates that uncoupling proteins (UCPs) can decrease ROS production by lowering the potential of the inner mitochondrial membrane, in which case the correlation between metabolic rate and ROS levels would be a negative rather than positive. In this study, we examined energy intake, oxidative stress levels, antioxidant activity and the expression of UCPs in brown adipose tissue (BAT), and in the liver, heart, skeletal muscle and brain, of striped hamsters (Cricetulus barabensis) acclimated to either 5 °C or 32.5 °C. The energy intake of hamsters acclimated to 5 °C increased by 70.7%, whereas the energy intake of hamsters acclimated to 32.5 °C decreased by 31.3%, relative to hamsters kept at room temperature (21 °C) (P<0.05). Malonadialdehyde (MDA) levels, total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX) activity in BAT significantly decreased in 5 °C group, but increased in 32.5 °C group, relative to the 21 °C group. Neither ROS levels (i.e. H2O2 levels), nor antioxidants in skeletal muscle, liver, heart or brain tissue, were affected by temperature. UCP1 expression in BAT was significantly up-regulated in 5 °C group, but down-regulated in 32.5 °C group, relative to the 21 °C group. UCP3 expression of skeletal muscle was also up-regulated significantly in hamsters acclimated to 5 °C. These results suggest that the relationship between ROS levels and metabolic rate was negative, rather than positive. UCP1 expression in BAT may have played a role in lowering ROS levels. PMID:26244518

  14. Reduced gene expression levels after chronic exposure to high concentrations of air pollutants.

    PubMed

    Rossner, Pavel; Tulupova, Elena; Rossnerova, Andrea; Libalova, Helena; Honkova, Katerina; Gmuender, Hans; Pastorkova, Anna; Svecova, Vlasta; Topinka, Jan; Sram, Radim J

    2015-10-01

    We analyzed the ability of particulate matter (PM) and chemicals adsorbed onto it to induce diverse gene expression profiles in subjects living in two regions of the Czech Republic differing in levels and sources of the air pollution. A total of 312 samples from polluted Ostrava region and 154 control samples from Prague were collected in winter 2009, summer 2009 and winter 2010. The highest concentrations of air pollutants were detected in winter 2010 when the subjects were exposed to: PM of aerodynamic diameter <2.5μm (PM2.5) (70 vs. 44.9μg/m(3)); benzo[a]pyrene (9.02 vs. 2.56ng/m(3)) and benzene (10.2 vs. 5.5μg/m(3)) in Ostrava and Prague, respectively. Global gene expression analysis of total RNA extracted from leukocytes was performed using Illumina Expression BeadChips microarrays. The expression of selected genes was verified by quantitative real-time PCR (qRT-PCR). Gene expression profiles differed by locations and seasons. Despite lower concentrations of air pollutants a higher number of differentially expressed genes and affected KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways was found in subjects from Prague. In both locations immune response pathways were affected, in Prague also neurodegenerative diseases-related pathways. Over-representation of the latter pathways was associated with the exposure to PM2.5. The qRT-PCR analysis showed a significant decrease in expression of APEX, ATM, FAS, GSTM1, IL1B and RAD21 in subjects from Ostrava, in a comparison of winter 2010 and summer 2009. In Prague, an increase in gene expression was observed for GADD45A and PTGS2. In conclusion, high concentrations of pollutants in Ostrava were not associated with higher number of differentially expressed genes, affected KEGG pathways and expression levels of selected genes. This observation suggests that chronic exposure to air pollution may result in reduced gene expression response with possible negative health consequences. PMID:26298100

  15. Decreased blood riboflavin levels are correlated with defective expression of RFT2 gene in gastric cancer

    PubMed Central

    Eli, Maynur; Li, De-Sheng; Zhang, Wei-Wei; Kong, Bing; Du, Chen-Song; Wumar, Maimaitiaili; Mamtimin, Batur; Sheyhidin, Ilyar; Hasim, Ayshamgul

    2012-01-01

    AIM: To investigate the relationship between blood riboflavin levels and riboflavin transporter 2 (RFT2) gene expression in gastric carcinoma (GC) development. METHODS: High-performance liquid chromatography was used to detect blood riboflavin levels in patients with GC. Real-time fluorogenic quantitative polymerase chain reaction and immunohistochemistry were used to analyze the expression of RFT2 mRNA and protein in samples from 60 GC patients consisting of both tumor and normal tissue. RESULTS: A significant decrease in the RFT2 mRNA levels was detected in GC samples compared with those in the normal mucous membrane (0.398 ± 0.149 vs 1.479 ± 0.587; P = 0.040). Tumors exhibited low RFT2 protein expression (75%, 16.7%, 8.3% and 0% for no RFT2 staining, weak staining, medium staining and strong staining, respectively), which was significantly lower than that in the normal mucous membrane (10%, 16.7%, 26.7% and 46.7% for no RFT2 staining, weak staining, medium staining and strong staining, respectively; P < 0.05). Tumors with low RFT2 expression were significantly associated with tumor stage and histological grade. Moreover, a significantly decrease in Uyghur patients was observed compared with Han patients. However, other parameters-gender, tumor location and lymph node metastasis-showed no significant relationship with RFT2 expression. Blood riboflavin levels were reverse correlated with development of GC (1.2000 ± 0.97 569 ng/mL in high tumor stage patients vs 2.5980 ± 1.31 129 ng/mL in low tumor stage patients; P < 0.05). A positive correlation of plasma riboflavin levels with defective expression of RFT2 protein was found in GC patients (χ2 = 2.619; P = 0.019). CONCLUSION: Defective expression of RFT2 is associated with the development of GC and this may represent a mechanism underlying the decreased plasma riboflavin levels in GC. PMID:22791947

  16. Role of EGFR expression levels in the regulation of integrin function by EGF.

    PubMed

    Vial, Daniel; McKeown-Longo, Paula J

    2016-06-01

    Activation of β1 integrins in dormant tumor cells has been linked to metastatic progression, suggesting that therapies designed to maintain β1 integrins in an inactive state may be useful in the prevention of metastatic disease. Our earlier studies have demonstrated that EGF regulates the activation state of the α5β1 integrin in EGFR overexpressing tumor cells through an ERK/p90RSK signaling pathway. Activation of this pathway by EGF resulted in the filamin A dependent inactivation of the α5β1 integrin receptor for fibronectin. The current study was designed to address the role of EGFR overexpression in the regulation of α5β1 integrin activation state by EGF. Lentiviral knockdown of EGFR coupled with limited dilution cloning was used to develop A431 squamous carcinoma cell lines expressing high, moderate, and low levels of EGFR. Inactivation of α5β1 integrin by EGF was shown to correlate with both the level of EGFR expression and the extent of p90RSK phosphorylation, but not with the level of ERK phosphorylation, suggesting that high levels of EGFR promote α5β1 integrin inactivation through sustained activation of p90RSK. Treatment of cells with EGFR kinase inhibitor resulted in a reactivation of the integrin which could be reversed with the phosphatase inhibitor, menadione. Taken together, these findings indicate that p90RSK may function to maintain dormancy in tumor cells expressing high levels of EGFR. © 2015 Wiley Periodicals, Inc. PMID:26053065

  17. The level of cytokines and expression of caspase genes in rheumatoid arthritis.

    PubMed

    Malysheva, I E; Topchieva, L V; Barysheva, O Y; Kurbatova, I V; Vasykova, O A; Vezikova, N N; Marusenko, I M; Nemova, N N

    2016-05-01

    The level of TNFα and IL6 in the blood plasma of patients with rheumatoid arthritis (RA) who received antiinflammatory therapy with methotrexate (MT) was significantly lower than in the patients without MT treatment. The level of caspase 6 and 9 gene transcripts in peripheral blood lymphocytes in patients with rheumatoid arthritic diagnosed for the first time and in patients with MT treatment were not significantly different. At the same time, the level of caspase 3 mRNA expression was significantly higher in the cells of the RA patients with MT therapy compared to the patients without MT therapy. PMID:27417728

  18. The Expression of c-Myb Correlates with the Levels of Rhabdomyosarcoma-specific Marker Myogenin

    PubMed Central

    Kaspar, Petr; Zikova, Martina; Bartunek, Petr; Sterba, Jaroslav; Strnad, Hynek; Kren, Leos; Sedlacek, Radislav

    2015-01-01

    The transcription factor c-Myb is required for modulation of progenitor cells in several tissues, including skeletal muscle and its upregulation is observed in many human malignancies. Rhabdomyosarcomas (RMS) are a heterogeneous group of mesodermal tumors with features of developing skeletal muscle. Several miRNAs are downregulated in RMS, including miR-150, a negative regulator of c-Myb expression. Using the C2C12 myoblast cell line, a cellular model of skeletal muscle differentiation, we showed that miR-150 controls c-Myb expression mainly at the level of translation. We hypothesized that a similar mechanism of c-Myb regulation operates in RMS tumors. We examined expression of c-Myb by immunohistochemistry and revealed c-Myb positivity in alveolar and embryonal tumors, the two most common subgroups of RMS. Furthermore, we showed direct correlation between c-Myb production and myogenin expression. Interestingly, high myogenin levels indicate poor prognosis in RMS patients. c-Myb could, therefore, contribute to the tumor phenotype by executing its inhibitory role in skeletal muscle differentiation. We also showed that c-Myb protein is abundant in migratory C2C12 myoblasts and its ectopic expression potentiates cell motility. In summary, our results implicate that metastatic properties of some RMS subtypes might be linked to c-Myb function. PMID:26462877

  19. The Expression of c-Myb Correlates with the Levels of Rhabdomyosarcoma-specific Marker Myogenin.

    PubMed

    Kaspar, Petr; Zikova, Martina; Bartunek, Petr; Sterba, Jaroslav; Strnad, Hynek; Kren, Leos; Sedlacek, Radislav

    2015-01-01

    The transcription factor c-Myb is required for modulation of progenitor cells in several tissues, including skeletal muscle and its upregulation is observed in many human malignancies. Rhabdomyosarcomas (RMS) are a heterogeneous group of mesodermal tumors with features of developing skeletal muscle. Several miRNAs are downregulated in RMS, including miR-150, a negative regulator of c-Myb expression. Using the C2C12 myoblast cell line, a cellular model of skeletal muscle differentiation, we showed that miR-150 controls c-Myb expression mainly at the level of translation. We hypothesized that a similar mechanism of c-Myb regulation operates in RMS tumors. We examined expression of c-Myb by immunohistochemistry and revealed c-Myb positivity in alveolar and embryonal tumors, the two most common subgroups of RMS. Furthermore, we showed direct correlation between c-Myb production and myogenin expression. Interestingly, high myogenin levels indicate poor prognosis in RMS patients. c-Myb could, therefore, contribute to the tumor phenotype by executing its inhibitory role in skeletal muscle differentiation. We also showed that c-Myb protein is abundant in migratory C2C12 myoblasts and its ectopic expression potentiates cell motility. In summary, our results implicate that metastatic properties of some RMS subtypes might be linked to c-Myb function. PMID:26462877

  20. Analysis of gene expression levels in individual bacterial cells without image segmentation

    SciTech Connect

    Kwak, In Hae; Son, Minjun; Hagen, Stephen J.

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

  1. Androgen Receptors in a Cichlid Fish, Astatotilapia burtoni: Structure, Localization, and Expression Levels

    PubMed Central

    HARBOTT, LENE K.; BURMEISTER, SABRINA S.; WHITE, RICHARD B.; VAGELL, MIKE; FERNALD, RUSSELL D.

    2009-01-01

    Androgens are an important output of the hypothalamic-pituitary-gonadal (HPG) axis that controls reproduction in all vertebrates. In male teleosts two androgens, testosterone and 11-ketotestosterone, control sexual differentiation and development in juveniles and reproductive behavior in adults. Androgenic signals provide feedback at many levels of the HPG axis, including the hypothalamic neurons that synthesize and release gonadotropin-releasing hormone 1 (GnRH1), but the precise cellular site of androgen action in the brain is not known. Here we describe two androgen receptor subtypes, ARα and ARβ, in the cichlid Astatotilapia burtoni and show that these subtypes are differentially located throughout the adult brain in nuclei known to function in the control of reproduction. ARα was expressed in the ventral part of the ventral telencephalon, the preoptic area (POA) of the hypothalamus and the ventral hypothalamus, whereas ARβ was more widely expressed in the dorsal and ventral telencephalon, the POA, and the ventral and dorsal hypothalamus. We provide the first evidence in any vertebrate that the GnRH1-releasing neurons, which serve as the central control point of the HPG axis, express both subtypes of AR. Using quantitative real-time PCR, we show that A. burtoni AR subtypes have different expression levels in adult tissue, with ARα showing significantly higher expression than ARβ in the pituitary, and ARβ expressed at a higher level than ARα in the anterior and middle brain. These data provide important insight into the role of androgens in regulating the vertebrate reproductive axis. PMID:17614300

  2. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

    PubMed

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven C L; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing. PMID:25621616

  3. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

    PubMed Central

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven CL; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing. PMID:25621616

  4. Population-level expression variability of mitochondrial DNA-encoded genes in humans

    PubMed Central

    Wang, Gang; Yang, Ence; Mandhan, Ishita; Brinkmeyer-Langford, Candice L; Cai, James J

    2014-01-01

    Human mitochondria contain multiple copies of a circular genome made up of double-stranded DNA (mtDNA) that encodes proteins involved in cellular respiration. Transcript abundance of mtDNA-encoded genes varies between human individuals, yet the level of variation in the general population has not been systematically assessed. In the present study, we revisited large-scale RNA sequencing data generated from lymphoblastoid cell lines of HapMap samples of European and African ancestry to estimate transcript abundance and quantify expression variation for mtDNA-encoded genes. In both populations, we detected up to over 100-fold difference in mtDNA gene expression between individuals. The marked variation was not due to differences in mtDNA copy number between individuals, but was shaped by the transcription of hundreds of nuclear genes. Many of these nuclear genes were co-expressed with one another, resulting in a module-enriched co-expression network. Significant correlations in expression between genes of the mtDNA and nuclear genomes were used to identify factors involved with the regulation of mitochondrial functions. In conclusion, we determined the baseline amount of variability in mtDNA gene expression in general human populations and cataloged a complete set of nuclear genes whose expression levels are correlated with those of mtDNA-encoded genes. Our findings will enable the integration of information from both mtDNA and nuclear genetic systems, and facilitate the discovery of novel regulatory pathways involving mitochondrial functions. PMID:24398800

  5. Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato

    PubMed Central

    Haroldsen, Victor M.; Chi-Ham, Cecilia L.; Kulkarni, Shashank; Lorence, Argelia; Bennett, Alan B.

    2012-01-01

    Vitamin C (l-ascorbate, AsA) is an essential nutrient required in key metabolic functions in humans and must be obtained from the diet, mainly from fruits and vegetables. Given its importance in human health and plant physiology we sought to examine the role of the ascorbate recycling enzymes monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) in tomato (Solanum lycopersicum), an economically important fruit crop. Cytosolic-targeted tomato genes Mdhar and Dhar were cloned and over-expressed under a constitutive promoter in tomato var. Micro-Tom. Lines with increased protein levels and enzymatic activity were identified and examined. Mature green and red ripe fruit from DHAR over-expressing lines had a 1.6 fold increase in AsA content in plants grown under relatively low light conditions (150 µmol m−2 s−1). Conversely, MDHAR over-expressers had significantly reduced AsA levels in mature green fruits by 0.7 fold. Neither over-expressing line had altered levels of AsA in foliar tissues. These results underscore a complex regulation of the AsA pool size in tomato. PMID:21875809

  6. Genome-level analysis of genetic regulation of liver gene expression networks

    SciTech Connect

    Gatti, Daniel; Maki, Akira; Chesler, Elissa J; Kirova, Roumyana; Kosyk, Oksana; Lu, Lu; Manly, Kenneth; Matthews, Douglas B.; Qu, Yanhua; Williams, Robert; Perkins, Andy; Langston, Michael A; Threadgill, David; Rusyn, Ivan

    2007-01-01

    Liver is the primary site for metabolism of nutrients, drugs and chemical agents. While metabolic pathways are complex and tightly regulated, genetic variation among individuals, reflected in variation in gene expression levels, introduces complexity into research on liver disease. This study aimed to dissect genetic networks that control liver gene expression by combining largescale quantitative mRNA expression analysis with genetic mapping in a reference population of BXD recombinant inbred mouse strains for which extensive SNP, haplotype and phenotypic data is publicly available. We profiled gene expression in livers of naive mice of both sexes from C57BL/6J, DBA/2J, B6D2F1, and 37 BXD strains using Agilent oligonucleotide microarrays. This data was used to map quantitative trait loci (QTLs) responsible for variation in expression of about 19,000 transcripts. We identified polymorphic cis- and trans-acting loci, including several loci that control expression of large numbers of genes in liver, by comparing the physical transcript position with the location of the controlling QTL. The data is available through a public web-based resource (www.genenetwork.org) that allows custom data mining, identification of co-regulated transcripts and correlated phenotypes, cross-tissue and -species comparisons, as well as testing of a broad array of hypotheses.

  7. Tumor redox metabolism correlation with the expression level of red fluorescent protein

    NASA Astrophysics Data System (ADS)

    Sha, Shuang; Wang, Anle; Lin, Qiaoya; Zhang, Zhihong

    2015-03-01

    The redox metabolism is variable and complicated with the progress of tumor development. Whether the tumor redox state will affect the exogenous gene expression or not, are still not clear now . To investigate the relationship between tumor endogenous redox state and the exogenous gene expression level, a far red fluorescent protein fRFP was used to monitor tumor cells proliferation and as an exogenous protein expression in tumors. NADH (nicotinamide adenine dinucleotide) and Fp (flavin protein) are two important coenzymes in the mitochondria respiratory chain, which can be as a standard representation for redox metabolism state. Three tumor subcutaneous models (melanoma, human pancreatic carcinoma and nasopharyngeal carcinoma) were used to observe their redox state and protein expression by our home-made redox scanner. The results showed that the distribution of fRFP fluorescent protein expression in the inner tumor regions are heterogeneous, and the fluorescent intensity of fRFP and the fluorescent intensity of NADH have high correlation. In addition, we also found the linear coefficient in three tumors are different, the value of coefficient is (R2 = 0.966 and R2 = 0.943) in melanoma, (R2 = 0.701 and R2 = 0.942) in human pancreatic carcinoma, and (R2 = 0.994) in nasopharyngeal carcinoma, respectively. From these results, we consider that the exogenous protein expression of fRFP in tumor had some relationship with the tumor redox state of NADH.

  8. Enhanced human papillomavirus type 8 oncogene expression levels are crucial for skin tumorigenesis in transgenic mice

    SciTech Connect

    Hufbauer, M.; Lazic, D.; Akguel, B.; Brandsma, J.L.; Pfister, H.; Weissenborn, S.J.

    2010-08-01

    Human papillomavirus 8 (HPV8) is involved in skin cancer development in epidermodysplasia verruciformis patients. Transgenic mice expressing HPV8 early genes (HPV8-CER) developed papillomas, dysplasias and squamous cell carcinomas. UVA/B-irradiation and mechanical wounding of HPV8-CER mouse skin led to prompt papilloma induction in about 3 weeks. The aim of this study was to analyze the kinetics and level of transgene expression in response to skin irritations. Transgene expression was already enhanced 1 to 2 days after UVA/B-irradiation or tape-stripping and maintained during papilloma development. The enhanced transgene expression could be assigned to UVB and not to UVA. Papilloma development was thus always paralleled by an increased transgene expression irrespective of the type of skin irritation. A knock-down of E6 mRNA by tattooing HPV8-E6-specific siRNA led to a delay and a lower incidence of papilloma development. This indicates that the early increase of viral oncogene expression is crucial for induction of papillomatosis.

  9. RNA-Seq workflow: gene-level exploratory analysis and differential expression

    PubMed Central

    Love, Michael I.; Anders, Simon; Kim, Vladislav; Huber, Wolfgang

    2015-01-01

    Here we walk through an end-to-end gene-level RNA-Seq differential expression workflow using Bioconductor packages. We will start from the FASTQ files, show how these were aligned to the reference genome, and prepare a count matrix which tallies the number of RNA-seq reads/fragments within each gene for each sample. We will perform exploratory data analysis (EDA) for quality assessment and to explore the relationship between samples, perform differential gene expression analysis, and visually explore the results. PMID:26674615

  10. [The expression level of adhesion molecules on neutrophils depending at segmentation of their nuclei].

    PubMed

    Kashutin, S L; Danilov, S I; Vereshchagina, E N; Kluchareva, S V

    2013-11-01

    The article deals with results of detection of expression level of adhesion molecules on neutrophils and segmentation of their nuclei. It is established that in conditions of absence of antigen stimulation neutrophils of circulating pool express molecules of L-selectin in 53.34%, LFA-1 molecules in 65.64%, ICAM-1 in 40.51%, LE4-3 in 58.72% and PECAM-1 in 59.74%. The full readiness to realization of phase of sliding, strong adhesion and immediately transmigration itselfis detected in neutrophils with five segments in nucleus. PMID:24640111

  11. Reduced mRNA expression levels of MBD2 and MBD3 in gastric carcinogenesis.

    PubMed

    Pontes, Thaís Brilhante; Chen, Elizabeth Suchi; Gigek, Carolina Oliveira; Calcagno, Danielle Queiroz; Wisnieski, Fernanda; Leal, Mariana Ferreira; Demachki, Samia; Assumpção, Paulo Pimentel; Artigiani, Ricardo; Lourenço, Laércio Gomes; Burbano, Rommel Rodriguez; Arruda Cardoso Smith, Marília

    2014-04-01

    Aberrant methylation has been reported in several neoplasias, including gastric cancer. The methyl-CpG-binding domain (MBD) family proteins have been implicated in the chromatin remodeling process, leading to the modulation of gene expression. To evaluate the role of MBD2 and MBD3 in gastric carcinogenesis and the possible association with clinicopathological characteristics, we assessed the mRNA levels and promoter methylation patterns in gastric tissues. In this study, MBD2 and MBD3 mRNA levels were determined by RT-qPCR in 28 neoplastic and adjacent nonneoplastic and 27 gastritis and non-gastritis samples. The promoter methylation status was determined by bisulfite sequencing, and we found reduced MBD2 and MBD3 levels in the neoplastic samples compared with the other groups. Moreover, a strong correlation between the MBD2 and MBD3 expression levels was observed in each set of paired samples. Our data also showed that the neoplastic tissues exhibited higher MBD2 promoter methylation than the other groups. Interestingly, the non-gastritis group was the only one with positive methylation in the MBD3 promoter region. Furthermore, a weak correlation between gene expression and methylation was observed. Therefore, our data suggest that DNA methylation plays a minor role in the regulation of MBD2 and MBD3 expression, and the presence of methylation at CpGs that interact with transcription factor complexes might also be involved in the modulation of these genes. Moreover, reduced mRNA expression of MBD2 and MBD3 is implicated in gastric carcinogenesis, and thus, further investigations about these genes should be conducted for a better understanding of the role of abnormal methylation involved in this neoplasia. PMID:24338710

  12. Expression Levels of LCORL Are Associated with Body Size in Horses

    PubMed Central

    Metzger, Julia; Schrimpf, Rahel; Philipp, Ute; Distl, Ottmar

    2013-01-01

    Body size is an important characteristic for horses of various breeds and essential for the classification of ponies concerning the limit value of 148 cm (58.27 inches) height at the withers. Genome-wide association analyses revealed the highest associated quantitative trait locus for height at the withers on horse chromosome (ECA) 3 upstream of the candidate gene LCORL. Using 214 Hanoverian horses genotyped on the Illumina equine SNP50 BeadChip and 42 different horse breeds across all size ranges, we confirmed the highly associated single nucleotide polymorphism BIEC2-808543 (−log10P = 8.3) and the adjacent gene LCORL as the most promising candidate for body size. We investigated the relative expression levels of LCORL and its two neighbouring genes NCAPG and DCAF16 using quantitative real-time PCR (RT-qPCR). We could demonstrate a significant association of the relative LCORL expression levels with the size of the horses and the BIEC2-808543 genotypes within and across horse breeds. In heterozygous C/T-horses expression levels of LCORL were significantly decreased by 40% and in homozygous C/C-horses by 56% relative to the smaller T/T-horses. Bioinformatic analyses indicated that this SNP T>C mutation is disrupting a putative binding site of the transcription factor TFIID which is important for the transcription process of genes involved in skeletal bone development. Thus, our findings suggest that expression levels of LCORL play a key role for body size within and across horse breeds and regulation of the expression of LCORL is associated with genetic variants of BIEC2-808543. This is the first functional study for a body size regulating polymorphism in horses and a further step to unravel the mechanisms for understanding the genetic regulation of body size in horses. PMID:23418579

  13. Change in Prostaglandin Expression Levels and Synthesizing Activities in Dry Eye Disease

    PubMed Central

    Shim, Jongwoo; Park, Changhun; Lee, Hyun Soo; Park, Min Soo; Lim, Hyung Taek; Chauhan, Sunil; Dana, Reza; Lee, Hyon; Lee, Hyung Keun

    2013-01-01

    Objective To investigate the expression level of prostaglandins (PGs) and their de novo synthesis in dry eye (DE) disease. Design Cross-sectional case-control study and in vivo mouse experimental study. Participants Forty-six eyes from 23 DE patients and 33 eyes from 17 age- and sex-matched controls were studied. Also, DE-induced murine eyes were compared with control eyes. Methods Patients completed a symptom questionnaire using a 100-mm visual analog scale (VAS). Nanoliquid chromatography tandem mass spectrometry was used for the quantification of PGE2 and PGD2. A DE disease environmental chamber was used to induce DE in mice. One week after induction, enzyme expressions of cyclooxygenase-1, cyclooxygenase-2 (COX-2), PG E synthase (PGES), and PG D synthase (PGDS) in the lacrimal glands, meibomian glands, and corneas were examined using immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). Main Outcome Measures The mean PGE2 and PGD2 levels in the tears of DE patients were measured and compared with symptom severity scores. Immunohistochemistry staining patterns and qRT-PCR data of DE mice were quantified. Results The mean PGE2 level in the tears of DE patients (2.72±3.42 ng/ml) was significantly higher than that in the control group (0.88±0.83 ng/ml; P = 0.003). However, the mean PGD2 level in the tears of DE patients (0.11 ±0.22 ng/ml) was significantly lower (0.91 ±3.28 ng/ml; P = 0.028). The mean PGE2-to-PGD2 ratio correlated strongly with VAS scoring (P = 0.008). In DE mice, COX-2 mRNA was significantly higher in ocular surface tissue and lacrimal glands. Furthermore, PGES mRNA was significantly higher in ocular surface tissue, whereas PGDS mRNA was decreased. Immunohistochemistry staining showed elevated COX-2 expression in the lacrimal glands, meibomian glands, corneas, and conjunctivas. Furthermore, PGES expression was found in periductal infiltrated cells of the lacrimal glands and conjunctival epithelium. Also, PGDS

  14. Interplay between selenium levels, selenoprotein expression, and replicative senescence in WI-38 human fibroblasts.

    PubMed

    Yona, Legrain; Zahia, Touat-Hamici; Laurent, Chavatte

    2014-10-01

    Selenium is an essential trace element, which is incorporated as selenocysteine into at least 25 selenoproteins using a unique translational UGA-recoding mechanism. Selenoproteins are important enzymes involved in antioxidant defense, redox homeostasis, and redox signaling pathways. Selenium levels decline during aging, and its deficiency is associated with a marked increase in mortality for people over 60 years of age. Here, we investigate the relationship between selenium levels in the culture medium, selenoprotein expression, and replicative life span of human embryonic lung fibroblast WI-38 cells. Selenium levels regulate the entry into replicative senescence and modify the cellular markers characteristic for senescent cells. Whereas selenium supplementation extends the number of population doublings, its deficiency impairs the proliferative capacity of WI-38 cells. We observe that the expression of several selenoproteins involved in antioxidant defense is specifically affected in response to cellular senescence. Their expression is selectively controlled by the modulation of mRNA levels and translational recoding efficiencies. Our data provide novel mechanistic insights into how selenium impacts the replicative life span of mammalian cells by identifying several selenoproteins as new targets of senescence. PMID:26461317

  15. Nonlinear Dynamics in Gene Regulation Promote Robustness and Evolvability of Gene Expression Levels

    PubMed Central

    Steinacher, Arno; Bates, Declan G.; Akman, Ozgur E.; Soyer, Orkun S.

    2016-01-01

    Cellular phenotypes underpinned by regulatory networks need to respond to evolutionary pressures to allow adaptation, but at the same time be robust to perturbations. This creates a conflict in which mutations affecting regulatory networks must both generate variance but also be tolerated at the phenotype level. Here, we perform mathematical analyses and simulations of regulatory networks to better understand the potential trade-off between robustness and evolvability. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics, through the creation of regions presenting sudden changes in phenotype with small changes in genotype. For genotypes embedding low levels of nonlinearity, robustness and evolvability correlate negatively and almost perfectly. By contrast, genotypes embedding nonlinear dynamics allow expression levels to be robust to small perturbations, while generating high diversity (evolvability) under larger perturbations. Thus, nonlinearity breaks the robustness-evolvability trade-off in gene expression levels by allowing disparate responses to different mutations. Using analytical derivations of robustness and system sensitivity, we show that these findings extend to a large class of gene regulatory network architectures and also hold for experimentally observed parameter regimes. Further, the effect of nonlinearity on the robustness-evolvability trade-off is ensured as long as key parameters of the system display specific relations irrespective of their absolute values. We find that within this parameter regime genotypes display low and noisy expression levels. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics. Our results provide a possible solution to the robustness-evolvability trade-off, suggest an explanation for

  16. Nonlinear Dynamics in Gene Regulation Promote Robustness and Evolvability of Gene Expression Levels.

    PubMed

    Steinacher, Arno; Bates, Declan G; Akman, Ozgur E; Soyer, Orkun S

    2016-01-01

    Cellular phenotypes underpinned by regulatory networks need to respond to evolutionary pressures to allow adaptation, but at the same time be robust to perturbations. This creates a conflict in which mutations affecting regulatory networks must both generate variance but also be tolerated at the phenotype level. Here, we perform mathematical analyses and simulations of regulatory networks to better understand the potential trade-off between robustness and evolvability. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics, through the creation of regions presenting sudden changes in phenotype with small changes in genotype. For genotypes embedding low levels of nonlinearity, robustness and evolvability correlate negatively and almost perfectly. By contrast, genotypes embedding nonlinear dynamics allow expression levels to be robust to small perturbations, while generating high diversity (evolvability) under larger perturbations. Thus, nonlinearity breaks the robustness-evolvability trade-off in gene expression levels by allowing disparate responses to different mutations. Using analytical derivations of robustness and system sensitivity, we show that these findings extend to a large class of gene regulatory network architectures and also hold for experimentally observed parameter regimes. Further, the effect of nonlinearity on the robustness-evolvability trade-off is ensured as long as key parameters of the system display specific relations irrespective of their absolute values. We find that within this parameter regime genotypes display low and noisy expression levels. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics. Our results provide a possible solution to the robustness-evolvability trade-off, suggest an explanation for

  17. Expression of Different Levels of Ethanologenic Enzymes from Zymomonas mobilis in Recombinant Strains of Escherichia coli†

    PubMed Central

    Ingram, L. O.; Conway, T.

    1988-01-01

    The expression of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II in Escherichia coli converted this organism from the production of organic acids to the production of ethanol. Ethanol was produced during both anaerobic and aerobic growth. The extent to which these ethanologenic enzymes were expressed correlated with the extent of ethanol production. The replacement of organic acids with ethanol as a metabolic product during aerobic and anaerobic growth resulted in dramatic increases in final cell density, indicating that these acids (and the associated decline in pH) are more damaging than the production of ethanol. Of the plasmids examined, the best plasmid for growth and ethanol production expressed pyruvate decarboxylase and alcohol dehydrogenase II at levels of 6.5 and 2.5 IU/mg of total cell protein, respectively. PMID:16347553

  18. Differential effects on ARF stability by normal vs. oncogenic levels of c-Myc expression

    PubMed Central

    Chen, Delin; Kon, Ning; Zhong, Jiayun; Zhang, Pingzhao; Yu, Long; Gu, Wei

    2013-01-01

    SUMMARY ARF suppresses aberrant cell growth upon c-Myc overexpression through activating p53 responses. Nevertheless, the precise mechanism by which ARF specifically, restrains the oncogenic potential of c-Myc without affecting its normal physiological function is not well understood. Here, we show that low levels of c-Myc expression stimulate cell proliferation whereas high levels inhibit through activating the ARF-p53 response. Although the mRNA levels of ARF are induced under both scenarios, the accumulation of ARF protein occurs only when ULF-mediated degradation of ARF is inhibited by c-Myc overexpression. Moreover, the levels of ARF are reduced through ULF-mediated ubiquitination upon DNA damage. Blocking ARF degradation by c-Myc overexpression dramatically stimulates the apoptotic responses. Our study reveals that ARF stability control is crucial for differentiating normal (low) vs. oncogenic (high) levels of c-Myc expression and suggests that differential effects on ULF- mediated ARF ubiquitination by c-Myc levels act as a barrier in oncogene-induced stress responses. PMID:23747016

  19. Integrated Exon Level Expression Analysis of Driver Genes Explain Their Role in Colorectal Cancer

    PubMed Central

    Aziz, Mohammad Azhar; Periyasamy, Sathish; Al Yousef, Zeyad; AlAbdulkarim, Ibrahim; Al Otaibi, Majed; Alfahed, Abdulaziz; Alasiri, Glowi

    2014-01-01

    Integrated analysis of genomic and transcriptomic level changes holds promise for a better understanding of colorectal cancer (CRC) biology. There is a pertinent need to explain the functional effect of genome level changes by integrating the information at the transcript level. Using high resolution cytogenetics array, we had earlier identified driver genes by ‘Genomic Identification of Significant Targets In Cancer (GISTIC)’ analysis of paired tumour-normal samples from colorectal cancer patients. In this study, we analyze these driver genes at three levels using exon array data – gene, exon and network. Gene level analysis revealed a small subset to experience differential expression. These results were reinforced by carrying out separate differential expression analyses (SAM and LIMMA). ATP8B1 was found to be the novel gene associated with CRC that shows changes at cytogenetic, gene and exon levels. Splice index of 29 exons corresponding to 13 genes was found to be significantly altered in tumour samples. Driver genes were used to construct regulatory networks for tumour and normal groups. There were rearrangements in transcription factor genes suggesting the presence of regulatory switching. The regulatory pattern of AHR gene was found to have the most significant alteration. Our results integrate data with focus on driver genes resulting in highly enriched novel molecules that need further studies to establish their role in CRC. PMID:25335079

  20. High-level transient expression of ER-targeted human interleukin 6 in Nicotiana benthamiana.

    PubMed

    Nausch, Henrik; Mikschofsky, Heike; Koslowski, Roswitha; Meyer, Udo; Broer, Inge; Huckauf, Jana

    2012-01-01

    Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T(0) transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T(2) plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein. PMID:23152824

  1. Zinc fortification decreases ZIP1 gene expression of some adolescent females with appropriate plasma zinc levels.

    PubMed

    Méndez, Rosa O; Santiago, Alejandra; Yepiz-Plascencia, Gloria; Peregrino-Uriarte, Alma B; de la Barca, Ana M Calderón; García, Hugo S

    2014-06-01

    Zinc homeostasis is achieved after intake variation by changes in the expression levels of zinc transporters. The aim of this study was to evaluate dietary intake (by 24-h recall), absorption, plasma zinc (by absorption spectrophotometry) and the expression levels (by quantitative PCR), of the transporters ZIP1 (zinc importer) and ZnT1 (zinc exporter) in peripheral white blood cells from 24 adolescent girls before and after drinking zinc-fortified milk for 27 day. Zinc intake increased (p < 0.001) from 10.5 ± 3.9 mg/day to 17.6 ± 4.4 mg/day, and its estimated absorption from 3.1 ± 1.2 to 5.3 ± 1.3 mg/day. Mean plasma zinc concentration remained unchanged (p > 0.05) near 150 µg/dL, but increased by 31 µg/dL (p < 0.05) for 6/24 adolescents (group A) and decreased by 25 µg/dL (p < 0.05) for other 6/24 adolescents (group B). Expression of ZIP1 in blood leukocytes was reduced 1.4-fold (p < 0.006) in group A, while for the expression of ZnT1 there was no difference after intervention (p = 0.39). An increase of dietary zinc after 27-days consumption of fortified-milk did not increase (p > 0.05) the plasma level of adolescent girls but for 6/24 participants from group A in spite of the formerly appropriation, which cellular zinc uptake decreased as assessed by reduction of the expression of ZIP1. PMID:24922175

  2. Zinc Fortification Decreases ZIP1 Gene Expression of Some Adolescent Females with Appropriate Plasma Zinc Levels

    PubMed Central

    Méndez, Rosa O.; Santiago, Alejandra; Yepiz-Plascencia, Gloria; Peregrino-Uriarte, Alma B.; de la Barca, Ana M. Calderón; García, Hugo S.

    2014-01-01

    Zinc homeostasis is achieved after intake variation by changes in the expression levels of zinc transporters. The aim of this study was to evaluate dietary intake (by 24-h recall), absorption, plasma zinc (by absorption spectrophotometry) and the expression levels (by quantitative PCR), of the transporters ZIP1 (zinc importer) and ZnT1 (zinc exporter) in peripheral white blood cells from 24 adolescent girls before and after drinking zinc-fortified milk for 27 day. Zinc intake increased (p < 0.001) from 10.5 ± 3.9 mg/day to 17.6 ± 4.4 mg/day, and its estimated absorption from 3.1 ± 1.2 to 5.3 ± 1.3 mg/day. Mean plasma zinc concentration remained unchanged (p > 0.05) near 150 µg/dL, but increased by 31 µg/dL (p < 0.05) for 6/24 adolescents (group A) and decreased by 25 µg/dL (p < 0.05) for other 6/24 adolescents (group B). Expression of ZIP1 in blood leukocytes was reduced 1.4-fold (p < 0.006) in group A, while for the expression of ZnT1 there was no difference after intervention (p = 0.39). An increase of dietary zinc after 27-days consumption of fortified-milk did not increase (p > 0.05) the plasma level of adolescent girls but for 6/24 participants from group A in spite of the formerly appropriation, which cellular zinc uptake decreased as assessed by reduction of the expression of ZIP1. PMID:24922175

  3. Effect of heat stress and feeding phosphorus levels on pig electron transport chain gene expression.

    PubMed

    Weller, M M D C A; Alebrante, L; Campos, P H R F; Saraiva, A; Silva, B A N; Donzele, J L; Oliveira, R F M; Silva, F F; Gasparino, E; Lopes, P S; Guimarães, S E F

    2013-12-01

    The purpose of this study was to evaluate the effect of temperature and different levels of available phosphorus (aP) on the expression of nine genes encoding electron transport chain proteins in the Longissimus dorsi (LD) muscle of pigs. Two trials were carried out using 48 high-lean growth pigs from two different growth phases: from 15 to 30 kg (phase 1) and from 30 to 60 kg (phase 2). Pigs from growth phase 1 were fed with three different levels of dietary aP (0.107%, 0.321% or 0.535%) and submitted either to a thermoneutral (24°C and RH at 76%) or to a heat stress (34°C and RH at 70%) environment. Pigs from growth phase 2 were fed with three different levels of dietary aP (0.116%, 0.306% or 0.496%) and submitted either to a thermoneutral (22ºC and RH at 77%) or to a heat stress (32ºC and RH at 73%) environment. Heat stress decreased (P<0.001) average daily feed intake at both growth phases. At 24°C, pigs in phase 1 fed the 0.321% aP diet had greater average daily gain and feed conversion (P<0.05) than those fed the 0.107% or 0.535% while, at 34°C pigs fed the 0.535% aP had the best performance (P<0.05). Pigs from phase 2 fed the 0.306% aP had best performance in both thermal environments. Gene expression profile was analyzed by quantitative real-time polymerase chain reaction. Irrespective of growing phase, the expression of six genes was lower (P<0.05) at high temperature than at thermoneutrality. The lower expression of these genes under high temperatures evidences the effects of heat stress by decreasing oxidative metabolism, through adaptive physiological mechanisms in order to reduce heat production. In pigs from phase 1, six genes were differentially expressed across aP levels (P<0.05) in the thermoneutral and one gene in the heat stress. In pigs from phase 2, two genes were differentially expressed across aP levels (P<0.05) in both thermal environments. These data revealed strong evidence that phosphorus and thermal environments are key factors to

  4. Maternal High Fat Diet Affects Offspring’s Vitamin K-Dependent Proteins Expression Levels

    PubMed Central

    Lanham, Stuart; Cagampang, Felino R.; Oreffo, Richard O. C.

    2015-01-01

    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. PMID:26381752

  5. Th17 expression and IL-17 levels in laryngeal squamous cell carcinoma patients.

    PubMed

    Li, Fu-Jun; Cai, Zhi-Jian; Yang, Fei; Zhang, Shou-De; Chen, Ming

    2016-05-01

    Conclusion The Th17 cell frequency in peripheral blood and levels of IL-17 showed significant differences between patients with laryngeal squamous cell carcinoma and those with vocal cords polyps. Serum levels of IL-17 were correlated with laryngocarcinoma staging. Objectives To investigate associations among the frequency of Th17 cells, levels of IL-17, and laryngeal squamous cell carcinoma. Method Eighty in-patients with laryngeal squamous cell carcinoma and 114 in-patients with polypus of the vocal cord were enrolled. Th17 cell frequencies in peripheral blood and serum levels of IL-17 were measured by flow cytometry and enzyme-linked immunosorbent assay, respectively. The tissue expression levels of IL-17 mRNA transcripts and protein were measured using quantitative RT-PCR or immunohistochemical detection, respectively. Results Th17 cell frequencies in peripheral blood and serum concentrations of IL-17 were significantly higher in patients with laryngocarcinoma compared with those in patients with polyps (p < 0.01 for both Th17 cells and IL-17 levels). Serum concentrations of IL-17 were significantly higher in patients with advanced laryngocarcinoma than in patients with early laryngocarcinoma (p < 0.01). The mRNA and protein levels of IL-17 were significantly higher in laryngocarcinoma tissues than in adjacent normal tissues (p < 0.01 for mRNA levels, p < 0.05 for protein levels). PMID:27052965

  6. Regional and cellular expression of CYP2D6 in human brain: higher levels in alcoholics.

    PubMed

    Miksys, Sharon; Rao, Yushu; Hoffmann, Ewa; Mash, Deborah C; Tyndale, Rachel F

    2002-09-01

    Cytochrome P450 (CYP) 2D6 is expressed in liver, brain and other extrahepatic tissues where it metabolizes a range of centrally acting drugs and toxins. As ethanol can induce CYP2D in rat brain, we hypothesized that CYP2D6 expression is higher in brains of human alcoholics. We examined regional and cellular expression of CYP2D6 mRNA and protein by RT-PCR, Southern blotting, slot blotting, immunoblotting and immunocytochemistry. A significant correlation was found between mean mRNA and CYP2D6 protein levels across 13 brain regions. Higher expression was detected in 13 brain regions of alcoholics (n = 8) compared to nonalcoholics (n = 5) (anovap < 0.0001). In hippocampus this was localized in CA1-3 pyramidal cells and dentate gyrus granular neurons. In cerebellum this was localized in Purkinje cells and their dendrites. Both of these brain regions, and these same cell-types, are known to be susceptible to alcohol damage. For one case, a poor metabolizer (CYP2D6*4/*4), there was no detectable CYP2D6 protein, confirming the specificity of the antibody used. These data suggest that in alcoholics elevated brain CYP2D6 expression may contribute to altered sensitivity to centrally acting drugs and to the mediation of neurotoxic and behavioral effects of alcohol. PMID:12354285

  7. High expression levels of MAGE-A9 are correlated with unfavorable survival in lung adenocarcinoma

    PubMed Central

    Zhai, Xiaolu; Xu, Liqin; Zhang, Siya; Zhu, Huijun; Mao, Guoxin; Huang, Jianfei

    2016-01-01

    A variety of melanoma-associated antigen-A (MAGE-A) protein are commonly detected in lung cancers. Their biological function is not well characterized but may involve cell cycle progression and the regulation of apoptosis. We hypothesized that MAGE-A9 is involved in the regulation of apoptosis. To test this hypothesis, we evaluated MAGE-A9 protein expression by immunohistochemical staining and we assessed the relationship between the expression of MAGE-A9 and clinical pathological parameters. In addition, we investigated the effect of MAGE-A9 down-regulation in lung adenocarcinoma. The results showed that a high expression level of MAGE-A9 protein in lung adenocarcinoma tumor cells was related to larger tumor diameter (P = 0.013) and poor differentiation (P = 0.029). Cox regression analysis revealed that the expression of MAGE-A9 in lung adenocarcinoma tumor cells (P < 0.001) is an independent prognostic factor in five-year survival rates. NSCLC cells with silenced MAGE-A9 had decreased cell proliferation, migration and invasion in cell culture compared to corresponding control cells. The NSCLC cells showing down-regulated MAGE-A9 induced the expression of apoptosis-associated proteins. In addition, MAGE-A9 was associated with resistance to conventional chemotherapeutic agents. Our findings provide evidence that MAGE-A9 could be a potential therapeutic target in NSCLC. PMID:26717042

  8. High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

    PubMed Central

    Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

    2016-01-01

    Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

  9. The progestin levonorgestrel disrupts gonadotropin expression and sex steroid levels in pubertal roach (Rutilus rutilus).

    PubMed

    Kroupova, H K; Trubiroha, A; Lorenz, C; Contardo-Jara, V; Lutz, I; Grabic, R; Kocour, M; Kloas, W

    2014-09-01

    The aim of the present study was to investigate the effects of the synthetic progestin levonorgestrel (LNG) on the reproductive endocrine system of a teleost fish, the roach (Rutilus rutilus). Pubertal roach were exposed for 28 days in a flow-through system to four concentrations of LNG (3, 31, 312, and 3124 ng/l). Both males and females treated with 3124 ng/l LNG exhibited the upregulated levels of vitellogenin and oestrogen receptor 1 mRNA in the liver. At the same concentration, LNG caused a significant upregulation of the mRNA expression of the gene encoding luteinising hormone β-subunit (lhβ) and the suppression of the mRNA expression of the gene encoding follicle-stimulating hormone β-subunit (fshβ) in the pituitary of both male and female roach. A lower LNG concentration (312 ng/l) suppressed mRNA expression of fshβ in males only. Females treated with 3124 ng/l LNG exhibited significantly lower plasma 11-ketotestosterone (11-KT) and oestradiol (E2) concentrations, whereas their testosterone (T) level was higher compared with the control. Females exposed to 312 ng/l LNG presented significantly lower plasma E2 concentrations. Males exposed to ≥31 ng/l LNG exhibited significantly reduced 11-KT levels. As determined through a histological analysis, the ovaries of females were not affected by LNG exposure, whereas the testes of males exposed to 31 and 312 ng/l LNG exhibited a significantly higher percentage of spermatogonia B compared with the control. The results of the present study demonstrate that LNG disrupts the reproductive system of pubertal roach by affecting the pituitary gonadotropin expression and the sex steroid levels. This disruption was determined to occur in males after exposure to an environmentally relevant concentration (31 ng/l). Moreover, the highest tested concentration of LNG (3124 ng/l) exerted an oestrogenic effect on fish of both sexes. PMID:24893273

  10. Role of Increased Guanosine Triphosphate Cyclohydrolase-1 Expression and Tetrahydrobiopterin Levels upon T Cell Activation

    PubMed Central

    Chen, Wei; Li, Li; Brod, Torben; Saeed, Omar; Thabet, Salim; Jansen, Thomas; Dikalov, Sergey; Weyand, Cornelia; Goronzy, Jorg; Harrison, David G.

    2011-01-01

    Tetrahydrobiopterin (BH4) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O2˙̄) rather than NO. The rate-limiting enzyme for BH4 production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH4 levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH4. Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH4 levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH4. Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH4 accumulation, reduced NO production, and increased T cell O2˙̄ production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH2 polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH4 in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH4 levels, NO production, and modulating T cell cytokine production. PMID:21343293

  11. Single chromosome transcriptional profiling reveals chromosome-level regulation of gene expression

    PubMed Central

    Levesque, Marshall J.; Raj, Arjun

    2013-01-01

    Here we report iceFISH, a multiplex imaging method for measuring gene expression and chromosome structure simultaneously on single chromosomes. We demonstrate that chromosomal translocations can alter transcription chromosome-wide, finding substantial differences in transcriptional frequency between genes located on a translocated chromosome in comparison to the normal chromosome in the same cell. Examination of correlations between genes on a single chromosome revealed a cis chromosome-level transcriptional interaction spanning 14.3 megabases. PMID:23416756

  12. Opposite Effects on Spodoptera littoralis Larvae of High Expression Level of a Trypsin Proteinase Inhibitor in Transgenic Plants1

    PubMed Central

    De Leo, Francesca; Bonadé-Bottino, Michel A.; Ceci, Luigi R.; Gallerani, Raffaele; Jouanin, Lise

    1998-01-01

    This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI. PMID:9808744

  13. Transgene Expression Levels Determine the Immunogenicity of Transduced Hematopoietic Grafts in Partially Myeloablated Mice

    PubMed Central

    Eixarch, Herena; Gómez, Alba; Kádár, Elisabeth; George, Mónica; Martínez, Nuria; Espejo, Carmen; Pétriz, Jordi; Gimeno, Ramon; Barquinero, Jordi

    2009-01-01

    We investigated whether transgene expression levels influence the immunogenicity of transduced hematopoietic grafts upon transplantation into partially myeloablated mice. To this aim, bone marrow cells (BMCs) transduced with retroviral vectors driving green fluorescent protein (GFP) expression either at high (high-EGFP) or low levels (low-EGFP) were transplanted into congenic recipients conditioned with sublethal doses of total body irradiation (TBI) or busulfan. Virtually all recipients showed evidence of donor engraftment 4 weeks after transplantation. However, as opposed to recipients receiving low-EGFP transduced grafts, the risk of rejecting the EGFP+ cells by 30 days after transplantation was significantly higher in mice conditioned with busulfan and receiving high-EGFP transduced grafts. Anti-EGFP cellular immune responses were demonstrated in high-EGFP-treated mice conditioned with busulfan by interferon-γ (IFN-γ), enzyme-linked immunospot assay (ELISPOT), and cytotoxic T lymphocyte (CTL) assays, in contrast to that observed in mice transplanted with low-EGFP BMC. These results show for the first time that transgene expression levels can be critical for the immunogenicity of gene-modified hematopoietic grafts, especially in immunocompetent or in partially immunosuppressed recipients. These results have profound implications in vector choice and in the design of gene therapy (GT) protocols. PMID:19707185

  14. Endogenous oxytocin levels are associated with the perception of emotion in dynamic body expressions in schizophrenia.

    PubMed

    Strauss, Gregory P; Keller, William R; Koenig, James I; Sullivan, Sara K; Gold, James M; Buchanan, Robert W

    2015-03-01

    Lower endogenous oxytocin levels have been associated with impaired social cognition in schizophrenia, particularly facial affect identification. Little is known about the relationship between oxytocin and other forms of emotion perception. In the current study, 41 individuals with schizophrenia (SZ) and 22 demographically matched healthy controls (CN) completed a forced-choice affective body expression classification task. Stimuli included dynamic videos of male and female actors portraying 4 discrete emotions: happiness, sadness, anger, and neutral. Plasma oxytocin levels were determined via radioimmunoassay. Results indicated that SZ had significantly higher plasma oxytocin concentrations than CN. SZ were also less accurate at identifying expressions of happiness and sadness; however, there were no group differences for anger or neutral stimuli. A group×sex interaction was also present, such that female CN were more accurate than male CN, whereas male SZ were more accurate than female SZ. Higher endogenous oxytocin levels were associated with better total recognition in both SZ and CN; this association was specific to females in SZ. Findings indicate that sex plays an important role in identifying emotional expressions in body gestures in SZ, and that individual differences in endogenous oxytocin predict emotion perception accuracy. PMID:25620121

  15. Endogenous Oxytocin Levels Are Associated with the Perception of Emotion in Dynamic Body Expressions in Schizophrenia

    PubMed Central

    Strauss, Gregory P.; Keller, William R.; Koenig, James I.; Sullivan, Sara K.; Gold, James M.; Buchanan, Robert W.

    2015-01-01

    Lower endogenous oxytocin levels have been associated with impaired social cognition in schizophrenia, particularly facial affect identification. Little is known about the relationship between oxytocin and other forms of emotion perception. In the current study, 41 individuals with schizophrenia (SZ) and 22 demographically matched healthy controls (CN) completed a forced-choice affective body expression classification task. Stimuli included dynamic videos of male and female actors portraying 4 discrete emotions: happiness, sadness, anger, and neutral. Plasma oxytocin levels were determined via radioimmunoassay. Results indicated that SZ had significantly higher plasma oxytocin concentrations than CN. SZ were also less accurate at identifying expressions of happiness and sadness; however, there were no group differences for anger or neutral stimuli. A group x sex interaction was also present, such that female CN were more accurate than male CN, whereas male SZ were more accurate than female SZ. Higher endogenous oxytocin levels were associated with better total recognition in both SZ and CN; this association was specific to females in SZ. Findings indicate that sex plays an important role in identifying emotional expressions in body gestures in SZ, and that individual differences in endogenous oxytocin predict emotion perception accuracy. PMID:25620121

  16. Earth Mover's Distance (EMD): A True Metric for Comparing Biomarker Expression Levels in Cell Populations.

    PubMed

    Orlova, Darya Y; Zimmerman, Noah; Meehan, Stephen; Meehan, Connor; Waters, Jeffrey; Ghosn, Eliver E B; Filatenkov, Alexander; Kolyagin, Gleb A; Gernez, Yael; Tsuda, Shanel; Moore, Wayne; Moss, Richard B; Herzenberg, Leonore A; Walther, Guenther

    2016-01-01

    Changes in the frequencies of cell subsets that (co)express characteristic biomarkers, or levels of the biomarkers on the subsets, are widely used as indices of drug response, disease prognosis, stem cell reconstitution, etc. However, although the currently available computational "gating" tools accurately reveal subset frequencies and marker expression levels, they fail to enable statistically reliable judgements as to whether these frequencies and expression levels differ significantly between/among subject groups. Here we introduce flow cytometry data analysis pipeline which includes the Earth Mover's Distance (EMD) metric as solution to this problem. Well known as an informative quantitative measure of differences between distributions, we present three exemplary studies showing that EMD 1) reveals clinically-relevant shifts in two markers on blood basophils responding to an offending allergen; 2) shows that ablative tumor radiation induces significant changes in the murine colon cancer tumor microenvironment; and, 3) ranks immunological differences in mouse peritoneal cavity cells harvested from three genetically distinct mouse strains. PMID:27008164

  17. Codon optimization for high level expression of human bone morphogenetic protein-2 in Escherichia coli.

    PubMed

    Retnoningrum, Debbie S; Pramesti, H T; Santika, P Y; Valerius, O; Asjarie, S; Suciati, T

    2012-08-01

    Codons in the open reading frame (ORF) encoding for human bone morphogenetic protein-2 (hBMP-2) were optimized to reach high level expression in Escherichia coli. The optimization was done by the computer programs DNA works and DNA Star according to Thermodynamically Balanced Inside Out (TBIO) approach. The ORF consisting of 342 base pairs (bp) was assembled using two-steps Polymerase Chain Reaction, cloned into a pGEM-T vector with a mutation rate of 6.38 bp per kb and transformed into E. coli JM109. After a DNA sequence confirmation, mutation-free ORF was subcloned into pET32b and transformed into E. coli BL21(DE3). The rhBMP-2 was produced as a thioredoxin-his-tag fusion protein at relatively high level, approximately 60% of total intracellular proteins as inclusion bodies (IB), with a yield of 1.39 g per liter culture. Solubilization of IB gave soluble monomer rhBMP-2 with a recovery of 13.6% and refolding of soluble rhBMP-2 produced dimeric forms with a yield of 8.7%. The size and identity of the purified rhBMP-2 was confirmed by nano-LC-MS/MS2 analysis. Our work demonstrates for the first time that by using TBIO approach, a codon-optimized ORF encoding for rhBMP-2 protein can be expressed at high level in E. coli expression system. PMID:22691543

  18. Relationship between expression levels and atherogenesis in scavenger receptor Class B, Type I Transgenics

    SciTech Connect

    Ueda, Yukihiko; Gong, Elaine; Royer, Lori; Cooper, Philip N.; Francone, Omar L; Rubin, Edward M.

    2000-03-15

    Both in vitro and in vivo studies of SR-BI have implicated it as a likely participant in the metabolism of HDL cholesterol. To investigate SR-BI's effect on atherogenesis we examined two lines of SR-BI transgenic mice with high (10-fold increases) and low (2-fold increases) in SR-BI expression in an inbred mouse background hemizygous for a human apo B transgene. Unlike non-HDL cholesterol levels which minimally differed in the various groups of animals, HDL cholesterol levels were inversely related to SR-BI expression. Mice with the low expression SR-BI transgene had a 50% reduction in HDL cholesterol while the high expression SR-BI transgene was associated with two-fold decreases in HDL as well as dramatic alterations in HDL composition and size including the near absence of a-migrating particles as determined by 2-dimensional electrophoresis. The low expression SR-BI/apo B transgenics had more than a two-fold decrease in the development of diet induced fatty streak lesions compared t o the apo B transgenics (4448{+-}1908 {mu}m2/aorta to 10133 {+-} 4035 {mu}m2/aorta; p<0.001), while the high expression SR-BI/apo B transgenics had an atherogenic response similar to that of the apo B transgenics (14692{+-}7238 {mu}m2/aorta) but three-fold greater than the low SR-BI/apo B mice (p<0.001). The prominent anti-atherogenic effect of moderate SR-BI expression provides in vivo support for the hypothesis that HDL functions to inhibit atherogenesis through its interactions with SR-BI in facilitating reverse cholesterol transport. The failure of the high SR-BI/apo B transgenics to have similar or even greater reductions in atherogenesis suggests that the changes resulting from extremely high SR-BI expression including dramatic changes in lipoproteins may have both pro- and anti-atherogenic consequences illustrating the complexity of the relationship between SR-BI and atherogenesis.

  19. Relationships between cell cycle regulator gene copy numbers and protein expression levels in Schizosaccharomyces pombe.

    PubMed

    Chino, Ayako; Makanae, Koji; Moriya, Hisao

    2013-01-01

    We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe using the "genetic tug-of-war" (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted. PMID:24019917

  20. Altered Hypoxia-inducible factor-1 alpha expression levels correlate with coronary vessel anomalies

    PubMed Central

    Wikenheiser, Jamie; Wolfram, Julie A.; Gargesha, Madhusudhana; Yang, Ke; Karunamuni, Ganga; Wilson, David L.; Semenza, Gregg L.; Agani, Faton; Fisher, Steven A.; Ward, Nicole; Watanabe, Michiko

    2009-01-01

    The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5 positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor-1 alpha (HIF-1α), the oxygen-sensitive component of the hypoxia inducible factor-1 (HIF-1) heterodimer. This led to our hypothesis that there is a “template” of hypoxic tissue that determines the stereotyped pattern of the major coronary vessels. In this study we disturbed this template by altering ambient oxygen levels (hypoxia 15%; hyperoxia 75-40%) during the early phases of avian coronary vessel development, in order to alter tissue hypoxia, HIF-1α protein expression and its downstream target genes without high mortality. We also altered HIF-1α gene expression in the embryonic outflow tract cardiomyocytes by injecting an adenovirus containing a constitutively active form of HIF-1α (AdCA5). We assayed for coronary anomalies using anti-alpha-smooth muscle actin immunohistology. When incubated under abnormal oxygen levels or injected with a low titer of the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicated that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF-1 regulation of gene expression. PMID:19777592

  1. Expression Level of Genes Coding for Cell Adhesion Molecules of Cadherin Group in Colorectal Cancer Patients

    PubMed Central

    Lorenc, Zbigniew; Opiłka, Mieszko Norbert; Kruszniewska-Rajs, Celina; Rajs, Antoni; Waniczek, Dariusz; Starzewska, Małgorzata; Lorenc, Justyna; Mazurek, Urszula

    2015-01-01

    Background Colorectal Cancer (CRC) is one of the most frequently diagnosed neoplasms and also one of the main death causes. Cell adhesion molecules are taking part in specific junctions, contributing to tissue integrality. Lower expression of the cadherins may be correlated with poorer differentiation of the CRC, and its more aggressive phenotype. The aim of the study is to designate the cadherin genes potentially useful for the diagnostics, prognostics, and the treatment of CRC. Material/Method Specimens were collected from 28 persons (14 female and 14 male), who were operated for CRC. The molecular analysis was performed using oligonucleotide microarrays, mRNA used was collected from adenocarcinoma, and macroscopically healthy tissue. The results were validated using qRT-PCR technique. Results Agglomerative hierarchical clustering of normalized mRNA levels has shown 4 groups with statistically different gene expression. The control group was divided into 2 groups, the one was appropriate control (C1), the second (C2) had the genetic properties of the CRC, without pathological changes histologically and macroscopically. The other 2 groups were: LSC (Low stage cancer) and HSC (High stage cancer). Consolidated results of the fluorescency of all of the differential genes, designated two coding E-cadherin (CDH1) with the lower expression, and P-cadherin (CDH3) with higher expression in CRC tissue. Conclusions The levels of genes expression are different for several groups of cadherins, and are related with the stage of CRC, therefore could be potentially the useful marker of the stage of the disease, also applicable in treatment and diagnostics of CRC. PMID:26167814

  2. Effects of nutritional level of concentrate-based diets on meat quality and expression levels of genes related to meat quality in Hainan black goats.

    PubMed

    Wang, Dingfa; Zhou, Luli; Zhou, Hanlin; Hou, Guanyu; Shi, Liguang; Li, Mao; Huang, Xianzhou; Guan, Song

    2015-02-01

    The present study investigated the effects of the nutritional levels of diets on meat quality and related gene expression in Hainan black goat. Twenty-four goats were divided into six dietary treatments and were fed a concentrate-based diet with two levels of crude protein (CP) (15% or 17%) and three levels of digestive energy (DE) (11.72, 12.55 or 13.39 MJ/kg DM) for 90 days. Goats fed the concentrate-based diet with 17% CP had significantly (P < 0.05) higher average daily gains (ADG) and better feed conversion rates (FCR). The pH 24h value tended to decrease (P < 0.05) with increasing DE levels. The tenderness of Longissimus dorsi muscle (LD) and Semimembranosus muscle (SM) reduced with increasing CP levels (P < 0.05). With increasing DE levels, tenderness was increased (P < 0.05). The heart fatty acid-binding protein (H-FABP) mRNA expression levels in LD and SM increased with increasing DE levels (P < 0.05), but decreased with increasing CP levels (P < 0.05). The calpastatin (CAST) and μ-calpain mRNA expressions levels in LD and SM were affected significantly (P < 0.05) by CP and DE levels in the diet. Therefore, the nutritional levels of diets affect meat quality and expression levels of genes associated with meat quality in Hainan black goats. PMID:25039653

  3. SOCS1/3 Expression Levels in HSV-1-Infected, Cytokine-Polarized and -Unpolarized Macrophages

    PubMed Central

    Reichard, Adam Craig; Cheemarla, Nagarjuna Reddy

    2015-01-01

    Macrophage subtypes are characterized as proinflammatory (M1) or immunomodulatory and tissue remodeling (M2). Since macrophages play a pivotal role in controlling Herpes simplex virus type-1 (HSV-1) replication, effects of HSV-1 by 24 h of infection were determined in murine J774A.1 macrophages unpolarized (M0) or polarized to either an M1 or M2 phenotype. Morphology, cell viability, and expression of CD14 (co-receptor for lipopolysaccharide), CD86 (B7.2-immune co-stimulatory molecule), and suppressors of cytokine signaling (SOCS1 and SOCS3) were determined. M1 macrophages were flattened and vacuolated, while M2 cells appeared elongated with a few vacuoles. Compared with unpolarized M0 cells, M1 cells showed a 31% decrease in viability, a 2-fold increase in the number of CD14+-CD86+ cells, no change in SOCS1 expression, and an 11-fold decrease in SOCS3 expression. M2 cells exhibited a 9% decrease in viability, a 26.0% decrease in the number of CD14+-CD86+ cells, and no change in SOCS1/SOCS3 expression levels compared with M0 cells. After HSV-1 infection, all phenotypes appeared rounded, cell viabilities decreased as did numbers of M1 cells expressing CD14 and CD86. At 24 h after infection, M0 control and M2 cells showed greater virus yield than did the M1 cells, presumably reflecting the loss of viable M1 cells. SOCS1 expression was predominant in uninfected M1-polarized cells and in virus-infected control (M0) cells. SOCS1/SOCS3 expression ratio was 7:1 in uninfected M1 macrophages and approached 1:1 in M1 cells at 24 h after infection with HSV-1. In contrast, little differences were seen in SOCS1/SOCS3 expression ratios in uninfected M2-polarized cells or virus-infected M2 cells. These observations suggest that SOCS1/SOCS3 expression ratios can be used to characterize HSV-1-infected and uninfected macrophages. PMID:24956148

  4. Levels of Expression and Immunogenicity of Attenuated Salmonella enterica Serovar Typhimurium Strains Expressing Escherichia coli Mutant Heat-Labile Enterotoxin

    PubMed Central

    Covone, M. Giuseppina; Brocchi, Marcelo; Palla, Emanuela; da Silveira, W. Dias; Rappuoli, Rino; Galeotti, Cesira L.

    1998-01-01

    The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat-labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed. Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in S. typhimurium Δcya Δcrp Δasd strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated Δcya Δcrp UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in S. typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence. PMID:9423862

  5. Rabies virus (RV) glycoprotein expression levels are not critical for pathogenicity of RV.

    PubMed

    Wirblich, Christoph; Schnell, Matthias J

    2011-01-01

    Previous comparisons of different rabies virus (RV) strains suggested an inverse relationship between pathogenicity and the amount of glycoprotein produced in infected cells. In order to provide more insight into this relationship, we pursued an experimental approach that allowed us to alter the glycoprotein expression level without altering the glycoprotein sequence, thereby eliminating the contribution of amino acid changes to differences in viral virulence. To this end, we constructed an infectious clone of the highly pathogenic rabies virus strain CVS-N2c and replaced its cognate glycoprotein gene with synthetic versions in which silent mutations were introduced to replace wild-type codons with the most or least frequently used synonymous codons. A recombinant N2c variant containing the fully codon-optimized G gene and three variants carrying a partially codon-deoptimized G gene were recovered on mouse neuroblastoma cells and shown to express 2- to 3-fold more and less glycoprotein, respectively, than wild-type N2c. Pathogenicity studies in mice revealed the WT-N2c virus to be the most pathogenic strain. Variants containing partially codon-deoptimized glycoprotein genes or the codon-optimized gene were less pathogenic than WT-N2c but still caused significant mortality. We conclude that the expression level of the glycoprotein gene does have an impact on pathogenicity but is not a dominant factor that determines pathogenicity. Thus, strategies such as changes in codon usage that aim solely at altering the expression level of the glycoprotein gene do not suffice to render a pathogenic rabies virus apathogenic and are not a viable and safe approach for attenuation of a pathogenic strain. PMID:21068252

  6. Rabies Virus (RV) Glycoprotein Expression Levels Are Not Critical for Pathogenicity of RV▿

    PubMed Central

    Wirblich, Christoph; Schnell, Matthias J.

    2011-01-01

    Previous comparisons of different rabies virus (RV) strains suggested an inverse relationship between pathogenicity and the amount of glycoprotein produced in infected cells. In order to provide more insight into this relationship, we pursued an experimental approach that allowed us to alter the glycoprotein expression level without altering the glycoprotein sequence, thereby eliminating the contribution of amino acid changes to differences in viral virulence. To this end, we constructed an infectious clone of the highly pathogenic rabies virus strain CVS-N2c and replaced its cognate glycoprotein gene with synthetic versions in which silent mutations were introduced to replace wild-type codons with the most or least frequently used synonymous codons. A recombinant N2c variant containing the fully codon-optimized G gene and three variants carrying a partially codon-deoptimized G gene were recovered on mouse neuroblastoma cells and shown to express 2- to 3-fold more and less glycoprotein, respectively, than wild-type N2c. Pathogenicity studies in mice revealed the WT-N2c virus to be the most pathogenic strain. Variants containing partially codon-deoptimized glycoprotein genes or the codon-optimized gene were less pathogenic than WT-N2c but still caused significant mortality. We conclude that the expression level of the glycoprotein gene does have an impact on pathogenicity but is not a dominant factor that determines pathogenicity. Thus, strategies such as changes in codon usage that aim solely at altering the expression level of the glycoprotein gene do not suffice to render a pathogenic rabies virus apathogenic and are not a viable and safe approach for attenuation of a pathogenic strain. PMID:21068252

  7. Sphingomyelinase-Like Phosphodiesterase 3b Expression Levels Determine Podocyte Injury Phenotypes in Glomerular Disease

    PubMed Central

    Yoo, Tae-Hyun; Pedigo, Christopher E.; Guzman, Johanna; Correa-Medina, Mayrin; Wei, Changli; Villarreal, Rodrigo; Mitrofanova, Alla; Leclercq, Farah; Faul, Christian; Li, Jing; Kretzler, Matthias; Nelson, Robert G.; Lehto, Markku; Forsblom, Carol; Groop, Per-Henrik; Reiser, Jochen; Burke, George William

    2015-01-01

    Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVβ3 integrin-dependent migration in vitro. Furthermore, αVβ3 integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVβ3 integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVβ3 integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target. PMID:24925721

  8. Sphingomyelinase-like phosphodiesterase 3b expression levels determine podocyte injury phenotypes in glomerular disease.

    PubMed

    Yoo, Tae-Hyun; Pedigo, Christopher E; Guzman, Johanna; Correa-Medina, Mayrin; Wei, Changli; Villarreal, Rodrigo; Mitrofanova, Alla; Leclercq, Farah; Faul, Christian; Li, Jing; Kretzler, Matthias; Nelson, Robert G; Lehto, Markku; Forsblom, Carol; Groop, Per-Henrik; Reiser, Jochen; Burke, George William; Fornoni, Alessia; Merscher, Sandra

    2015-01-01

    Diabetic kidney disease (DKD) is the most common cause of ESRD in the United States. Podocyte injury is an important feature of DKD that is likely to be caused by circulating factors other than glucose. Soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor found to be elevated in the serum of patients with FSGS and causes podocyte αVβ3 integrin-dependent migration in vitro. Furthermore, αVβ3 integrin activation occurs in association with decreased podocyte-specific expression of acid sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) in kidney biopsy specimens from patients with FSGS. However, whether suPAR-dependent αVβ3 integrin activation occurs in diseases other than FSGS and whether there is a direct link between circulating suPAR levels and SMPDL3b expression in podocytes remain to be established. Our data indicate that serum suPAR levels are also elevated in patients with DKD. However, unlike in FSGS, SMPDL3b expression was increased in glomeruli from patients with DKD and DKD sera-treated human podocytes, where it prevented αVβ3 integrin activation by its interaction with suPAR and led to increased RhoA activity, rendering podocytes more susceptible to apoptosis. In vivo, inhibition of acid sphingomyelinase reduced proteinuria in experimental DKD but not FSGS, indicating that SMPDL3b expression levels determined the podocyte injury phenotype. These observations suggest that SMPDL3b may be an important modulator of podocyte function by shifting suPAR-mediated podocyte injury from a migratory phenotype to an apoptotic phenotype and that it represents a novel therapeutic glomerular disease target. PMID:24925721

  9. Association of Sirtuin 1 (SIRT1) Gene SNPs and Transcript Expression Levels With Severe Obesity

    PubMed Central

    Clark, Stephen J.; Falchi, Mario; Olsson, Bob; Jacobson, Peter; Cauchi, Stéphane; Balkau, Beverley; Marre, Michel; Lantieri, Olivier; Andersson, Johanna C.; Jernås, Margareta; Aitman, Timothy J.; Richardson, Sylvia; Sjöström, Lars; Wong, Hang Y.; Carlsson, Lena M. S.; Froguel, Philippe; Walley, Andrew J.

    2013-01-01

    Recent studies have reported associations of sirtuin 1 (SIRT1) single nucleotide polymorphisms (SNPs) to both obesity and BMI. This study was designed to investigate association between SIRT1 SNPs, SIRT1 gene expression and obesity. Case-control analyses were performed using 1,533 obese subjects (896 adults, BMI >40 kg/m2 and 637 children, BMI >97th percentile for age and sex) and 1,237 nonobese controls, all French Caucasians. Two SNPs (in high linkage disequilibrium (LD), r2 = 0.96) were significantly associated with adult obesity, rs33957861 (P value = 0.003, odds ratio (OR) = 0.75, confidence interval (CI) = 0.61–0.92) and rs11599176 (P value: 0.006, OR = 0.74, CI = 0.61–0.90). Expression of SIRT1 mRNA was measured in BMI-discordant siblings from 154 Swedish families. Transcript expression was significantly correlated to BMI in the lean siblings (r2 = 0.13, P value = 3.36 × 10−7) and lower SIRT1 expression was associated with obesity (P value = 1.56 × 10−35). There was also an association between four SNPs (rs11599176, rs12413112, rs33957861, and rs35689145) and BMI (P values: 4 × 10−4, 6 × 10−4, 4 × 10−4, and 2 × 10−3) with the rare allele associated with a lower BMI. However, no SNP was associated with SIRT1 transcript expression level. In summary, both SNPs and SIRT1 gene expression are associated with severe obesity. PMID:21760635

  10. Gene family level comparative analysis of gene expression in mammals validates the ortholog conjecture.

    PubMed

    Rogozin, Igor B; Managadze, David; Shabalina, Svetlana A; Koonin, Eugene V

    2014-04-01

    The ortholog conjecture (OC), which is central to functional annotation of genomes, posits that orthologous genes are functionally more similar than paralogous genes at the same level of sequence divergence. However, a recent study challenged the OC by reporting a greater functional similarity, in terms of Gene Ontology (GO) annotations and expression profiles, among within-species paralogs compared with orthologs. These findings were taken to indicate that functional similarity of homologous genes is primarily determined by the cellular context of the genes, rather than evolutionary history. However, several subsequent studies suggest that GO annotations and microarray data could artificially inflate functional similarity between paralogs from the same organism. We sought to test the OC using approaches distinct from those used in previous studies. Analysis of a large RNAseq data set from multiple human and mouse tissues shows that expression similarity (correlations coefficients, rank's, or Z-scores) between orthologs is substantially greater than that for between-species paralogs with the same sequence divergence, in agreement with the OC and the results of recent detailed analyses. These findings are further corroborated by a fine-grain analysis in which expression profiles of orthologs and paralogs were compared separately for individual gene families. Expression profiles of within-species paralogs are more strongly correlated than profiles of orthologs but it is shown that this is caused by high background noise, that is, correlation between profiles of unrelated genes in the same organism. Z-scores and rank scores show a nonmonotonic dependence of expression profile similarity on sequence divergence. This complexity of gene expression evolution after duplication might be at least partially caused by selection for protein dosage rebalancing following gene duplication. PMID:24610837

  11. Repeated exposure to neurotoxic levels of chlorpyrifos alters hippocampal expression of neurotrophins and neuropeptides.

    PubMed

    Lee, Young S; Lewis, John A; Ippolito, Danielle L; Hussainzada, Naissan; Lein, Pamela J; Jackson, David A; Stallings, Jonathan D

    2016-01-18

    Chlorpyrifos (CPF), an organophosphorus pesticide (OP), is one of the most widely used pesticides in the world. Subchronic exposures to CPF that do not cause cholinergic crisis are associated with problems in cognitive function (i.e., learning and memory deficits), but the biological mechanism(s) underlying this association remain speculative. To identify potential mechanisms of subchronic CPF neurotoxicity, adult male Long Evans (LE) rats were administered CPF at 3 or 10mg/kg/d (s.c.) for 21 days. We quantified mRNA and non-coding RNA (ncRNA) expression profiles by RNA-seq, microarray analysis and small ncRNA sequencing technology in the CA1 region of the hippocampus. Hippocampal slice immunohistochemistry was used to determine CPF-induced changes in protein expression and localization patterns. Neither dose of CPF caused overt clinical signs of cholinergic toxicity, although after 21 days of exposure, cholinesterase activity was decreased to 58% or 13% of control levels in the hippocampus of rats in the 3 or 10mg/kg/d groups, respectively. Differential gene expression in the CA1 region of the hippocampus was observed only in the 10mg/kg/d dose group relative to controls. Of the 1382 differentially expressed genes identified by RNA-seq and microarray analysis, 67 were common to both approaches. Differential expression of six of these genes (Bdnf, Cort, Crhbp, Nptx2, Npy and Pnoc) was verified in an independent CPF exposure study; immunohistochemistry demonstrated that CRHBP and NPY were elevated in the CA1 region of the hippocampus at 10mg/kg/d CPF. Gene ontology enrichment analysis suggested association of these genes with receptor-mediated cell survival signaling pathways. miR132/212 was also elevated in the CA1 hippocampal region, which may play a role in the disruption of neurotrophin-mediated cognitive processes after CPF administration. These findings identify potential mediators of CPF-induced neurobehavioral deficits following subchronic exposure to CPF at

  12. High-level soluble expression of Serratia marcescens H30 lipase in Escherichia coli.

    PubMed

    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. PMID:24852607

  13. High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

    PubMed

    Hebben, Matthias; Brants, Jan; Birck, Catherine; Samama, Jean-Pierre; Wasylyk, Bohdan; Spehner, Danièle; Pradeau, Karine; Domi, Arban; Moss, Bernard; Schultz, Patrick; Drillien, Robert

    2007-12-01

    Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production. PMID:17892951

  14. Identification of a gene, FMP21, whose expression levels are involved in thermotolerance in Saccharomyces cerevisiae

    PubMed Central

    2014-01-01

    Elucidation of the mechanism of high temperature tolerance in yeasts is important for the molecular breeding of high temperature-tolerant yeasts that can be used in bioethanol production. We identified genes whose expression is correlated with the degree of thermotolerance in Saccharomyces cerevisiae by DNA microarray analysis. Gene expression profiles of three S. cerevisiae strains showing different levels of thermotolerance were compared, and we chose three of them as candidate genes. Among these genes, FMP21 was investigated as a thermotolerance-related gene in S. cerevisiae by comparing the growth at high temperature with the gene expression in eight strains. The expression ratio of FMP21 at 37°C was correlated with the doubling time ratio at a coefficient of determination of 0.787. The potential involvement of the Fmp21 in the thermotolerance of yeasts was evaluated. The FMP21 deletion variant showed a decreased respiratory growth rate and increased thermosensitivity. Furthermore, the overexpression of FMP21 improved thermotolerance in yeasts. In conclusion, the function of Fmp21 is important for thermotolerance in yeasts. PMID:25177541

  15. Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding

    PubMed Central

    Chappell, Paul E; Meziane, El Kahina; Harrison, Michael; Magiera, Łukasz; Hermann, Clemens; Mears, Laura; Wrobel, Antoni G; Durant, Charlotte; Nielsen, Lise Lotte; Buus, Søren; Ternette, Nicola; Mwangi, William; Butter, Colin; Nair, Venugopal; Ahyee, Trudy; Duggleby, Richard; Madrigal, Alejandro; Roversi, Pietro; Lea, Susan M; Kaufman, Jim

    2015-01-01

    Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. DOI: http://dx.doi.org/10.7554/eLife.05345.001 PMID:25860507

  16. Codon influence on protein expression in E. coli correlates with mRNA levels.

    PubMed

    Boël, Grégory; Letso, Reka; Neely, Helen; Price, W Nicholson; Wong, Kam-Ho; Su, Min; Luff, Jon D; Valecha, Mayank; Everett, John K; Acton, Thomas B; Xiao, Rong; Montelione, Gaetano T; Aalberts, Daniel P; Hunt, John F

    2016-01-21

    Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

  17. Expression levels of neuroimmune biomarkers in hypothalamus of allergic mice after phthalate exposure.

    PubMed

    Win-Shwe, Tin-Tin; Yanagisawa, Rie; Koike, Eiko; Nitta, Hiroshi; Takano, Hirohisa

    2013-10-01

    Previously, we demonstrated that maternal exposure to phthalates enhances atopic dermatitis in male mouse offspring. However, whether phthalate exposure affects neuroimmune biomarkers in allergic mice has not yet been studied. Di-(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DINP) are environmental chemicals that are commonly used as plasticizers. This study was designed to investigate the expression levels of neuroimmune biomarkers in the hypothalamus of a murine model of allergic asthma after phthalate exposure throughout juvenility until adulthood. Six-week-old C3H/HeJ Jcl male mice were treated with DEHP or DINP (0, 0.02, 0.4 or 8 nmol per body per week) and ovalbumin (OVA; 1 µg per body per 2 weeks) for 7 weeks intratracheally. On the day after the completion of the phthalate and OVA treatment, the hypothalamus from each mouse was collected, and the mRNA expression levels of neuroimmune biomarkers were examined using a real-time RT-PCR analysis. The mRNA expression levels of the proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α, the chemokine CCL3, the transcription factor nuclear factor (NF)-κB, the oxidative stress marker heme-oxygenase (HO)1, a nerve growth factor, and the microglia marker Iba1 were remarkably up-regulated in the hypothalami of mice treated with 8 nmol of DEHP in the presence of the allergen. However, no significant changes were observed, except for reductions in the TNF-α and CCL2 mRNA levels, in mice exposed to DINP combined with the allergen. This study is the first report to show that high-dose DEHP exposure throughout juvenility until adulthood may induce neuroinflammation by modulating neuroimmune biomarkers in the hypothalami of allergic mice. PMID:23148021

  18. Bis-(2-ethylhexyl) Phthalate Increases Insulin Expression and Lipid Levels in Drosophila melanogaster.

    PubMed

    Cao, Hao; Wiemerslage, Lyle; Marttila, Petra S K; Williams, Michael J; Schiöth, Helgi B

    2016-09-01

    Bis-(2-ethylhexyl) phthalate (DEHP) is one of the most widely used plasticizers, and human beings are exposed to DEHP via polyvinyl chloride (PVC) materials, medical equipment and even drinking water. While DEHP has been implicated to influence metabolism and endocrine functions, important questions remain about the molecular mechanisms of these effects. We employed the model organism Drosophila melanogaster and examined physiological, molecular and behavioural effects from DEHP-contaminated food. We found that DEHP, at levels comparable to human exposure, made male flies more resistant to starvation and increased lipid levels, while decreasing circulating carbohydrates. Moreover, DEHP-fed male flies had higher expression levels of an insulin-like peptide known to regulate metabolism, as well as the insulin receptor. Our results suggest that long-term DEHP feeding may induce diabetes-like dysfunctions. These findings provide a molecular background of how DEHP may have detrimental effects on metabolic functions. PMID:27009472

  19. Recurrent selection for transgene expression levels in maize results in proxy selection for a native gene with the same promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High expression levels of a transgene can be very useful, making a transgene easier to evaluate for safety and efficacy. High expression levels can also increase the economic benefit of the production of high value proteins in transgenic plants. The goal of this research is to determine if recurre...

  20. GENE EXPRESSION PROFILES IN ARSENIC-TREATED MCF-7 BREAST CANCER CELLS EXPRESSING DIFFERENT LEVELS OF HSP70

    EPA Science Inventory

    Gene expression profiles in arsenic-treated MCF-7 breast cancer cells expressing different levels of HSP70

    Gail Nelson, Susan Hester, Ernest Winkfield, Jill Barnes, James Allen
    Environmental Carcinogenesis Division, NHEERL, ORD, US Environmental Protection Agency, Rese...

  1. LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.

    PubMed

    Needham, M; Gooding, C; Hudson, K; Antoniou, M; Grosveld, F; Hollis, M

    1992-03-11

    We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells. The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells. Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter. In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors. The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction. Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection. PMID:1549512

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

    PubMed

    Ozawa, Masayuki; Himaki, Takehiro; Ookutsu, Shoji; Mizobe, Yamato; Ogawa, Junki; Miyoshi, Kazuchika; Yabuki, Akira; Fan, Jianglin; Yoshida, Mitsutoshi

    2015-01-01

    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. PMID:26147378

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

    PubMed Central

    Ozawa, Masayuki; Himaki, Takehiro; Ookutsu, Shoji; Mizobe, Yamato; Ogawa, Junki; Miyoshi, Kazuchika; Yabuki, Akira; Fan, Jianglin; Yoshida, Mitsutoshi

    2015-01-01

    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. PMID:26147378

  4. Differential hormonal and gene expression dynamics in two inbred sunflower lines with contrasting dormancy level.

    PubMed

    Roselló, Paula L; Vigliocco, Ana E; Andrade, Andrea M; Riera, Natalí V; Calafat, Mario; Molas, María L; Alemano, Sergio G

    2016-05-01

    Seed germination and dormancy are tightly regulated by hormone metabolism and signaling pathway. We investigated the endogenous content of abscisic acid (ABA), its catabolites, and gibberellins (GAs), as well as the expression level of certain ABA and GAs metabolic and signaling genes in embryo of dry and imbibed cypselas of inbred sunflower (Helianthus annuus L., Asteraceae) lines: B123 (dormant) and B91 (non-dormant). Under our experimental conditions, the expression of RGL2 gene might be related to the ABA peak in B123 line at 3 h of imbibition. Indeed, RGL2 transcripts are absent in dry and early embedded cypselas of the non-dormant line B91. ABA increase was accompanied by a significant ABA-Glucosyl ester (ABA-GE) and phaseic acid (PA) (two ABA catabolites) decrease in B123 line (3 h) which indicates that ABA metabolism seems to be more active in this line, and that it would be involved in the imposition and maintenance of sunflower seed dormancy, as it has been reported for many species. Finally, an increase of bioactive GAs (GA1 and GA3) occurs at 12 h of imbibition in both lines after a decrease in ABA content. This study shows the first report about the RGL2 tissue-specific gene expression in sunflower inbred lines with contrasting dormancy level. Furthermore, our results provide evidence that ABA and GAs content and differential expression of metabolism and signaling genes would be interacting in seed dormancy regulation through a mechanism of action related to embryo itself. PMID:26934102

  5. The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli

    PubMed Central

    Latifi, Ali Mohammad; Khajeh, Khosro; Farnoosh, Gholamreza; Hassanpour, Kazem; Khodi, Samaneh

    2015-01-01

    Background: Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process. Objectives: In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b). Materials and Methods: The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd. Results: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band. Conclusions: The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step. PMID:26870308

  6. Developmental changes in the expression level of connexin36 in the rat retina.

    PubMed

    Kovács-Öller, Tamás; Raics, Katalin; Orbán, József; Nyitrai, Miklós; Völgyi, Béla

    2014-11-01

    Connexin36 (Cx36) is the major gap junction forming protein in the brain and the retina; thus, alterations in its expression indicate changes in the corresponding circuitry. Many structural changes occur in the early postnatal retina before functional neuronal circuits are finalized, including those that incorporate gap junctions. To reveal the time-lapse formation of inner retinal gap junctions, we examine the developing postnatal rat retina from birth (P0) to young adult age (P20) and follow the expression of Cx36 in the mRNA and protein levels. We found a continuous elevation in the expression of both the Cx36 transcript and protein between P0 and P20 and a somewhat delayed Cx36 plaque formation throughout the inner plexiform layer (IPL) starting at P10. By using tristratificated calretinin positive (CaR(+)) fibers in the IPL as a guide, we detected a clear preference of Cx36 plaques for the ON sublamina from the earliest time of detection. This distributional preference became more pronounced at P15 and P20 due to the emergence and widespread expression of large (>0.1 μm(2)) Cx36 plaques in the ON sublamina. Finally, we showed that parvalbumin-positive (PV(+)) AII amacrine cell dendrites colocalize with Cx36 plaques as early as P10 in strata 3 and 4, whereas colocalizations in stratum 5 became characteristic only around P20. We conclude that Cx36 expression in the rat IPL displays a characteristic succession of changes during retinogenesis reflecting the formation of the underlying electrical synaptic circuitry. In particular, AII cell gap junctions, first formed with ON cone bipolar cells and later with other AII amacrine cells, accounted for the observed Cx36 expressional changes. PMID:25110193

  7. Levels of expression of CD19 and CD20 in chronic B cell leukaemias.

    PubMed Central

    Ginaldi, L; De Martinis, M; Matutes, E; Farahat, N; Morilla, R; Catovsky, D

    1998-01-01

    AIMS: To investigate whether the antigen levels of the B cell lineage markers CD19 and CD20 can distinguish between normal and neoplastic B cells or characterise distinct expression patterns among the chronic B cell leukaemias. METHODS: Peripheral blood cells from 70 patients with B cell disorders and 17 healthy donors were analysed by quantitative flow cytometry. Direct immunofluorescence staining was performed with phycoerythrin conjugated CD19 and CD20 monoclonal antibodies. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values into number of antigen molecules/cell, expressed as antibody binding capacity (ABC). RESULTS: CD19 and CD20 ABC values in leukaemic B cells differed from those of normal blood B lymphocytes. The results identified distinct profiles of CD19 and CD20 expression in the various types of B cell leukaemias. In all leukaemias studied except hairy cell leukaemia (HCL), CD19 expression was significantly lower than the mean (SD) value in normal B cells (22 (7) x 10(3) molecules/cell), as follows: chronic lymphocytic leukaemia (CLL), 13 (7) x 10(3); B prolymphocytic leukaemia (B-PLL), 16 (9) x 10(3); splenic lymphoma with villous lymphocytes (SLVL), 15 (11) x 10(3); mantle cell lymphoma (MCL), 10 (7) x 10(3). In HCL there was strong CD19 expression (38 (16) x 10(3)). In contrast, the level of expression of membrane CD20 was higher than the mean (SD) value in normal B cells (94 (16) x 10(3) molecules/cell) in MCL (123 (51) x 10(3)); B-PLL (129 (47) x 10(3)); SLVL (167 (72) x 10(3)); and HCL (312 (110) x 10(3)); while it was significantly lower (65 (11) x 10(3)) in CLL compared with normal B cells and the other B cell leukaemias. CONCLUSIONS: Quantitative determination of CD19 and CD20 may provide useful diagnostic information for the study of B lymphoproliferative disorders. PMID:9708202

  8. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Parekh, R N; Wittrup, K D

    1997-01-01

    The relationship between expression level and secretion of bovine pancreatic trypsin inhibitor (BPTI) was determined in Saccharomyces cerevisiae using a tunable amplifiable delta integration vector. Optimal secretory productivity of 15 mg of BPTI/g cell dry weight yields 180 mg/L secreted active BPTI in test-tube cultures, an order of magnitude increase over 2 mu plasmid-directed secretion. Maximum productivity is determined by the protein folding capacity of the endoplasmic reticulum (ER). Unfolded protein accumulates in the ER as synthesis increases, until a physiological instability is reached and secretion decreases precipitously despite high BPTI mRNA levels. Optimal specific productivity of a standard laboratory strain of S. cerevisiae is double that reported for secretion of BPTI by Pichia pastoris, indicating that efficient utilization of S. cerevisiae's available secretory capacity can eliminate apparent differences among yeast species in their capacity for heterologous protein secretion. Although not generally recognized, the existence of an optimum synthesis level for secretion is apparently a general feature of eucaryotic expression systems and could be of substantial significance for maximization of protein secretion in mammalian and insect cell culture. PMID:9104035

  9. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    PubMed

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism. PMID:22003434

  10. High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates

    PubMed Central

    Schneider, Anina; Weier, Manuela; Sweep, Fred C. G. J.; Le Roy, Didier; Bernhagen, Jürgen; Calandra, Thierry; Giannoni, Eric

    2016-01-01

    The vulnerability to infection of newborns is associated with a limited ability to mount efficient immune responses. High concentrations of adenosine and prostaglandins in the fetal and neonatal circulation hamper the antimicrobial responses of newborn immune cells. However, the existence of mechanisms counterbalancing neonatal immunosuppression has not been investigated. Remarkably, circulating levels of macrophage migration inhibitory factor (MIF), a proinflammatory immunoregulatory cytokine expressed constitutively, were 10-fold higher in newborns than in children and adults. Newborn monocytes expressed high levels of MIF and released MIF upon stimulation with Escherichia coli and group B Streptococcus, the leading pathogens of early-onset neonatal sepsis. Inhibition of MIF activity or MIF expression reduced microbial product-induced phosphorylation of p38 and ERK1/2 mitogen-activated protein kinases and secretion of cytokines. Recombinant MIF used at newborn, but not adult, concentrations counterregulated adenosine and prostaglandin E2-mediated inhibition of ERK1/2 activation and TNF production in newborn monocytes exposed to E. coli. In agreement with the concept that once infection is established high levels of MIF are detrimental to the host, treatment with a small molecule inhibitor of MIF reduced systemic inflammatory response, bacterial proliferation, and mortality of septic newborn mice. Altogether, these data provide a mechanistic explanation for how newborns may cope with an immunosuppressive environment to maintain a certain threshold of innate defenses. However, the same defense mechanisms may be at the expense of the host in conditions of severe infection, suggesting that MIF could represent a potential attractive target for immune-modulating adjunctive therapies for neonatal sepsis. PMID:26858459

  11. Characterization of changes in gene expression and biochemical pathways at low levels of benzene exposure.

    PubMed

    Thomas, Reuben; Hubbard, Alan E; McHale, Cliona M; Zhang, Luoping; Rappaport, Stephen M; Lan, Qing; Rothman, Nathaniel; Vermeulen, Roel; Guyton, Kathryn Z; Jinot, Jennifer; Sonawane, Babasaheb R; Smith, Martyn T

    2014-01-01

    Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML). Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC), we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across four airborne concentration ranges (from <1 ppm to >10 ppm) compared with 42 subjects with non-workplace ambient exposure levels. Here, we further characterize these dose-dependent effects with continuous benzene exposure in all 125 study subjects. We estimated air benzene exposure levels in the 42 environmentally-exposed subjects from their unmetabolized urinary benzene levels. We used a novel non-parametric, data-adaptive model selection method to estimate the change with dose in the expression of each gene. We describe non-parametric approaches to model pathway responses and used these to estimate the dose responses of the AML pathway and 4 other pathways of interest. The response patterns of majority of genes as captured by mean estimates of the first and second principal components of the dose-response for the five pathways and the profiles of 6 AML pathway response-representative genes (identified by clustering) exhibited similar apparent supra-linear responses. Responses at or below 0.1 ppm benzene were observed for altered expression of AML pathway genes and CYP2E1. Together, these data show that benzene alters disease-relevant pathways and genes in a dose-dependent manner, with effects apparent at doses as low as 100 ppb in air. Studies with extensive exposure assessment of subjects exposed in the low-dose range between 10 ppb and 1 ppm are needed to confirm these findings. PMID:24786086

  12. Characterization of Changes in Gene Expression and Biochemical Pathways at Low Levels of Benzene Exposure

    PubMed Central

    Thomas, Reuben; Hubbard, Alan E.; McHale, Cliona M.; Zhang, Luoping; Rappaport, Stephen M.; Lan, Qing; Rothman, Nathaniel; Vermeulen, Roel; Guyton, Kathryn Z.; Jinot, Jennifer; Sonawane, Babasaheb R.; Smith, Martyn T.

    2014-01-01

    Benzene, a ubiquitous environmental pollutant, causes acute myeloid leukemia (AML). Recently, through transcriptome profiling of peripheral blood mononuclear cells (PBMC), we reported dose-dependent effects of benzene exposure on gene expression and biochemical pathways in 83 workers exposed across four airborne concentration ranges (from <1 ppm to >10 ppm) compared with 42 subjects with non-workplace ambient exposure levels. Here, we further characterize these dose-dependent effects with continuous benzene exposure in all 125 study subjects. We estimated air benzene exposure levels in the 42 environmentally-exposed subjects from their unmetabolized urinary benzene levels. We used a novel non-parametric, data-adaptive model selection method to estimate the change with dose in the expression of each gene. We describe non-parametric approaches to model pathway responses and used these to estimate the dose responses of the AML pathway and 4 other pathways of interest. The response patterns of majority of genes as captured by mean estimates of the first and second principal components of the dose-response for the five pathways and the profiles of 6 AML pathway response-representative genes (identified by clustering) exhibited similar apparent supra-linear responses. Responses at or below 0.1 ppm benzene were observed for altered expression of AML pathway genes and CYP2E1. Together, these data show that benzene alters disease-relevant pathways and genes in a dose-dependent manner, with effects apparent at doses as low as 100 ppb in air. Studies with extensive exposure assessment of subjects exposed in the low-dose range between 10 ppb and 1 ppm are needed to confirm these findings. PMID:24786086

  13. Strategies for achieving high-level expression of genes in Escherichia coli.

    PubMed Central

    Makrides, S C

    1996-01-01

    Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli. PMID:8840785

  14. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

    PubMed

    Masser, Anna E; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan; Andréasson, Claes

    2016-05-01

    Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE ), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  15. High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates.

    PubMed

    Roger, Thierry; Schneider, Anina; Weier, Manuela; Sweep, Fred C G J; Le Roy, Didier; Bernhagen, Jürgen; Calandra, Thierry; Giannoni, Eric

    2016-02-23

    The vulnerability to infection of newborns is associated with a limited ability to mount efficient immune responses. High concentrations of adenosine and prostaglandins in the fetal and neonatal circulation hamper the antimicrobial responses of newborn immune cells. However, the existence of mechanisms counterbalancing neonatal immunosuppression has not been investigated. Remarkably, circulating levels of macrophage migration inhibitory factor (MIF), a proinflammatory immunoregulatory cytokine expressed constitutively, were 10-fold higher in newborns than in children and adults. Newborn monocytes expressed high levels of MIF and released MIF upon stimulation with Escherichia coli and group B Streptococcus, the leading pathogens of early-onset neonatal sepsis. Inhibition of MIF activity or MIF expression reduced microbial product-induced phosphorylation of p38 and ERK1/2 mitogen-activated protein kinases and secretion of cytokines. Recombinant MIF used at newborn, but not adult, concentrations counterregulated adenosine and prostaglandin E2-mediated inhibition of ERK1/2 activation and TNF production in newborn monocytes exposed to E. coli. In agreement with the concept that once infection is established high levels of MIF are detrimental to the host, treatment with a small molecule inhibitor of MIF reduced systemic inflammatory response, bacterial proliferation, and mortality of septic newborn mice. Altogether, these data provide a mechanistic explanation for how newborns may cope with an immunosuppressive environment to maintain a certain threshold of innate defenses. However, the same defense mechanisms may be at the expense of the host in conditions of severe infection, suggesting that MIF could represent a potential attractive target for immune-modulating adjunctive therapies for neonatal sepsis. PMID:26858459

  16. Endothelin A and B receptors change their expression levels during development of human placental villi.

    PubMed

    Cervar, M; Huppertz, B; Barth, S; Hahn, T; Weiss, U; Kaufmann, P; Desoye, G

    2000-01-01

    Endothelin receptors have recently been found in non-vascular tissues including the human placenta. The present study investigated developmental changes in location and expression levels of endothelin A and B receptors (ETA-R, ETB-R) in human placentae and isolated trophoblast by comparing first and third trimester tissues. In the first trimester all cells and tissues were immunolabelled for ETA-R and ETB-R, whereas in third trimester placentae the syncytiotrophoblast (ETA-R, ETB-R) and macrophages (ETA-R) were unstained. Immunoblotting for both receptors revealed up to three bands at 33-35, 50 and 75 kDa, respectively, which were differentially present in the first and third trimester. Pre-adsorption of antibodies with oligopeptides used for antigen-generation weakened the immunoreactions. ETA-R protein levels decreased (P< 0.05) in total villous tissue and isolated trophoblast, whereas ETB-R was unchanged. ETB-R transcripts (RT-PCR) prevailed in both stages and tissues, but in contrast to the protein levels its preponderance decreased from first trimester to term in villous tissue (P< 0.01), because of a four to five-fold increase in ETA-R and only a two-fold (P< 0.05) increase in ETB-R mRNA levels (P< 0.01). We conclude that ET receptor location, intracellular processing and expression levels in human villous tissue change between the first and third trimester. This may reflect changing functions of ET-1 during placental development. PMID:10940204

  17. Characterization of protein expression levels with label-free detected reverse phase protein arrays.

    PubMed

    Guo, Xuexue; Deng, Yihong; Zhu, Chenggang; Cai, Junlong; Zhu, Xiangdong; Landry, James P; Zheng, Fengyun; Cheng, Xunjia; Fei, Yiyan

    2016-09-15

    In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies. PMID:27372609

  18. Topology-optimized multiple-disk resonators obtained using level set expression incorporating surface effects.

    PubMed

    Fujii, Garuda; Ueta, Tsuyoshi; Mizuno, Mamoru; Nakamura, Masayuki

    2015-05-01

    Topology-optimized designs of multiple-disk resonators are presented using level-set expression that incorporates surface effects. Effects from total internal reflection at the surfaces of the dielectric disks are precisely simulated by modeling clearly defined dielectric boundaries during topology optimization. The electric field intensity in optimal resonators increases to more than four and a half times the initial intensity in a resonant state, whereas in some cases the Q factor increases by three and a half times that for the initial state. Wavelength-scale link structures between neighboring disks improve the performance of the multiple-disk resonators. PMID:25969226

  19. Regulatory Nexus of Synthesis and Degradation Deciphers Cellular Nrf2 Expression Levels

    PubMed Central

    Suzuki, Takafumi; Shibata, Tatsuhiro; Takaya, Kai; Shiraishi, Kouya; Kohno, Takashi; Kunitoh, Hideo; Tsuta, Koji; Furuta, Koh; Goto, Koichi; Hosoda, Fumie; Sakamoto, Hiromi; Motohashi, Hozumi

    2013-01-01

    Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine. PMID:23572560

  20. Regulatory nexus of synthesis and degradation deciphers cellular Nrf2 expression levels.

    PubMed

    Suzuki, Takafumi; Shibata, Tatsuhiro; Takaya, Kai; Shiraishi, Kouya; Kohno, Takashi; Kunitoh, Hideo; Tsuta, Koji; Furuta, Koh; Goto, Koichi; Hosoda, Fumie; Sakamoto, Hiromi; Motohashi, Hozumi; Yamamoto, Masayuki

    2013-06-01

    Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine. PMID:23572560

  1. Nasal Associated Lymphoid Tissue of the Syrian Golden Hamster Expresses High Levels of PrPC

    PubMed Central

    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. PMID:25642714

  2. Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

    PubMed Central

    Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.; Williams, Kenneth H.; McCue, Lee Ann; Handley, Kim M.; Miller, Chris S.; Giloteaux, Ludovic; Montgomery, Alison P.; Lovley, Derek R.; Banfield, Jillian F.; Long, Philip E.; Lipton, Mary S.

    2013-01-01

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment. PMID:23472107

  3. Fluctuations in species-level protein expression occur during element and nutrient cycling in the subsurface.

    PubMed

    Wilkins, Michael J; Wrighton, Kelly C; Nicora, Carrie D; Williams, Kenneth H; McCue, Lee Ann; Handley, Kim M; Miller, Chris S; Giloteaux, Ludovic; Montgomery, Alison P; Lovley, Derek R; Banfield, Jillian F; Long, Philip E; Lipton, Mary S

    2013-01-01

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment. PMID:23472107

  4. Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

    SciTech Connect

    Wilkins, Michael J.; Wrighton, Kelly C.; Nicora, Carrie D.; Williams, Kenneth H.; McCue, Lee Ann; Handley, Kim M.; Miller, C. S.; Giloteaux, L.; Montgomery, A. P.; Lovley, Derek R.; Banfield, Jillian F.; Long, Philip E.; Lipton, Mary S.

    2013-03-05

    While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.

  5. Metabolic syndrome influences cardiac gene expression pattern at the transcript level in male ZDF rats

    PubMed Central

    2013-01-01

    Background Metabolic syndrome (coexisting visceral obesity, dyslipidemia, hyperglycemia, and hypertension) is a prominent risk factor for cardiovascular morbidity and mortality, however, its effect on cardiac gene expression pattern is unclear. Therefore, we examined the possible alterations in cardiac gene expression pattern in male Zucker Diabetic Fatty (ZDF) rats, a model of metabolic syndrome. Methods Fasting blood glucose, serum insulin, cholesterol and triglyceride levels were measured at 6, 16, and 25 wk of age in male ZDF and lean control rats. Oral glucose tolerance test was performed at 16 and 25 wk of age. At week 25, total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 14921 genes. Expression of selected genes was confirmed by qRT-PCR. Results Fasting blood glucose, serum insulin, cholesterol and triglyceride levels were significantly increased, glucose tolerance and insulin sensitivity were impaired in ZDF rats compared to leans. In hearts of ZDF rats, 36 genes showed significant up-regulation and 49 genes showed down-regulation as compared to lean controls. Genes with significantly altered expression in the heart due to metabolic syndrome includes functional clusters of metabolism (e.g. 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2; argininosuccinate synthetase; 2-amino-3-ketobutyrate-coenzyme A ligase), structural proteins (e.g. myosin IXA; aggrecan1), signal transduction (e.g. activating transcription factor 3; phospholipase A2; insulin responsive sequence DNA binding protein-1) stress response (e.g. heat shock 70kD protein 1A; heat shock protein 60; glutathione S-transferase Yc2 subunit), ion channels and receptors (e.g. ATPase, (Na+)/K+ transporting, beta 4 polypeptide; ATPase, H+/K+ transporting, nongastric, alpha polypeptide). Moreover some other genes with no definite functional clusters were also changed such as e.g. S100 calcium binding protein A3; ubiquitin carboxy-terminal hydrolase L1; interleukin

  6. High levels of homocysteine downregulate apolipoprotein E expression via nuclear factor kappa B

    PubMed Central

    Trusca, Violeta G; Mihai, Adina D; Fuior, Elena V; Fenyo, Ioana M; Gafencu, Anca V

    2016-01-01

    AIM: To investigate the effect of high homocysteine (Hcy) levels on apolipoprotein E (apoE) expression and the signaling pathways involved in this gene regulation. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to assess apoE expression in cells treated with various concentrations (50-500 μmol/L) of Hcy. Calcium phosphate-transient transfections were performed in HEK-293 and RAW 264.7 cells to evaluate the effect of Hcy on apoE regulatory elements [promoter and distal multienhancer 2 (ME2)]. To this aim, plasmids containing the proximal apoE promoter [(-500/+73)apoE construct] alone or in the presence of ME2 [ME2/(-500/+73)apoE construct] to drive the expression of the reporter luciferase gene were used. Co-transfection experiments were carried out to investigate the downstream effectors of Hcy-mediated regulation of apoE promoter by using specific inhibitors or a dominant negative form of IKβ. In other co-transfections, the luciferase reporter was under the control of synthetic promoters containing multiple specific binding sites for nuclear factor kappa B (NF-κB), activator protein-1 (AP-1) or nuclear factor of activated T cells (NFAT). Chromatin immunoprecipitation (ChIP) assay was accomplished to detect the binding of NF-κB p65 subunit to the apoE promoter in HEK-293 treated with 500 μmol/L Hcy. As control, cells were incubated with similar concentration of cysteine. NF-κB p65 proteins bound to DNA were immunoprecipitated with anti-p65 antibodies and DNA was identified by PCR using primers amplifying the region -100/+4 of the apoE gene. RESULTS: RT-PCR revealed that high levels of Hcy (250-750 μmol/L) induced a 2-3 fold decrease in apoE mRNA levels in HEK-293 cells, while apoE gene expression was not significantly affected by treatment with lower concentrations of Hcy (100 μmol/L). Immunoblotting data provided additional evidence for the negative role of Hcy in apoE expression. Hcy decreased apoE promoter

  7. Pipasic: similarity and expression correction for strain-level identification and quantification in metaproteomics

    PubMed Central

    Penzlin, Anke; Lindner, Martin S.; Doellinger, Joerg; Dabrowski, Piotr Wojtek; Nitsche, Andreas; Renard, Bernhard Y.

    2014-01-01

    Motivation: Metaproteomic analysis allows studying the interplay of organisms or functional groups and has become increasingly popular also for diagnostic purposes. However, difficulties arise owing to the high sequence similarity between related organisms. Further, the state of conservation of proteins between species can be correlated with their expression level, which can lead to significant bias in results and interpretation. These challenges are similar but not identical to the challenges arising in the analysis of metagenomic samples and require specific solutions. Results: We introduce Pipasic (peptide intensity-weighted proteome abundance similarity correction) as a tool that corrects identification and spectral counting-based quantification results using peptide similarity estimation and expression level weighting within a non-negative lasso framework. Pipasic has distinct advantages over approaches only regarding unique peptides or aggregating results to the lowest common ancestor, as demonstrated on examples of viral diagnostics and an acid mine drainage dataset. Availability and implementation: Pipasic source code is freely available from https://sourceforge.net/projects/pipasic/. Contact: RenardB@rki.de Supplementary information: Supplementary data are available at Bioinformatics online PMID:24931978

  8. Direct effect of acaricides on pathogen loads and gene expression levels in honey bees Apis mellifera.

    PubMed

    Boncristiani, Humberto; Underwood, Robyn; Schwarz, Ryan; Evans, Jay D; Pettis, Jeffery; vanEngelsdorp, Dennis

    2012-05-01

    The effect of using acaricides to control varroa mites has long been a concern to the beekeeping industry due to unintended negative impacts on honey bee health. Irregular ontogenesis, suppression of immune defenses, and impairment of normal behavior have been linked to pesticide use. External stressors, including parasites and the pathogens they vector, can confound studies on the effects of pesticides on the metabolism of honey bees. This is the case of Varroa destructor, a mite that negatively affects honey bee health on many levels, from direct parasitism, which diminishes honey bee productivity, to vectoring and/or activating other pathogens, including many viruses. Here we present a gene expression profile comprising genes acting on diverse metabolic levels (detoxification, immunity, and development) in a honey bee population that lacks the influence of varroa mites. We present data for hives treated with five different acaricides; Apiguard (thymol), Apistan (tau-fluvalinate), Checkmite (coumaphos), Miteaway (formic acid) and ApiVar (amitraz). The results indicate that thymol, coumaphos and formic acid are able to alter some metabolic responses. These include detoxification gene expression pathways, components of the immune system responsible for cellular response and the c-Jun amino-terminal kinase (JNK) pathway, and developmental genes. These could potentially interfere with the health of individual honey bees and entire colonies. PMID:22212860

  9. Expression of DMP-1 in the human pulp tissue using low level laser therapy

    NASA Astrophysics Data System (ADS)

    Lourenço Neto, Natalino; Teixeira Marques, Nádia Carolina; Fernandes, Ana Paula; Oliveira Rodini, Camila; Cruvinel Silva, Thiago; Moreira Machado, Maria Aparecida Andrade; Marchini Oliveira, Thais

    2015-09-01

    This study aimed to evaluate the effects of low-level laser therapy (LLLT) on DMP-1 expression in pulp tissue repair of human primary teeth. Twenty mandibular primary molars were randomly assigned into the following groups: Group I—Buckley’s Formocresol (FC); Group II—Calcium Hydroxide (CH); Group III—LLLT + CH and Group IV—LLLT + Zinc oxide/Eugenol. The teeth at the regular exfoliation period were extracted for histological analysis and immunolocalization of DMP-1. Descriptive analysis was performed on the dentin pulp complex. Histopathological assessment showed internal resorption in group FC. Groups CH and LLLT + CH provided better pulpal repair due to the absence of inflammation and the formation of hard tissue barrier. These two groups presented odontoblastic layer expressing DMP-1. According to this study, low level laser therapy preceding the use of calcium hydroxide exhibited satisfactory bio-inductive activity on pulp tissue repair of human primary teeth. However, other histological and cellular studies are needed to confirm the laser tissue action and efficacy.

  10. High level expression of peptides and proteins using cytochrome b5 as a fusion host.

    PubMed

    Mitra, Ashima; Chakrabarti, Kalyan Sundar; Shahul Hameed, M S; Srinivas, Kalyan V; Senthil Kumar, Ganesan; Sarma, Siddhartha P

    2005-05-01

    A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. The ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., alpha hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, NaV1.2. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel purification protocol has been designed to purify the fusion proteins using metal affinity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome b5 based fusion system for the production of peptides and small proteins (<15 kDa). PMID:15802225

  11. Neuroblastoma patient outcomes, tumor differentiation, and ERK activation are correlated with expression levels of the ubiquitin ligase UBE4B

    PubMed Central

    Woodfield, Sarah E.; Guo, Rong Jun; Liu, Yin; Major, Angela M.; Hollingsworth, Emporia Faith; Indiviglio, Sandra; Whittle, Sarah B.; Mo, Qianxing; Bean, Andrew J.; Ittmann, Michael; Lopez-Terrada, Dolores; Zage, Peter E.

    2016-01-01

    Background UBE4B is an E3/E4 ubiquitin ligase whose gene is located in chromosome 1p36.22. We analyzed the associations of UBE4B gene and protein expression with neuroblastoma patient outcomes and with tumor prognostic features and histology. Methods We evaluated the association of UBE4B gene expression with neuroblastoma patient outcomes using the R2 Platform. We screened neuroblastoma tumor samples for UBE4B protein expression using immunohistochemistry. FISH for UBE4B and 1p36 deletion was performed on tumor samples. We then evaluated UBE4B expression for associations with prognostic factors and with levels of phosphorylated ERK in neuroblastoma tumors and cell lines. Results Low UBE4B gene expression is associated with poor outcomes in patients with neuroblastoma and with worse outcomes in all patient subgroups. UBE4B protein expression was associated with neuroblastoma tumor differentiation, and decreased UBE4B protein levels were associated with high-risk features. UBE4B protein levels were also associated with levels of phosphorylated ERK. Conclusions We have demonstrated associations between UBE4B gene expression and neuroblastoma patient outcomes and prognostic features. Reduced UBE4B protein expression in neuroblastoma tumors was associated with high-risk features, a lack of differentiation, and with ERK activation. These results suggest UBE4B may contribute to the poor prognosis of neuroblastoma tumors with 1p36 deletions and that UBE4B expression may mediate neuroblastoma differentiation. PMID:27014418

  12. Endogenous galectin-3 expression levels modulate immune responses in galectin-3 transgenic mice.

    PubMed

    Chaudhari, Aparna D; Gude, Rajiv P; Kalraiya, Rajiv D; Chiplunkar, Shubhada V

    2015-12-01

    Galectin-3 (Gal-3), a β-galactoside-binding mammalian lectin, is involved in cancer progression and metastasis. However, there is an unmet need to identify the underlying mechanisms of cancer metastasis mediated by endogenous host galectin-3. Galectin-3 is also known to be an important regulator of immune responses. The present study was aimed at analysing how expression of endogenous galectin-3 regulates host immunity and lung metastasis in B16F10 murine melanoma model. Transgenic Gal-3(+/-) (hemizygous) and Gal-3(-/-) (null) mice exhibited decreased levels of Natural Killer (NK) cells and lower NK mediated cytotoxicity against YAC-1 tumor targets, compared to Gal-3(+/+) (wild-type) mice. On stimulation, Gal-3(+/-) and Gal-3(-/-) mice splenocytes showed increased T cell proliferation than Gal-3(+/+) mice. Intracellular calcium flux was found to be lower in activated T cells of Gal-3(-/-) mice as compared to T cells from Gal-3(+/+) and Gal-3(+/-) mice. In Gal-3(-/-) mice, serum Th1, Th2 and Th17 cytokine levels were found to be lowest, exhibiting dysregulation of pro-inflammatory and anti-inflammatory cytokines balance. Marked decrease in serum IFN-γ levels and splenic IFN-γR1 (IFN-γ Receptor 1) expressing T and NK cell percentages were observed in Gal-3(-/-) mice. On recombinant IFN-γ treatment of splenocytes in vitro, Suppressor of Cytokine Signaling (SOCS) 1 and SOCS3 protein expression was higher in Gal-3(-/-) mice compared to that in Gal-3(+/+) and Gal-3(+/-) mice; suggesting possible attenuation of Signal Transducer and Activator of Transcription (STAT) 1 mediated IFN-γ signaling in Gal-3(-/-) mice. The ability of B16F10 melanoma cells to form metastatic colonies in the lungs of Gal-3(+/+) and Gal-3(-/-) mice remained comparable, whereas it was found to be reduced in Gal-3(+/-) mice. Our data indicates that complete absence of endogenous host galectin-3 facilitates lung metastasis of B16F10 cells in mice, which may be contributed by dysregulated immune

  13. TREM-2 Receptor Expression Increases with 25(OH)D Vitamin Serum Levels in Patients with Pulmonary Sarcoidosis

    PubMed Central

    Bucova, Maria; Suchankova, Magda; Tibenska, Elena; Tedlova, Eva; Demian, Juraj; Majer, Ivan; Novosadova, Helena; Tedla, Miroslav

    2015-01-01

    TREM-1 and TREM-2 molecules are members of the TREM transmembrane glycoproteins. In our previous study we identified increased expressions of TREM-1 and TREM-2 receptors in pulmonary sarcoidosis (PS). Only a few studies concerning the association between vitamin D and TREM receptor expression can be found. The aim of our current study was to determine the association between the levels of an inactive form of 25(OH)D vitamin and TREM-1 and TREM-2 receptor expressions. We have detected low levels of 25(OH)D vitamin in 79% of PS patients. Only 21% of patients had normal serum level of 25(OH)D vitamin with values clustered within the low-normal range. The most striking findings were the increased TREM-2 expressions on myeloid cells surfaces in BALF of PS patients with normal 25(OH)D vitamin serum levels compared with those with its decreased levels. The total number of TREM-2 positive cells was 5.7 times higher and the percentage of TREM-2 positive cells was also significantly increased in BALF of PS patients with normal compared to PS patients with low 25(OH)D vitamin serum levels. A significant correlation between total TREM-2 expression and vitamin D levels has been detected too. However, we have not detected similar differences in TREM-1expression and 25(OH)D vitamin serum levels. PMID:26166951

  14. TREM-2 Receptor Expression Increases with 25(OH)D Vitamin Serum Levels in Patients with Pulmonary Sarcoidosis.

    PubMed

    Bucova, Maria; Suchankova, Magda; Tibenska, Elena; Tedlova, Eva; Demian, Juraj; Majer, Ivan; Novosadova, Helena; Tedla, Miroslav

    2015-01-01

    TREM-1 and TREM-2 molecules are members of the TREM transmembrane glycoproteins. In our previous study we identified increased expressions of TREM-1 and TREM-2 receptors in pulmonary sarcoidosis (PS). Only a few studies concerning the association between vitamin D and TREM receptor expression can be found. The aim of our current study was to determine the association between the levels of an inactive form of 25(OH)D vitamin and TREM-1 and TREM-2 receptor expressions. We have detected low levels of 25(OH)D vitamin in 79% of PS patients. Only 21% of patients had normal serum level of 25(OH)D vitamin with values clustered within the low-normal range. The most striking findings were the increased TREM-2 expressions on myeloid cells surfaces in BALF of PS patients with normal 25(OH)D vitamin serum levels compared with those with its decreased levels. The total number of TREM-2 positive cells was 5.7 times higher and the percentage of TREM-2 positive cells was also significantly increased in BALF of PS patients with normal compared to PS patients with low 25(OH)D vitamin serum levels. A significant correlation between total TREM-2 expression and vitamin D levels has been detected too. However, we have not detected similar differences in TREM-1expression and 25(OH)D vitamin serum levels. PMID:26166951

  15. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters

    PubMed Central

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-01-01

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise. PMID:27098003

  16. High-level expression and characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production

    PubMed Central

    2013-01-01

    Background Production of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called “China wood oil” is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase as catalyst. However, the high cost of R. oryzae lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from R. oryzae reached 1334 U/mL in Pichia pastoris, which is still not optimistic for industry applications. Results The prosequence of lipases from Rhizopus sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from R. oryzae was constructed by replacing the prosequence with that from the R. chinensis lipase and expressed in P. pastoris. The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C. Conclusions This is the first report on improving the expression level of the lipase from R. oryzae by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw

  17. VNN1 Gene Expression Levels and the G-137T Polymorphism Are Associated with HDL-C Levels in Mexican Prepubertal Children

    PubMed Central

    Jacobo-Albavera, Leonor; Aguayo-de la Rosa, Pablo I.; Villarreal-Molina, Teresa; Villamil-Ramírez, Hugo; León-Mimila, Paola; Romero-Hidalgo, Sandra; López-Contreras, Blanca E.; Sánchez-Muñoz, Fausto; Bojalil, Rafael; González-Barrios, Juan Antonio; Aguilar-Salinas, Carlos A.; Canizales-Quinteros, Samuel

    2012-01-01

    Background VNN1 gene expression levels and the G-137T polymorphism have been associated with high density lipoprotein cholesterol (HDL-C) levels in Mexican American adults. We aim to evaluate the contribution of VNN1 gene expression and the G-137T variant to HDL-C levels and other metabolic traits in Mexican prepubertal children. Methodology/Principal Findings VNN1 mRNA expression levels were quantified in peripheral blood leukocytes from 224 unrelated Mexican-Mestizo children aged 6–8 years (107 boys and 117 girls) and were genotyped for the G-137T variant (rs4897612). To account for population stratification, a panel of 10 ancestry informative markers was analyzed. After adjustment for admixture, the TT genotype was significantly associated with lower VNN1 mRNA expression levels (P = 2.9 × 10−5), decreased HDL-C levels (β = −6.19, P = 0.028) and with higher body mass index (BMI) z-score (β = 0.48, P = 0.024) in the total sample. In addition, VNN1 expression showed a positive correlation with HDL-C levels (r = 0.220; P = 0.017) and a negative correlation with BMI z-score (r = −0.225; P = 0.015) only in girls. Conclusion/Significance Our data suggest that VNN1 gene expression and the G-137T variant are associated with HDL-C levels in Mexican children, particularly in prepubertal girls. PMID:23185446

  18. Association of MAPT haplotypes with Alzheimer’s disease risk and MAPT brain gene expression levels

    PubMed Central

    2014-01-01

    Introduction MAPT encodes for tau, the predominant component of neurofibrillary tangles that are neuropathological hallmarks of Alzheimer’s disease (AD). Genetic association of MAPT variants with late-onset AD (LOAD) risk has been inconsistent, although insufficient power and incomplete assessment of MAPT haplotypes may account for this. Methods We examined the association of MAPT haplotypes with LOAD risk in more than 20,000 subjects (n-cases = 9,814, n-controls = 11,550) from Mayo Clinic (n-cases = 2,052, n-controls = 3,406) and the Alzheimer’s Disease Genetics Consortium (ADGC, n-cases = 7,762, n-controls = 8,144). We also assessed associations with brain MAPT gene expression levels measured in the cerebellum (n = 197) and temporal cortex (n = 202) of LOAD subjects. Six single nucleotide polymorphisms (SNPs) which tag MAPT haplotypes with frequencies greater than 1% were evaluated. Results H2-haplotype tagging rs8070723-G allele associated with reduced risk of LOAD (odds ratio, OR = 0.90, 95% confidence interval, CI = 0.85-0.95, p = 5.2E-05) with consistent results in the Mayo (OR = 0.81, p = 7.0E-04) and ADGC (OR = 0.89, p = 1.26E-04) cohorts. rs3785883-A allele was also nominally significantly associated with LOAD risk (OR = 1.06, 95% CI = 1.01-1.13, p = 0.034). Haplotype analysis revealed significant global association with LOAD risk in the combined cohort (p = 0.033), with significant association of the H2 haplotype with reduced risk of LOAD as expected (p = 1.53E-04) and suggestive association with additional haplotypes. MAPT SNPs and haplotypes also associated with brain MAPT levels in the cerebellum and temporal cortex of AD subjects with the strongest associations observed for the H2 haplotype and reduced brain MAPT levels (β = -0.16 to -0.20, p = 1.0E-03 to 3.0E-03). Conclusions These results confirm the previously reported MAPT H2 associations with LOAD risk in two large series, that this haplotype has the strongest

  19. Vitamin D Levels Are Associated with Expression of SLE, but Not Flare Frequency.

    PubMed

    Squance, Marline L; Reeves, Glenn E M; Tran, Huy A

    2014-01-01

    This study explores links between vitamin D deficiency (25(OH)D = 50 nmol/L) and serological autoimmunity (ANA > 1 : 80) and frequency of self-reported flares (SRF) in participants with clinical autoimmunity (SLE). 25(OH)D levels of 121 females were quantified and compared. The cohort consisted of 80 ACR defined SLE patients and 41 age and sex matched controls. Association analysis of log2 (25(OH)D) levels and ANA 80 positivity was undertaken via two-sample t-tests and regression models. Significant differences were found for 25(OH)D levels (mean: control 74 nmol/L (29.5 ng/ml); SLE 58 nmol/L (23.1 ng/ml), P = 0.04), 25(OH)D deficiency (P = 0.02). Regression models indicate that, for a twofold rise in 25(OH)D level, the odds ratio (OR) for ANA-positivity drops to 36% of the baseline OR. No link was found between SRF-days and 25(OH)D levels. Our results support links between vitamin D deficiency and expression of serological autoimmunity and clinical autoimmunity (SLE). However, no demonstrable association between 25(OH)D and SRF was confirmed, suggesting independent influences of other flare-inducing factors. Results indicate that SLE patients have high risk of 25(OH)D deficiency and therefore supplementation with regular monitoring should be considered as part of patient management. PMID:25506363

  20. Vitamin D Levels Are Associated with Expression of SLE, but Not Flare Frequency

    PubMed Central

    Squance, Marline L.; Reeves, Glenn E. M.; Tran, Huy A.

    2014-01-01

    This study explores links between vitamin D deficiency (25(OH)D = 50 nmol/L) and serological autoimmunity (ANA > 1 : 80) and frequency of self-reported flares (SRF) in participants with clinical autoimmunity (SLE). 25(OH)D levels of 121 females were quantified and compared. The cohort consisted of 80 ACR defined SLE patients and 41 age and sex matched controls. Association analysis of log2 (25(OH)D) levels and ANA 80 positivity was undertaken via two-sample t-tests and regression models. Significant differences were found for 25(OH)D levels (mean: control 74 nmol/L (29.5 ng/ml); SLE 58 nmol/L (23.1 ng/ml), P = 0.04), 25(OH)D deficiency (P = 0.02). Regression models indicate that, for a twofold rise in 25(OH)D level, the odds ratio (OR) for ANA-positivity drops to 36% of the baseline OR. No link was found between SRF-days and 25(OH)D levels. Our results support links between vitamin D deficiency and expression of serological autoimmunity and clinical autoimmunity (SLE). However, no demonstrable association between 25(OH)D and SRF was confirmed, suggesting independent influences of other flare-inducing factors. Results indicate that SLE patients have high risk of 25(OH)D deficiency and therefore supplementation with regular monitoring should be considered as part of patient management. PMID:25506363

  1. Chronic stress alters the expression levels of longevity-related genes in the rat hippocampus.

    PubMed

    Sánchez-Hidalgo, Ana C; Muñoz, Mario F; Herrera, Antonio J; Espinosa-Oliva, Ana M; Stowell, Rianne; Ayala, Antonio; Machado, Alberto; Venero, José L; de Pablos, Rocío M

    2016-07-01

    The molecular mechanisms underlying the negative effects of psychological stress on cellular stress during aging and neurodegenerative diseases are poorly understood. The main objective of this study was to test the effect of chronic psychological stress, and the consequent increase of circulating glucocorticoids, on several hippocampal genes involved in longevity. Sirtuin-1, p53, thioredoxin-interacting protein, and heat shock protein 70 were studied at the mRNA and protein levels in stressed and non-stressed animals. Stress treatment for 10 days decreased sirtuin-1 and heat shock protein 70 levels, but increased levels of p53, thioredoxin-interacting protein and the NADPH oxidase enzyme. Examination of protein expression following two months of stress treatment indicated that sirtuin-1 remained depressed. In contrast, an increase was observed for thioredoxin-interacting protein, heat shock protein 70, p53 and the NADPH oxidase enzyme. The effect of stress was reversed by mifepristone, a glucocorticoid receptor antagonist. These data suggest that chronic stress could contribute to aging in the hippocampus. PMID:27120255

  2. Criteria for acceptable levels of the Shinkansen Super Express train noise and vibration in residential areas

    NASA Astrophysics Data System (ADS)

    Yamanaka, K.; Nakagawa, T.; Kobayashi, F.; Kanada, S.; Tanahashi, M.; Muramatsu, T.; Yamada, S.

    1982-10-01

    A survey of 1187 housewives living in 18 areas along the Shinkansen Super Express (bullet train) railway was conducted by means of a self-administered health questionnaire (modified Cornell Medical Index). In addition, geographically corresponding measurements of noise level and vibration intensity were taken. The relationship of noise and vibration to positive responses (health complaints) related to bodily symptoms, illness and emotional disturbances was analyzed. The factors which correlated with an increase in the average number of positive responses included noise, vibration, age and health status. Such factors as marital status, educational level, part time work, duration of inhabitancy and occupation of the head of the houshold correlated poorly with the number of positive responses. Unhealthy respondents compared to healthy respondents are more frequently affected by noise and vibration. The rate of positive responses in the visual, respiratory, cardiovascular, digestive and nervous systems, sleep disturbances and emotional disturbances increased accordingly as noise and vibration increased. Combined effects of noise and vibration stimuli on the total number of positive responses (an indicator of general health) were found. This study has produced results indicating that the maximum permissible noise level should not exceed 70 dB(A) in the residential areas along the Shinkansen railway.

  3. CHIP buffers heterogeneous Bcl-2 expression levels to prevent augmentation of anticancer drug-resistant cell population.

    PubMed

    Tsuchiya, M; Nakajima, Y; Waku, T; Hiyoshi, H; Morishita, T; Furumai, R; Hayashi, Y; Kishimoto, H; Kimura, K; Yanagisawa, J

    2015-08-27

    Many types of cancer display heterogeneity in various features, including gene expression and malignant potential. This heterogeneity is associated with drug resistance and cancer progression. Recent studies have shown that the expression of a major protein quality control ubiquitin ligase, carboxyl terminus of Hsc70-interacting protein (CHIP), is negatively correlated with breast cancer clinicopathological stages and poor overall survival. Here we show that CHIP acts as a capacitor of heterogeneous Bcl-2 expression levels and prevents an increase in the anticancer drug-resistant population in breast cancer cells. CHIP knockdown in breast cancer cells increased variation in Bcl-2 expression levels, an antiapoptotic protein, among the cells. Our results also showed that CHIP knockdown increased the proportion of anticancer drug-resistant cells. These findings suggest that CHIP buffers variation in gene expression levels, affecting resistance to anticancer drugs. In single-cell clones derived from breast cancer cell lines, CHIP knockdown did not alter the variation in Bcl-2 expression levels and the proportion of anticancer drug-resistant cells. In contrast, when clonal cells were treated with a mutagen, the variation in Bcl-2 expression levels and proportion of anticancer drug-resistant cells were altered by CHIP knockdown. These results suggest that CHIP masks genetic variations to suppress heterogeneous Bcl-2 expression levels and prevents augmentation of the anticancer drug-resistant population of breast cancer cells. Because genetic variation is a major driver of heterogeneity, our results suggest that the degree of heterogeneity in expression levels is decided by a balance between genetic variation and the buffering capacity of CHIP. PMID:25435366

  4. Identification of APC mutations and evaluation of their expression level using a functional screening assay

    SciTech Connect

    Varesco, L.; Gismondi, V.; Bafico, A.

    1994-09-01

    A functional screen for chain-terminating mutations in the APC gene recently has been developed. It is based on the PCR and cloning of a segment of the gene in-frame with a colorimetric marker gene (lacz) followed by screening for the level of activity of the marker polypeptide (beta-galactosidase). This method scores colony number with different blue colors that are produced by bacteria containing normal and mutant APC segments. In the present work this method was used to screen the entire APC coding region by using eight primer pairs. DNA segments with known APC mutations at different positions in the gene were used as controls and were clearly identifiable with this assay. In addition, the entire APC coding region has been examined in 21 APC patients in whom PCR-SSCP did not identify an APC mutation. Novel mutations (n=14) were identified by the blue/white assay and were all confirmed by sequence analysis. This method also was used to quantitate the expression of paternal and maternal APC alleles taking advantage of an RsaI site polymorphism at position 1458 in a small number of informative individuals. Differential expression of some known mutant APC mRNAs was observed.

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

    PubMed

    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. PMID:24334117

  6. High-level expression and characterization of two serine protease inhibitors from Trichinella spiralis.

    PubMed

    Zhang, Zhaoxia; Mao, Yixian; Li, Da; Zhang, Yvhan; Li, Wei; Jia, Honglin; Zheng, Jun; Li, Li; Lu, Yixin

    2016-03-30

    Serine protease inhibitors (SPIs) play important roles in tissue homeostasis, cell survival, development, and host defense. So far, SPIs have been identified from various organisms, such as animals, plants, bacteria, poxviruses, and parasites. In this study, two SPIs (Tsp03044 and TspAd5) were identified from the genome of Trichinella spiralis and expressed in Escherichia coli. Sequence analysis revealed that these two SPIs contained essential structural motifs, which were well conserved within the tumor-infiltrating lymphocytes (TIL) and serpin superfamily. Based on protease inhibition assays, the recombinant Tsp03044 showed inhibitory effects on trypsin, α-chymotrypsin, and pepsin, while the recombinant TspAd5 could effectively inhibit the activities of α-chymotrypsin and pepsin. Both these inhibitors showed activity between 28 and 48 °C. The expression levels of the two SPIs were also determined at different developmental stages of the parasite with real-time PCR. Our results indicate that Tsp03044 and TspAd5 are functional serine protease inhibitors. PMID:26921036

  7. Low-level laser therapy promotes dendrite growth via upregulating brain-derived neurotrophic factor expression

    NASA Astrophysics Data System (ADS)

    Meng, Chengbo; He, Zhiyong; Xing, Da

    2014-09-01

    Downregulation of brain-derived neurotrophic factor (BDNF) in the hippocampus occurs early in the progression of Alzheimer's disease (AD). Since BDNF plays a critical role in neuronal survival and dendrite growth, BDNF upregulation may contribute to rescue dendrite atrophy and cell loss in AD. Low-level laser therapy (LLLT) has been demonstrated to regulate neuronal function both in vitro and in vivo. In the present study, we found that LLLT rescued neurons loss and dendritic atrophy via the increase of both BDNF mRNA and protein expression. In addition, dendrite growth was improved after LLLT, characterized by upregulation of PSD95 expression, and the increase in length, branching, and spine density of dendrites in hippocampal neurons. Together, these studies suggest that upregulation of BDNF with LLLT can ameliorate Aβ-induced neurons loss and dendritic atrophy, thus identifying a novel pathway by which LLLT protects against Aβ-induced neurotoxicity. Our research may provide a feasible therapeutic approach to control the progression of Alzheimer's disease.

  8. Soft mean spherical approximation for dusty plasma liquids: Level of accuracy and analytic expressions

    SciTech Connect

    Tolias, P.; Ratynskaia, S.; Angelis, U. de

    2015-08-15

    The soft mean spherical approximation is employed for the study of the thermodynamics of dusty plasma liquids, the latter treated as Yukawa one-component plasmas. Within this integral theory method, the only input necessary for the calculation of the reduced excess energy stems from the solution of a single non-linear algebraic equation. Consequently, thermodynamic quantities can be routinely computed without the need to determine the pair correlation function or the structure factor. The level of accuracy of the approach is quantified after an extensive comparison with numerical simulation results. The approach is solved over a million times with input spanning the whole parameter space and reliable analytic expressions are obtained for the basic thermodynamic quantities.

  9. Do fasudil and Y-27632 affect the level of transient receptor potential (TRP) gene expressions in breast cancer cell lines?

    PubMed

    Gogebakan, Bulent; Bayraktar, Recep; Suner, Ali; Balakan, Ozan; Ulasli, Mustafa; Izmirli, Muzeyyen; Oztuzcu, Serdar; Camci, Celaletdin

    2014-08-01

    Breast cancer (BC) is the most frequent cancer type in women, and the mortality rate is high especially in metastatic disease. Ion channels such as the transient receptor potential (TRP) channels correlate with malignant growth and cancer progression. Hence, some authors have suggested that the expression levels of TRP channels may be used as a marker in the diagnosis and predicting the prognosis of BC. Also, in some recent studies, targeting TRP channels are suggested as a novel treatment strategy in BC. The aim of this study was to investigate the effect of two Rho-kinase (ROCK) inhibitors, fasudil and Y-27632, on the expression levels of TRP channel genes in breast cancer cell lines (ZR-75-1, MCF7, and MDA-MB-231) and breast epithelial cell line (hTERT-HME1). The expression levels of TRP genes were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). We found that fasudil had reduced the TRPC1, TRPV2 expression levels in the ZR-75-1, MCF7, and MDA-MB-231 cell lines. On the other hand, fasudil and Y-27632 had reduced TRPM6 expression levels in all cell lines. Y-27632 increased the expression levels of TRPC7 in all cell lines. In conclusion, this is the first study demonstrating that the inhibition of ROCK pathway changes the expression levels of some TRP genes. Also, our study has firstly shown that the expression levels of the TRP genes which are suggested as a diagnostic and prognostic biomarker in BC, were changed with the treatment of fasudil and Y-27632. PMID:24839003

  10. Baseline Chromatin Modification Levels May Predict Interindividual Variability in Ozone-Induced Gene Expression.

    PubMed

    McCullough, Shaun D; Bowers, Emma C; On, Doan M; Morgan, David S; Dailey, Lisa A; Hines, Ronald N; Devlin, Robert B; Diaz-Sanchez, David

    2016-03-01

    Traditional toxicological paradigms have relied on factors such as age, genotype, and disease status to explain variability in responsiveness to toxicant exposure; however, these are neither sufficient to faithfully identify differentially responsive individuals nor are they modifiable factors that can be leveraged to mitigate the exposure effects. Unlike these factors, the epigenome is dynamic and shaped by an individual's environment. We sought to determine whether baseline levels of specific chromatin modifications correlated with the interindividual variability in their ozone (O3)-mediated induction in an air-liquid interface model using primary human bronchial epithelial cells from a panel of 11 donors. We characterized the relationship between the baseline abundance of 6 epigenetic markers with established roles as key regulators of gene expression-histone H3 lysine 4 trimethylation (H3K4me3), H3K27 acetylation (H3K27ac), pan-acetyl H4 (H4ac), histone H3K27 di/trimethylation (H3K27me2/3), unmodified H3, and 5-hydroxymethylcytosine (5-hmC)-and the variability in the O3-induced expression of IL-8, IL-6, COX2, and HMOX1. Baseline levels of H3K4me3, H3K27me2/3, and 5-hmC, but not H3K27ac, H4ac, and total H3, correlated with the interindividual variability in O3-mediated induction of HMOX1 and COX2. In contrast, none of the chromatin modifications that we examined correlated with the induction of IL-8 and IL-6. From these findings, we propose an "epigenetic seed and soil" model in which chromatin modification states between individuals differ in the relative abundance of specific modifications (the "soil") that govern how receptive the gene is to toxicant-mediated cellular signals (the "seed") and thus regulate the magnitude of exposure-related gene induction. PMID:26719369

  11. Exposure to Bisphenol AF disrupts sex hormone levels and vitellogenin expression in zebrafish.

    PubMed

    Yang, Xiaoxi; Liu, Yuchen; Li, Jia; Chen, Minjie; Peng, Di; Liang, Yong; Song, Maoyong; Zhang, Jie; Jiang, Guibin

    2016-03-01

    Bisphenol AF (BPAF) is widely used in food-contact products, electronic devices, and as a cross-linking reagent in fluoroelastomers. There are growing concerns about its toxicity and endocrine-disrupting effects based on its structural similarity with bisphenol A (BPA). The endocrine-disrupting effects of BPAF were studied by exposing 2-month-old zebrafish to 0, 0.05, 0.25, or 1 mg/L BPAF for 28 days and evaluating the effect on growth, histopathology, hormone levels, enzyme activity, and gene expression. The overall fitness was not significantly affected. There were no apparent alterations in the gills and intestine tissues of both sexes after BPAF exposure. However, exposure to 1 mg/L BPAF caused damage to the liver in the male fish, characterized by hepatocellular swelling and vacuolation. There was no obvious effect in the liver of female fish, suggesting that the hepatic toxicity of BPAF is gender dependent. Gonadal examination indicated that exposure to 1 mg/L BPAF caused induction of acellular areas in the testis and retardation of oocyte development in the ovary. BPAF exposure increased free triiodothyronine levels of females in a dose-dependent manner. In males, the testosterone levels decreased in a concentration-dependent manner. In contrast, estradiol levels increased in a concentration-dependent manner and were significantly higher in males exposed to 1 mg/L BPAF compared with the controls. In females, 0.05 and 0.25 mg/L BPAF caused an increase in testosterone levels. Furthermore, the estradiol levels increased in females exposed to 0.05 and 1 mg/L. We observed an upregulation of hepatic vitellogenin in both sexes and significantly higher levels in males exposed to 1 mg/L BPAF and females exposed to 0.25 mg/L BPAF, suggesting that BPAF has an estrogenic activity. Our results indicate that BPAF is an endocrine-disrupting chemical that exerts reproductive toxicity and estrogenic effects on zebrafish. PMID:25213402

  12. FNDC5 expression and circulating irisin levels are modified by diet and hormonal conditions in hypothalamus, adipose tissue and muscle.

    PubMed

    Varela-Rodríguez, B M; Pena-Bello, L; Juiz-Valiña, P; Vidal-Bretal, B; Cordido, F; Sangiao-Alvarellos, S

    2016-01-01

    Irisin is processed from fibronectin type III domain-containing protein 5 (FNDC5). However, a controversy exists concerning irisin origin, regulation and function. To elucidate the relationship between serum irisin and FNDC5 mRNA expression levels, we evaluated plasma irisin levels and FNDC5 gene expression in the hypothalamus, gastrocnemius muscle and different depots of adipose tissue in models of altered metabolism. In normal rats, blood irisin levels diminished after 48-h fast and with leptin, insulin and alloxan treatments, and serum irisin concentrations increased in diabetic rats after insulin treatment and acute treatments of irisin increased blood insulin levels. No changes were observed during long-term experiments with different diets. We suggested that levels of circulating irisin are the result of the sum of the irisin produced by different depots of adipose tissue and skeletal muscle. This study shows for the first time that there are differences in FNDC5 expression depending on white adipose tissue depots. Moreover, a considerable decrease in visceral and epididymal adipose tissue depots correlated with increased FNDC5 mRNA expression levels, probably in an attempt to compensate the decrease that occurs in their mass. Hypothalamic FNDC5 expression did not change for any of the tested diets but increased with leptin, insulin and metformin treatments suggesting that the regulation of central and peripheral FNDC5/irisin expression and functions are different. PMID:27432282

  13. FNDC5 expression and circulating irisin levels are modified by diet and hormonal conditions in hypothalamus, adipose tissue and muscle

    PubMed Central

    Varela-Rodríguez, B. M.; Pena-Bello, L.; Juiz-Valiña, P.; Vidal-Bretal, B.; Cordido, F.; Sangiao-Alvarellos, S.

    2016-01-01

    Irisin is processed from fibronectin type III domain-containing protein 5 (FNDC5). However, a controversy exists concerning irisin origin, regulation and function. To elucidate the relationship between serum irisin and FNDC5 mRNA expression levels, we evaluated plasma irisin levels and FNDC5 gene expression in the hypothalamus, gastrocnemius muscle and different depots of adipose tissue in models of altered metabolism. In normal rats, blood irisin levels diminished after 48-h fast and with leptin, insulin and alloxan treatments, and serum irisin concentrations increased in diabetic rats after insulin treatment and acute treatments of irisin increased blood insulin levels. No changes were observed during long-term experiments with different diets. We suggested that levels of circulating irisin are the result of the sum of the irisin produced by different depots of adipose tissue and skeletal muscle. This study shows for the first time that there are differences in FNDC5 expression depending on white adipose tissue depots. Moreover, a considerable decrease in visceral and epididymal adipose tissue depots correlated with increased FNDC5 mRNA expression levels, probably in an attempt to compensate the decrease that occurs in their mass. Hypothalamic FNDC5 expression did not change for any of the tested diets but increased with leptin, insulin and metformin treatments suggesting that the regulation of central and peripheral FNDC5/irisin expression and functions are different. PMID:27432282

  14. High-level expression, purification, and characterization of bifunctional ScFv-9R fusion protein.

    PubMed

    Zhang, Xiguang; Xie, Jiasen; Sun, Yan; Xu, Huijing; Du, Tonghua; Liu, Zixuan; Chen, Jinhui; Zheng, Zhong; Liu, Keqiang; Zhang, Jizhou; Kan, Mujie; Li, Xiaokun; Xiao, Yechen

    2014-06-01

    Fibroblast growth factor receptor 3 (FGFR3) is a noted proto-oncogene involved in the pathogenesis of many tumors, so more and more studies focus on the potential use of receptor kinase inhibitor and therapeutic antibodies against FGFR3. In this study, we designed a novel fusion protein containing the single-chain Fv (ScFv) against FGFR3 and 9-arginine, denoted as ScFv-9R. To achieve the high-level production and soluble expression, ScFv and ScFv-9R were fused with small ubiquitin-related modifier (Sumo) by polymerase chain reaction and expressed in Escherichia coli BL21 (DE3). The recombinant bacteria was induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside for 20 h at 20 °C; supernatants of Sumo-ScFv was harvested and purified by DEAE Sepharose FF and Ni-NTA orderly, and supernatants of Sumo-ScFv-9R was harvested and purified by Ni-NTA. After cleaved by the Sumo protease, the recombinant ScFv or ScFv-9R was released from the fusion protein, respectively. The purity of ScFv or ScFV-9R was shown to be higher than 90 %, and their yield reached 3-5 mg per liter of bacterial culture. In vitro data showed that ScFV-9R can attenuate the phosphorylation of FGFR3 and ERK in the absence or presence of FGF9. Gel retardation assay showed that 1 μg of ScFv-9R could efficiently bind to about 4 pmol siRNA. Fluorescent microscope analysis showed that ScFv-9R can efficiently bind and deliver siRNA into RT112 cells. In conclusion, we use Sumo fusion system to acquire high-level production, soluble expression, and bifunctional activity of ScFv-9R in E. coli. Our results also revealed that ScFv-9R, as a novel carrier, may have potential applications in antitumor studies and pharmaceutical development. PMID:24519456

  15. Diabetic retinopathy alters light-induced clock gene expression and dopamine levels in the mouse retina

    PubMed Central

    Lahouaoui, Hasna; Coutanson, Christine; Cooper, Howard M.; Bennis, Mohamed

    2016-01-01

    Purpose Diabetic retinopathy is one of the most common consequences of diabetes that affects millions of working-age adults worldwide and leads to progressive degeneration of the retina, visual loss, and blindness. Diabetes is associated with circadian disruption of the central and peripheral circadian clocks, but the mechanisms responsible for such alterations are unknown. Using a streptozotocin (STZ)-induced model of diabetes, we investigated whether diabetes alters 1) the circadian regulation of clock genes in the retina and in the central clocks, 2) the light response of clock genes in the retina, and/or 3) light-driven retinal dopamine (DA), a major output marker of the retinal clock. Methods To quantify circadian expression of clock and clock-controlled genes, retinas and suprachiasmatic nucleus (SCN) from the same animals were collected every 4 h in circadian conditions, 12 weeks post-diabetes. Induction of Per1, Per2, and c-fos mRNAs was quantified in the retina after the administration of a pulse of monochromatic light (480 nm, 1.17×1014 photons/cm2/s, 15 min) at circadian time 16. Gene expression was assessed with real-time reverse transcription PCR (RT–PCR). Pooled retinas from the control and STZ-diabetic mice were collected 2 h after light ON and light OFF (Zeitgeber time (ZT)2 and ZT14), and DA and its metabolite were analyzed with high-performance liquid chromatography (HPLC). Results We found variable effects of diabetes on the expression of clock genes in the retina and only slight differences in phase and/or amplitude in the SCN. c-fos and Per1 induction by a 480 nm light pulse was abolished in diabetic animals at 12 weeks post-induction of diabetes in comparison with the control mice, suggesting a deficit in light-induced neuronal activation of the retinal clock. Finally, we quantified a 56% reduction in the total number of tyrosine hydroxylase (TH) immunopositive cells, associated with a decrease in DA levels during the subjective day (ZT2

  16. MMP-2/MMP-9 plasma level and brain expression in cerebral amyloid angiopathy-associated hemorrhagic stroke.

    PubMed

    Hernandez-Guillamon, Mar; Martinez-Saez, Elena; Delgado, Pilar; Domingues-Montanari, Sophie; Boada, Cristina; Penalba, Anna; Boada, Mercè; Pagola, Jorge; Maisterra, Olga; Rodriguez-Luna, David; Molina, Carlos A; Rovira, Alex; Alvarez-Sabin, José; Ortega-Aznar, Arantxa; Montaner, Joan

    2012-03-01

    Cerebral amyloid angiopathy (CAA) is one of the main causes of intracerebral hemorrhage (ICH) in the elderly. Matrix metalloproteinases (MMPs) have been implicated in blood-brain barrier disruption and ICH pathogenesis. In this study, we determined the levels MMP-2 and MMP-9 in plasma and their brain expression in CAA-associated hemorrhagic stroke. Although MMP-2 and MMP-9 plasma levels did not differ among patients and controls, their brain expression was increased in perihematoma areas of CAA-related hemorrhagic strokes compared with contralateral areas and nonhemorrhagic brains. In addition, MMP-2 reactivity was found in β-amyloid (Aβ)-damaged vessels located far from the acute ICH and in chronic microbleeds. MMP-2 expression was associated to endothelial cells, histiocytes and reactive astrocytes, whereas MMP-9 expression was restricted to inflammatory cells. In summary, MMP-2 expression within and around Aβ-compromised vessels might contribute to the vasculature fatal fate, triggering an eventual bleeding. PMID:21707819

  17. Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration.

    PubMed

    Verburg, Melissa; Renes, Ingrid B; Van Nispen, Danielle J P M; Ferdinandusse, Sacha; Jorritsma, Marieke; Büller, Hans A; Einerhand, Alexandra W C; Dekker, Jan

    2002-11-01

    The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (-76%) and SGLT1 (-77%) and between I-FABP (-52%) and L-FABP (-45%). Decreases in GLUT5 (-53%), MUC2 (-43%), and TFF3 (-54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected. PMID:12417619

  18. Expression Profile of Antioxidant Enzymes in Hemocytes from Freshwater Prawn Macrobrachium rosenbergii Exposed to an Elevated Level of Copper.

    PubMed

    Guo, Hui; Miao, Yu-Tao; Xian, Jian-An; Qian, Kun; Wang, An-Li

    2015-10-01

    This study evaluated the expression level of antioxidant enzymes Cu, Zn-superoxide dismutase (Cu, Zn-SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) in hemocytes of Macrobrachium rosenbergii exposed to copper by real-time PCR (qRT-PCR). Results showed that the mRNA expression of Cu, Zn-SOD increased to reach a peak at 6 h, then recovered to its normal level at 48 h. CAT expression level was significantly increased at 12 h and reached a peak at 24 h, but recovered to its normal level later. GPx expression level was significantly increased at 6 h and reached the peak at 12 h. GST expression level was significantly induced from 12 to 24 h and then dropped to its normal level at 48 h. These results indicated that antioxidant enzymes were inducible, possibly for removing excessive reactive oxygen species to protect prawn from oxidative stress. PMID:26215459

  19. Molecular Basis for Different Levels of tet(M) Expression in Streptococcus pneumoniae Clinical Isolates

    PubMed Central

    Grohs, Patrick; Trieu-Cuot, Patrick; Podglajen, Isabelle; Grondin, Sophie; Firon, Arnaud; Poyart, Claire; Varon, Emmanuelle

    2012-01-01

    Seventy-four unrelated clinical isolates of Streptococcus pneumoniae harboring the tet(M) gene were studied. Seven strains with low tetracycline (Tc) MICs (0.25 to 0.5 μg/ml) were found to harbor truncated tet(M) alleles that were inactivated by different frameshift mutations. In contrast, five strains bore deletions in the tet(M) promoter region, among which four displayed increased Tc MICs (16 to 64 μg/ml). The same promoter mutations were detected in Tc-resistant mutants selected in vitro from various susceptible strains. Sequence analysis revealed that these deletions might impede the formation of the transcriptional attenuator located immediately upstream of tet(M). Expression in Enterococcus faecalis of a tet(M) reporter gene transcribed from these promoter mutants conferred a level of Tc resistance similar to that observed in the parental S. pneumoniae strains. These results show that different levels of Tc susceptibility found in clinical isolates of S. pneumoniae can be explained by frameshift mutations within tet(M) and by alterations of the upstream transcriptional attenuator. PMID:22802249

  20. BA11 FKBP5 expression levels correlate with dendritic spine density in postmortem PTSD and controls

    PubMed Central

    Young, Keith A.; Thompson, Peter M.; Cruz, Dianne A.; Williamson, Douglas E.; Selemon, Lynn D.

    2015-01-01

    Genetic variants of the immunophilin FKBP5 have been implicated in susceptibility to post-traumatic stress disorder (PTSD) and other stress-related disorders. We examined the relationship between mushroom, stubby, thin and filopodial spine densities measured with Golgi staining and FKBP5 gene expression in the medial orbitofrontal cortex (BA11) in individuals diagnosed with PTSD and normal controls (n = 8/8). ANCOVA revealed PTSD cases had a significantly elevated density of stubby spines (29%, P < 0.037) and a trend for a reduction in mushroom spine density (25%, p < 0.082). Levels of FKBP5 mRNA were marginally elevated in the PTSD cases (z = 1.94, p = 0.053) and levels correlated inversely with mushroom (Spearman's rho = −0.83, p < 0.001) and overall spine density (rho = −0.75, p < 0.002) and directly with stubby spine density (rho = 0.55, p < 0.027). These data suggest that FKBP5 may participate in a cellular pathway modulating neuronal spine density changes in the brain, and that this pathway may be dysregulated in PTSD. PMID:26844242

  1. Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels.

    PubMed

    Yelin, Ronit; Schyr, Racheli Ben-Haroush; Kot, Hadas; Zins, Sharon; Frumkin, Ayala; Pillemer, Graciela; Fainsod, Abraham

    2005-03-01

    Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental malformations caused by alcohol consumption during pregnancy. Fetal Alcohol Syndrome (FAS), the strongest manifestation of FASD, results in short stature, microcephally and facial dysmorphogenesis including microphthalmia. Using Xenopus embryos as a model developmental system, we show that ethanol exposure recapitulates many aspects of FAS, including a shortened rostro-caudal axis, microcephally and microphthalmia. Temporal analysis revealed that Xenopus embryos are most sensitive to ethanol exposure between late blastula and early/mid gastrula stages. This window of sensitivity overlaps with the formation and early function of the embryonic organizer, Spemann's organizer. Molecular analysis revealed that ethanol exposure of embryos induces changes in the domains and levels of organizer-specific gene expression, identifying Spemann's organizer as an early target of ethanol. Ethanol also induces a defect in convergent extension movements that delays gastrulation movements and may affect the overall length. We show that mechanistically, ethanol is antagonistic to retinol (Vitamin A) and retinal conversion to retinoic acid, and that the organizer is active in retinoic acid signaling during early gastrulation. The model suggests that FASD is induced in part by an ethanol-dependent reduction in retinoic acid levels that are necessary for the normal function of Spemann's organizer. PMID:15708568

  2. High levels of EGFR expression in tumor stroma are associated with aggressive clinical features in epithelial ovarian cancer

    PubMed Central

    Wang, Ke; Li, Dan; Sun, Lu

    2016-01-01

    Purpose The aim of this study was to investigate the clinical significance and biological function of epidermal growth factor receptor (EGFR) expressed in tumor stroma of epithelial ovarian cancer. Methods Immunohistological staining of EGFR was evaluated in 242 patients with epithelial ovarian cancer. The correlations of EGFR expression in tumor stroma with clinicopathological features and with the expression level of Ki-67 were analyzed by SPSS software. Kaplan–Meier analysis and the Cox proportional hazard model were used to analyze the effect of EGFR expression in tumor stroma on the prognosis of patients with epithelial ovarian cancer. Meanwhile, the activities of proliferation and migration of tumor cells were detected when EGFR overexpressed in stroma cells. Results EGFR expression in tumor stroma correlated significantly with clinical stage (χ2=7.002, P=0.008) and distant metastases (χ2=16.59, P<0.001). Furthermore, there was a significantly positive correlation between the level of EGFR expressed in tumor stroma and the level of Ki-67 expressed in tumor cells (χ2=6.120, P=0.013). Patients with high EGFR expression level in tumor stroma showed poor survival (P=0.002). Multivariate analysis showed that high expression of EGFR in tumor stroma was an independent predictor for epithelial ovarian cancer patients (hazard ratio =1.703; 95% confidence interval 1.125–2.578, P=0.012). Furthermore, stroma cells overexpressing EGFR could promote the proliferation and migration of adjacent tumor cells. Conclusion High expression of EGFR in tumor stroma correlates with aggressive clinical features in epithelial ovarian cancer, and is an independent prognostic factor. PMID:26855586

  3. Sustained high level transgene expression in mammalian cells mediated by the optimized piggyBac transposon system

    PubMed Central

    Chen, Xiang; Cui, Jing; Yan, Zhengjian; Zhang, Hongmei; Chen, Xian; Wang, Ning; Shah, Palak; Deng, Fang; Zhao, Chen; Geng, Nisha; Li, Melissa; Denduluri, Sahitya K.; Haydon, Rex C.; Luu, Hue H.; Reid, Russell R.; He, Tong-Chuan

    2015-01-01

    Sustained, high level transgene expression in mammalian cells, especially stem cells, may be desired in many cases for studying gene functions. Traditionally, stable transgene expression has been accomplished by using retroviral or lentiviral vectors. However, such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression. The piggyBac transposon has emerged as a promising non-viral vector system for efficient gene transfer into mammalian cells. Despite its inherent advantages over lentiviral and retroviral systems, piggyBac system has not been widely used, at least in part due to the limited availability of piggyBac vectors with manipulation flexibilities. Here, we seek to optimize piggyBac-mediated transgene expression and generate a more efficient, user-friendly piggyBac system. By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase (PBase), we demonstrate that adenovirus-mediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells, compared to that obtained from co-transfection of the CMV-PBase plasmid. We further determine the drug selection timeline to achieve optimal stable transgene expression. Moreover, we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors. Using the engineered tandem expression vector, we show that three transgenes can be simultaneously expressed in a single vector with high efficiency. Thus, these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained, high transgene expression. PMID:25815368

  4. Use of heat stress responsive gene expression levels for early selection of heat tolerant cabbage (Brassica oleracea L.).

    PubMed

    Park, Hyun Ji; Jung, Won Yong; Lee, Sang Sook; Song, Jun Ho; Kwon, Suk-Yoon; Kim, Hyeran; Kim, Chulwook; Ahn, Jun Cheul; Cho, Hye Sun

    2013-01-01

    Cabbage is a relatively robust vegetable at low temperatures. However, at high temperatures, cabbage has disadvantages, such as reduced disease tolerance and lower yields. Thus, selection of heat-tolerant cabbage is an important goal in cabbage breeding. Easier or faster selection of superior varieties of cabbage, which are tolerant to heat and disease and have improved taste and quality, can be achieved with molecular and biological methods. We compared heat-responsive gene expression between a heat-tolerant cabbage line (HTCL), "HO", and a heat-sensitive cabbage line (HSCL), "JK", by Genechip assay. Expression levels of specific heat stress-related genes were increased in response to high-temperature stress, according to Genechip assays. We performed quantitative RT-PCR (qRT-PCR) to compare expression levels of these heat stress-related genes in four HTCLs and four HSCLs. Transcript levels for heat shock protein BoHsp70 and transcription factor BoGRAS (SCL13) were more strongly expressed only in all HTCLs compared to all HSCLs, showing much lower level expressions at the young plant stage under heat stress (HS). Thus, we suggest that expression levels of these genes may be early selection markers for HTCLs in cabbage breeding. In addition, several genes that are involved in the secondary metabolite pathway were differentially regulated in HTCL and HSCL exposed to heat stress. PMID:23736694

  5. Role of serum TRAIL level and TRAIL apoptosis gene expression in multiple sclerosis and relation to brain atrophy.

    PubMed

    Tawdy, Mohamed H; Abd El Nasser, Maged M; Abd El Shafy, Sanaa S; Nada, Mona A F; El Sirafy, Mohamed Nasr I; Magd, Amany Hussien Abol

    2014-09-01

    One of the presumed pathological mechanisms of multiple sclerosis (MS) is the failure of apoptosis of autoreactive T lymphocytes. This study aimed to determine the relationship of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA gene expression ratio and serum TRAIL levels with MS and brain atrophy. This study was conducted on 53 relapsing-remitting Egyptian MS patients and 25 matched healthy volunteers. The expression of TRAIL in peripheral blood lymphocytes was analyzed by reverse transcription polymerase chain reaction, serum levels of soluble TRAIL (sTRAIL) were determined by enzyme-linked immunosorbent assay and brain MRI measured "black holes" and the bicaudate ratio as a measure of brain atrophy in all patients. The serum TRAIL level was lower in MS patients compared to controls but no difference was seen in the TRAIL mRNA gene expression ratio. No significant correlation was detected between the serum TRAIL level and the TRAIL mRNA expression ratio in either group. No statistically significant correlation was found between serum TRAIL levels or the TRAIL mRNA expression ratio with the number of black holes or the bicaudate ratio on MRI. Apoptosis of T lymphocytes is decreased in MS patients, which could be useful when designing treatments. There was no difference in the TRAIL mRNA gene expression ratio between MS patients and controls. PMID:24913933

  6. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level.

    PubMed

    Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-06-01

    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

  7. Circulating IGF1 regulates hippocampal IGF1 levels and brain gene expression during adolescence

    PubMed Central

    Yan, Han; Mitschelen, Matthew; Bixler, Georgina V; Brucklacher, Robert M; Farley, Julie A; Han, Song; Freeman, Willard M; Sonntag, William E

    2012-01-01

    GH and its anabolic mediator, IGF1, are important not only in somatic growth but also in the regulation of brain function. Even though GH treatment has been used clinically to improve body composition and exercise capacity in adults, its influence on central nervous system function has only recently been recognized. This is also the case for children with childhood-onset GH deficiency (GHD) where GH has been used to stimulate bone growth and enhance final adult height. Circulating IGF1 is transported across the blood–brain barrier and IGF1 and its receptors are also synthesized in the brain by neurons and glial and endothelial cells. Nevertheless, the relationship between circulating IGF1 and brain IGF1 remains unclear. This study, using a GH-deficient dwarf rat model and peripheral GH replacement, investigated the effects of circulating IGF1 during adolescence on IGF1 levels in the brain. Our results demonstrated that hippocampal IGF1 protein concentrations during adolescence are highly regulated by circulating IGF1, which were reduced by GHD and restored by systematic GH replacement. Importantly, IGF1 levels in the cerebrospinal fluid were decreased by GHD but not restored by GH replacement. Furthermore, analysis of gene expression using microarrays and RT-PCR indicated that circulating IGF1 levels did not modify the transcription of Igf1 or its receptor in the hippocampus but did regulate genes that are involved in microvascular structure and function, brain development, and synaptic plasticity, which potentially support brain structures involved in cognitive function during this important developmental period. PMID:21750148

  8. Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

    PubMed Central

    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

  9. Regulatory mechanisms, expression levels and proliferation effects of the FUS-DDIT3 fusion oncogene in liposarcoma.

    PubMed

    Åman, Pierre; Dolatabadi, Soheila; Svec, David; Jonasson, Emma; Safavi, Setareh; Andersson, Daniel; Grundevik, Pernilla; Thomsen, Christer; Ståhlberg, Anders

    2016-04-01

    Fusion oncogenes are among the most common types of oncogene in human cancers. The gene rearrangements result in new combinations of regulatory elements and functional protein domains. Here we studied a subgroup of sarcomas and leukaemias characterized by the FET (FUS, EWSR1, TAF15) family of fusion oncogenes, including FUS-DDIT3 in myxoid liposarcoma (MLS). We investigated the regulatory mechanisms, expression levels and effects of FUS-DDIT3 in detail. FUS-DDIT3 showed a lower expression than normal FUS at both the mRNA and protein levels, and single-cell analysis revealed a lack of correlation between FUS-DDIT3 and FUS expression. FUS-DDIT3 transcription was regulated by the FUS promotor, while its mRNA stability depended on the DDIT3 sequence. FUS-DDIT3 protein stability was regulated by protein interactions through the FUS part, rather than the leucine zipper containing DDIT3 part. In addition, in vitro as well as in vivo FUS-DDIT3 protein expression data displayed highly variable expression levels between individual MLS cells. Combined mRNA and protein analyses at the single-cell level showed that FUS-DDIT3 protein expression was inversely correlated to the expression of cell proliferation-associated genes. We concluded that FUS-DDIT3 is uniquely regulated at the transcriptional as well as the post-translational level and that its expression level is important for MLS tumour development. The FET fusion oncogenes are potentially powerful drug targets and detailed knowledge about their regulation and functions may help in the development of novel treatments. PMID:26865464

  10. High expression level of levansucrase from Bacillus licheniformis RN-01 and synthesis of levan nanoparticles.

    PubMed

    Nakapong, Santhana; Pichyangkura, Rath; Ito, Kazuo; Iizuka, Masaru; Pongsawasdi, Piamsook

    2013-03-01

    LsRN from Bacillus licheniformis was cloned and expressed in Escherichia coli. From a 1793 bp genomic sequence, the lsRN gene was found to be composed of a single 1446 bp ORF with a putative promoter consensus boxes and a ribosome-binding site. This ORF was predicted to encode for 482 amino acid residues. The LsRN was constitutively expressed at a relatively high level without sucrose induction. The enzyme was highly purified and an apparent size of 52 kDa with an optimum temperature and pH of 50 °C and 6.0 were determined. The wide range of M(w) of levan (1-600 kDa) was synthesized in a controlled reaction with two variable parameters: temperature and ionic strength. At high temperature (50 °C), LsRN synthesized high M(w) levan (612 kDa) as a major product while at low temperature (30 °C), low M(w) levan (11 kDa) was mainly synthesized. When 0.5M NaCl was added into the reaction, the major products at both temperatures were of the size 11 kDa. Moreover we report for the first time, an enzymatic synthesis of levan nanoparticles (NPs) by a single step reaction. The LsRN synthesized levan NPs as agglomerate with average particle size of 50 nm. The encapsulation of O-acetyl-α-tocopherol was carried out to demonstrate the applicable use of levan NPs. PMID:23219733

  11. Comparison of IgE expression at the mRNA and protein levels in vitro.

    PubMed Central

    Turner, K J; Creany, J; Coelen, R J; Cameron, K J; Holt, B J; Beilharz, M W

    1991-01-01

    The regulating effects of IL-4 and pokeweed mitogen on IgE synthesis in vitro by human peripheral blood leucocytes has been compared with the corresponding effect of these regulators on the expression of IgE mRNA. The latter was measured by dot blot hybridization with an oligonucleotide coding for a unique six amino acid region of the CH epsilon 2 domain. Specificity of the oligonucleotide probe was established by its inability to hybridize with RNA extracted from HMY-2 (IgG) and XQ-15 (IgM) secreting cell lines whilst producing intense signals with RNA extracted from the IgE secreting cell line U266. Whilst IgE mRNA was detected in RNA extracted from PBL of both atopic and control subjects, spontaneous IgE synthesis was restricted to atopic PBL. IL-4 increased both IgE mRNA and IgE synthesis in all PBL samples but PWM, while significantly increasing IgE mRNA expression either failed to modify IgE synthesis or actively suppressed it. The assay system employed to quantitate IgE synthesis in vitro was shown to be inhibited by both IgE binding factors and IgG anti-IgE autoantibodies which are produced in PBL cultures. IgE mRNA levels might therefore more accurately monitor the regulatory effects of IL-4 and PWM on IgE synthesis than quantitation of the IgE by radioimmunoassay. Images Figure 1 PMID:1783428

  12. Tumor Vascular Permeability to a Nanoprobe Correlates to Tumor-Specific Expression Levels of Angiogenic Markers

    PubMed Central

    Karathanasis, Efstathios; Chan, Leslie; Karumbaiah, Lohitash; McNeeley, Kathleen; D'Orsi, Carl J.; Annapragada, Ananth V.; Sechopoulos, Ioannis; Bellamkonda, Ravi V.

    2009-01-01

    Background Vascular endothelial growth factor (VEGF) receptor-2 is the major mediator of the mitogenic, angiogenic, and vascular hyperpermeability effects of VEGF on breast tumors. Overexpression of VEGF and VEGF receptor-2 is associated with the degree of pathomorphosis of the tumor tissue and unfavorable prognosis. In this study, we demonstrate that non-invasive quantification of the degree of tumor vascular permeability to a nanoprobe correlates with the VEGF and its receptor levels and tumor growth. Methodology/Principal Findings We designed an imaging nanoprobe and a methodology to detect the intratumoral deposition of a 100 nm-scale nanoprobe using mammography allowing measurement of the tumor vascular permeability in a rat MAT B III breast tumor model. The tumor vascular permeability varied widely among the animals. Notably, the VEGF and VEGF receptor-2 gene expression of the tumors as measured by qRT-PCR displayed a strong correlation to the imaging-based measurements of vascular permeability to the 100 nm-scale nanoprobe. This is in good agreement with the fact that tumors with high angiogenic activity are expected to have more permeable blood vessels resulting in high intratumoral deposition of a nanoscale agent. In addition, we show that higher intratumoral deposition of the nanoprobe as imaged with mammography correlated to a faster tumor growth rate. This data suggest that vascular permeability scales to the tumor growth and that tumor vascular permeability can be a measure of underlying VEGF and VEGF receptor-2 expression in individual tumors. Conclusions/Significance This is the first demonstration, to our knowledge, that quantitative imaging of tumor vascular permeability to a nanoprobe represents a form of a surrogate, functional biomarker of underlying molecular markers of angiogenesis. PMID:19513111

  13. Expression levels of JNK associated with polymorphic lactotransferrin haplotypes in human nasopharyngeal carcinoma

    PubMed Central

    Luo, Gengqiu; Zhou, Yanhong; Yi, Wei; Yi, Hong

    2016-01-01

    Lactotransferrin (LTF), a member of the transferrin family, serves a role in the innate immune response and is involved in anti-inflammatory, anti-microbial and anti-tumor activity. Alterations in the LTF gene are associated with an increased incidence of cancer. The LTF gene is polymorphic, and several common alleles may be observed in the general population. Our previous study identified a lower rate of occurrence of the ‘A-G-G-T’ haplotype (constructed with rs1126477, rs1126478, rs2073495 and rs9110) in nasopharyngeal carcinoma (NPC) patients compared with controls. In the present study, in order to elucidate a possible mechanism of LTF-mediated anti-tumor activity in NPC, the protein profiles of NPC and non-tumorous nasopharyngeal epithelium tissues with/without the ‘A-G-G-T’ haplotype were constructed using LTQ Orbitrap technology. The results revealed that c-Jun N-terminal kinase 2 (JNK2) was highly expressed in NPC tissues and non-tumor nasopharyngeal epithelium tissues without the ‘A-G-G-T’ haplotype. These results were confirmed by western blot analysis. Furthermore, microRNA (miRNA) microarray analysis was conducted to investigate the differential miRNA profiles of NPC and non-tumor nasopharyngeal epithelium tissues with/without the ‘A-G-G-T’ haplotype. It was observed that hsa-miR-1256 and hsa-miR-659, which are potentially targeted to the JNK2 gene, were downregulated in NPC tissues without the ‘A-G-G-T’ haplotype. Hsa-miR-298, another miRNA potentially targeted to the JNK2 gene, was downregulated in non-tumor nasopharyngeal epithelium tissues without the ‘A-G-G-T’ haplotype. In summary, these results suggested that the expression levels of JNK2 may be associated with polymorphic LTF haplotypes in human NPC. PMID:27446399

  14. High Levels of Expression of P-glycoprotein/Multidrug Resistance Protein Result in Resistance to Vintafolide.

    PubMed

    Guertin, Amy D; O'Neil, Jennifer; Stoeck, Alexander; Reddy, Joseph A; Cristescu, Razvan; Haines, Brian B; Hinton, Marlene C; Dorton, Ryan; Bloomfield, Alicia; Nelson, Melissa; Vetzel, Marilynn; Lejnine, Serguei; Nebozhyn, Michael; Zhang, Theresa; Loboda, Andrey; Picard, Kristen L; Schmidt, Emmett V; Dussault, Isabelle; Leamon, Christopher P

    2016-08-01

    Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR. PMID:27256377

  15. The expression of pre- and postcopulatory sexually selected traits reflects levels of dietary stress in guppies.

    PubMed

    Rahman, Md Moshiur; Turchini, Giovanni M; Gasparini, Clelia; Norambuena, Fernando; Evans, Jonathan P

    2014-01-01

    Environmental and ecological conditions can shape the evolution of life history traits in many animals. Among such factors, food or nutrition availability can play an important evolutionary role in moderating an animal's life history traits, particularly sexually selected traits. Here, we test whether diet quantity and/or composition in the form of omega-3 long chain polyunsaturated fatty acids (here termed 'n3LC') influence the expression of pre- and postcopulatory traits in the guppy (Poecilia reticulata), a livebearing poeciliid fish. We assigned males haphazardly to one of two experimental diets supplemented with n3LC, and each of these diet treatments was further divided into two diet 'quantity' treatments. Our experimental design therefore explored the main and interacting effects of two factors (n3LC content and diet quantity) on the expression of precopulatory (sexual behaviour and sexual ornamentation, including the size, number and spectral properties of colour spots) and postcopulatory (the velocity, viability, number and length of sperm) sexually selected traits. Our study revealed that diet quantity had significant effects on most of the pre- and postcopulatory traits, while n3LC manipulation had a significant effect on sperm traits and in particular on sperm viability. Our analyses also revealed interacting effects of diet quantity and n3LC levels on courtship displays, and the area of orange and iridescent colour spots in the males' colour patterns. We also confirmed that our dietary manipulations of n3LC resulted in the differential uptake of n3LC in body and testes tissues in the different n3LC groups. This study reveals the effects of diet quantity and n3LC on behavioural, ornamental and ejaculate traits in P. reticulata and underscores the likely role that diet plays in maintaining the high variability in these condition-dependent sexual traits. PMID:25170940

  16. The Expression of Pre- and Postcopulatory Sexually Selected Traits Reflects Levels of Dietary Stress in Guppies

    PubMed Central

    Rahman, Md. Moshiur; Turchini, Giovanni M.; Gasparini, Clelia; Norambuena, Fernando; Evans, Jonathan P.

    2014-01-01

    Environmental and ecological conditions can shape the evolution of life history traits in many animals. Among such factors, food or nutrition availability can play an important evolutionary role in moderating an animal's life history traits, particularly sexually selected traits. Here, we test whether diet quantity and/or composition in the form of omega-3 long chain polyunsaturated fatty acids (here termed ‘n3LC’) influence the expression of pre- and postcopulatory traits in the guppy (Poecilia reticulata), a livebearing poeciliid fish. We assigned males haphazardly to one of two experimental diets supplemented with n3LC, and each of these diet treatments was further divided into two diet ‘quantity’ treatments. Our experimental design therefore explored the main and interacting effects of two factors (n3LC content and diet quantity) on the expression of precopulatory (sexual behaviour and sexual ornamentation, including the size, number and spectral properties of colour spots) and postcopulatory (the velocity, viability, number and length of sperm) sexually selected traits. Our study revealed that diet quantity had significant effects on most of the pre- and postcopulatory traits, while n3LC manipulation had a significant effect on sperm traits and in particular on sperm viability. Our analyses also revealed interacting effects of diet quantity and n3LC levels on courtship displays, and the area of orange and iridescent colour spots in the males’ colour patterns. We also confirmed that our dietary manipulations of n3LC resulted in the differential uptake of n3LC in body and testes tissues in the different n3LC groups. This study reveals the effects of diet quantity and n3LC on behavioural, ornamental and ejaculate traits in P. reticulata and underscores the likely role that diet plays in maintaining the high variability in these condition-dependent sexual traits. PMID:25170940

  17. mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

    PubMed

    Bache, Matthias; Rot, Swetlana; Keßler, Jacqueline; Güttler, Antje; Wichmann, Henri; Greither, Thomas; Wach, Sven; Taubert, Helge; Söling, Ariane; Bilkenroth, Udo; Kappler, Matthias; Vordermark, Dirk

    2015-06-01

    The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme. PMID:25963717

  18. Expression of FOXO6 is Associated With Oxidative Stress Level and Predicts the Prognosis in Hepatocellular Cancer: A Comparative Study.

    PubMed

    Chen, Hai-Yong; Chen, Yao-Min; Wu, Jian; Yang, Fu-Chun; Lv, Zhen; Xu, Xiao-Feng; Zheng, Shu-Sen

    2016-05-01

    The aim of this study was to explore the association of Forkhead box O6 (FOXO6) expression with oxidative stress level and prognosis of hepatocellular cancer (HCC).The case group included tissues of HCC from 128 patients who were hospitalized in Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery of First Affiliated Hospital, School of Medicine, Zhejiang University. The control group included normal liver tissues from 74 patients. RT-PCR and Western blot were used to test expressions of FOXO6, heme oxygenase (HO)-1, glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). Dihydroethidium (DHE) was dyed to observe reactive oxygen species (ROS) level. Immunohistochemistry was used to test FOXO6 expression. FOXO6 was silenced in HepG2 cells to detect cell proliferation and apoptosis. The expressions of ROS, HO-1, GPx, SOD, CAT, p27, and cyclin D1 were also detected to further explore the possible mechanism.The expressions of FOXO6, HO-1, GPx, SOD, and CAT in HCC tissue was significantly higher than those in normal and adjacent HCC tissues (P <0.05). The tumor size, TNM stage, Alpha-fetoprotein (AFP) level, the presence or absence of hepatitis B surface antigen (HbsAg), and differentiation degree were related to FOXO6 expression level (all P <0.05). COX analysis showed that high FOXO6 expression, male, positive HBsAg, advanced TNM staging, high expression of AFP, and low degree of differentiation were all risk factors for prognosis in HCC (P <0.05). Compared with the blank group (C group, without transfection) and the negative control (NC) group, the mRNA expressions of ROS, FOXO6, HO-1, SOD, GPx, and CAT were decreased (P <0.05). si-RNA group had significantly decreased proliferation speed during 24 to 72 hours (P <0.05), whereas si-FOXO6 group had remarkably increased G0/G1 staged cells and decreased S-staged cells (P <0.05). The si-FOXO6 group showed notably increased apoptosis rate (P <0.05) and p27

  19. Sustaining Written Expression Quality through Leveled Procedural Facilitators in Secondary Students with and without Learning Disabilities

    ERIC Educational Resources Information Center

    Flanagan, Sara M.

    2012-01-01

    Students with and without learning disabilities (LD) struggle with the written expression process, from planning and organization (e.g., prewriting) to actually writing an essay. For students with LD, challenges in written expression are more intensive than their typical peers. Without effective written expression supports and instruction, such as…

  20. Low level expression of glycine receptor beta subunit transgene is sufficient for phenotype correction in spastic mice.

    PubMed Central

    Hartenstein, B; Schenkel, J; Kuhse, J; Besenbeck, B; Kling, C; Becker, C M; Betz, H; Weiher, H

    1996-01-01

    Mutations in inhibitory glycine receptor (GlyR) subunit genes are associated with neuromotor diseases in man and mouse. To use the potential of the mouse mutants as animal models of human disease, we altered GlyR levels in mutant mice and studied their phenotype. A transgene coding for the beta subunit of the rat GlyR was introduced into the genetic background of the spa mutation, which is characterized by low endogenous expression levels of the beta subunit and a dramatic neuromotor phenotype. The resulting transgenic mice expressed the beta subunit mRNA at intermediate levels, and their phenotype was rescued. This provides formal proof for the casual relationship between GlyR beta gene mutation and motor disease, and indicates that a low level of beta gene expression (25% of normal) is sufficient for proper functioning of glycinergic synapses. Images PMID:8635460

  1. CTLA-4 in mesothelioma patients: tissue expression, body fluid levels and possible relevance as a prognostic factor.

    PubMed

    Roncella, Silvio; Laurent, Stefania; Fontana, Vincenzo; Ferro, Paola; Franceschini, Maria Cristiana; Salvi, Sandra; Varesano, Serena; Boccardo, Simona; Vigani, Antonella; Morabito, Anna; Canessa, Pier Aldo; Giannoni, Ugo; Rosenberg, Ilan; Valentino, Alessandro; Fedeli, Franco; Merlo, Domenico Franco; Ceppi, Marcello; Riggio, Salvatore; Romani, Massimo; Saverino, Daniele; Poggi, Alessandro; Pistillo, Maria Pia

    2016-08-01

    CTLA-4 function as a negative regulator of T cell-mediated immune response is well established, whereas much less is known about the immunoregulatory role of its soluble isoform (sCTLA-4). No data are available on CTLA-4 expression and prognostic impact in malignant pleural mesothelioma (MPM). We investigated, by immunohistochemistry, CTLA-4 expression in tumor tissues and, by ELISA, sCTLA-4 levels in sera and matched pleural effusions from 45 MPM patients. Prognostic effect of CTLA-4 expression on overall survival (OS) was assessed through Cox regression and prognostic significance expressed as death rate ratio (HR). We found that 56.0 % of MPM tissues expressed CTLA-4 with variable intensity and percentage of positive cells estimated by the immunoreactive score. sCTLA-4 levels were significantly higher in sera (S-sCTLA-4) than in pleural effusions (PE-sCTLA-4) (geometric mean ratio = 2.70, P value = 0.020). CTLA-4 expression at the tissue level was higher in the epithelioid histological subtype than in the sarcomatoid, whereas at the serum level, it was higher in the sarcomatoid subtype. A homogeneous favorable prognostic effect was found for CTLA-4 overexpression in tissue, serum and pleural effusion. Interestingly, only the PE-sCTLA-4 was found to be a statistically significant positive prognostic factor (HR = 0.37, 95 % CI = 0.18-0.77, P value = 0.007). Indeed, PE-sCTLA-4 correlated with CTLA-4 expression in tissues, whereas this latter expression showed a weak association with OS. To confirm our findings, further experimental evidences obtained from a larger cohort of MPM patients are required. However, our results would indicate a positive correlation of PE-sCTLA-4 levels and OS in MPM patients. PMID:27207606

  2. Venus winds at cloud level from VIRTIS during the Venus Express mission

    NASA Astrophysics Data System (ADS)

    Hueso, Ricardo; Peralta, Javier; Sánchez-Lavega, Agustín.; Pérez-Hoyos, Santiago; Piccioni, Giuseppe; Drossart, Pierre

    2010-05-01

    The Venus Express (VEX) mission has been in orbit to Venus for almost four years now. The VIRTIS instrument onboard VEX observes Venus in two channels (visible and infrared) obtaining spectra and multi-wavelength images of the planet. Images in the ultraviolet range are used to study the upper cloud at 66 km while images in the infrared (1.74 μm) map the opacity of the lower cloud deck at 48 km. Here we present our latest results on the analysis of the global atmospheric dynamics at these cloud levels using a large selection over the full VIRTIS dataset. We will show the atmospheric zonal superrotation at these levels and the mean meridional motions. The zonal winds are very stable in the lower cloud at mid-latitudes to the tropics while it shows different signatures of variability in the upper cloud where solar tide effects are manifest in the data. While the upper clouds present a net meridional motion consistent with the upper branch of a Hadley cell the lower cloud present almost null global meridional motions at all latitudes but with particular features traveling both northwards and southwards in a turbulent manner depending on the cloud morphology on the observations. A particular important atmospheric feature is the South Polar vortex which might be influencing the structure of the zonal winds in the lower cloud at latitudes from the vortex location up to 55°S. Acknowledgements This work has been funded by the Spanish MICIIN AYA2009-10701 with FEDER support and Grupos Gobierno Vasco IT-464-07.

  3. Exenatide enhances cognitive performance and upregulates neurotrophic factor gene expression levels in diabetic mice.

    PubMed

    Gumuslu, Esen; Mutlu, Oguz; Celikyurt, Ipek K; Ulak, Guner; Akar, Furuzan; Erden, Faruk; Ertan, Merve

    2016-08-01

    Exenatide is a potent and selective agonist for the GLP-1 (glucagon-like peptide-1) receptor. Recent studies are focused on the effects of GLP-1 analogues on hippocampal neurogenesis, cognition, learning and memory functions. The aim of this study was to assess the effects of chronic exenatide treatment (0.1 μg/kg, s.c, twice daily for 2 weeks) on spatial memory functions by using the modified elevated plus maze (mEPM) test and emotional memory functions by using the passive avoidance (PA) test in streptozotocin/nicotinamide (STZ-NA)-induced diabetic mice. As the genes involved in neurite remodelling are among the primary targets of regulation, the effects of diabetes and chronic administration of exenatide on brain-derived neurotrophic factor (BDNF) and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) messenger ribonucleic acid (mRNA) levels in the hippocampus of mice were also determined using quantitative real-time polymerase chain reaction (RT-PCR). This study revealed that in the mEPM and PA tests, type-2 diabetes-induced mice exhibited significant impairment of learning and memory which were ameliorated by GLP-1 receptor agonist exenatide. Quantitative RT-PCR revealed that CREB and BDNF gene expression levels were downregulated in diabetic mice, and these alterations were increased by exenatide treatment. Since, exenatide improves cognitive ability in STZ/NA-induced diabetic mice and activates molecular mechanisms of memory storage in response to a learning experience, it may be a candidate for alleviation of mood and cognitive disorder. PMID:26935863

  4. Successful personalized chemotherapy for metastatic gastric cancer based on quantitative BRCA1 mRNA expression level: A case report

    PubMed Central

    HUANG, YING; WU, PUYUAN; LIU, BAORUI; DU, JUAN

    2016-01-01

    Personalized chemotherapy is based on the specific genetic profile of individual patients and is replacing the traditional ‘one size fits all’ medicine. Breast cancer 1 (BRCA1) plays a central role in the chemotherapy-induced DNA damage response. It has been repeatedly demonstrated that BRCA1 mRNA levels were negatively associated with cisplatin sensitivity, but positively associated with docetaxel sensitivity in patients with gastric cancer in experimental and clinical studies. This feature leads to customized chemotherapy based on the BRCA1 mRNA expression level and results in a high efficacy of treatment. The present study describes the case of a 77-year-old patient with metastatic gastric cancer who was treated with personalized chemotherapy based on quantitative BRCA1 mRNA expression level. This study and the available literature data suggest that the expression level of BRCA1 mRNA is dynamic to BRCA1-based chemotherapy. More importantly, de novo assessment of BRCA1 status is a preferable option for ciscisplatin- or docetaxel-resistant patients, since the expression levels of BRCA1 mRNA in certain patients may alter significantly following treatment. Therefore, BRCA1 expression should be assessed for predicting differential chemosensitivity and tailoring chemotherapy in gastric cancer. PMID:27313763

  5. [Expression levels of Slc7a11 in the skin of Kazakh sheep with different coat colors].

    PubMed

    Li, Hong-Tao; He, Xin; Zhou, Zhi-Yong; Zhao, Song-Hua; Zhang, Wen-Xiang; Liu, Gang; Zhao, Zong-Sheng; Jia, Bin

    2012-10-01

    Slc7a11 belongs to solute transporter gene family, encoding cystine/glutamate transporter xCT. It regulates switching between eumelanin and pheomelanin synthesis. In the present study, Real-time PCR was used to detect the mRNA expression levels of Slc7a11 in the skin of Kazakh lambs with different coat colors (black, brown and white), and then the prokaryotic expression plasmid PET-32a-sxCT was constructed to induce the expression of fusion protein. The target pro-tein was purified by Ni-NTA affinity chromatographic separation, and then was used to immunize rabbit in order to produce rabbit anti-sxCT polyclonal antibody. Finally, the expression levels of sxCT were detected in the skin of Kazakh lambs with different hair colors by Western blotting analysis. Results showed that the mRNA expression levels of Slc7a11 differed significantly in the skin of Kazakh lambs with different coat colors, with the highest level in brown coat color, followed by the black, and then the white. The sxCT protein was also detected in the skin of different coat colors by polyclonal antibody, with the highest level in brown coat color, followed by the black, and then the white. It is, therefore, concluded that slc7a11 gene might be associated with the phenotype of coat color in Kazakh sheep. PMID:23099788

  6. Global Analysis of Protein Expression and Phosphorylation Levels in Nicotine-Treated Pancreatic Stellate Cells.

    PubMed

    Paulo, Joao A; Gaun, Aleksandr; Gygi, Steven P

    2015-10-01

    Smoking is a risk factor in pancreatic disease; however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large-scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12 h in triplicate and compared alterations in protein expression and phosphorylation levels to mock-treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8100 proteins were quantified across all nine samples, of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease, and lysosomal proteins. In addition, we measured differences for ∼16 000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  7. Global analysis of protein expression and phosphorylation levels in nicotine-treated pancreatic stellate cells

    PubMed Central

    Paulo, Joao A.; Gaun, Aleksandr; Gygi, Steven P.

    2016-01-01

    Smoking is a risk factor in pancreatic disease, however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12hr in triplicate and compared alterations in protein expression and phosphorylation levels to mock treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8,100 proteins were quantified across all nine samples of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease and lysosomal proteins. In addition, we measured differences for ∼16,000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  8. Relative expression levels of the HLA class-I proteins in normal and HIV-infected cells 1

    PubMed Central

    Apps, Richard; Meng, Zhaojing; Del Prete, Gregory Q.; Lifson, Jeffrey D.; Zhou, Ming; Carrington, Mary

    2015-01-01

    The expression level of human leukocyte antigens (HLA) is known to influence pathological outcomes: pathogens downregulate HLA to evade host immune responses, host inflammatory reactions upregulate HLA, and differences between people in steady-state expression levels of HLA associate with disease susceptibility. Yet precise quantification of relative expression levels of the various HLA loci is difficult due to the tremendous polymorphism of HLA. We report relative expression levels of HLA-A, HLA-B, HLA-C and HLA-E proteins for the specific haplotype A*02:01, B*44:02, C*05:01, characterized using two independent methods based on flow cytometry and mass spectrometry. Peripheral blood lymphocytes from normal donors showed that HLA-A and HLA-B proteins are expressed at similar levels, which are 13-18 times higher than HLA-C by flow cytometry and 4-5 times higher than HLA-C by mass spectrometry, differences that may reflect variation in the conformation or location of proteins detected. HLA-E was detected at a level 25 times lower than that of HLA-C by mass spectrometry. Primary CD4+ T cells infected with HIV in vitro were also studied since HIV downregulates selective HLA types. HLA-A and -B were reduced on HIV-infected cells by a magnitude that varied between cells in an infected culture. Averaging all infected cells from an individual showed HLA-A to be 1-3 and HLA-B to be 2-5 times higher than HLA-C for different individuals by flow cytometry. These results quantify substantial differences in expression levels of the proteins from different HLA loci, which are very likely physiologically significant on both uninfected and HIV-infected cells. PMID:25754738

  9. HLA-G Level on Monocytoid Dendritic Cells Correlates with Regulatory T Cell Foxp3 Expression in Liver Transplant Tolerance

    PubMed Central

    Castellaneta, Antonino; Mazariegos, George V; Nayyar, Navdeep; Zeevi, Adriana; Thomson, Angus W

    2011-01-01

    Background Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule expressed as membrane-bound and soluble isoforms. Interaction of HLA-G with its receptor, immunoglobulin (Ig)-like transcript (ILT) 4 on dendritic cells (DC) down-regulates their T cell stimulatory ability. Methods We examined expression of HLA-G, ILT4, other immune regulatory molecules (inducible costimulator ligand and glucocorticoid-induced tumor necrosis factor-related receptor ligand), and the activation marker CMRF44 on circulating monocytoid (m) and plasmacytoid (p)DC by monoclonal antibody staining and flow cytometry. Three groups of stable liver transplant recipients,-operationally tolerant (TOL), prospective immunosuppressive drug weaning (PW) and maintenance immunosuppression (MI) were studied, together with healthy controls (HC). Serum HLA-G levels were measured by enzyme-linked immunosorbent assay. Results In TOL patients, mDC but not pDC expressed higher HLA-G than in MI patients or HC. In TOL patients, the incidence of CD4+CD25hiCD127− regulatory T cells (Treg) and the intensity of Treg forkhead box p3 (Foxp3) expression were significantly higher than in the MI group. HLA-G expression on circulating mDC correlated significantly with that of Foxp3 in the TOL group. There was no correlation between immunosuppressive drug (tacrolimus) dose or trough level and HLA-G expression or Treg frequency or Foxp3 expression. The incidence of patients with circulating HLA-G levels >100ng/ml was highest in the TOL group, although statistical significance was not achieved. Conclusions Higher HLA-G expression on circulating mDC in TOL recipients compared with MI or HC, suggests a possible role of HLA-G in immune regulation possibly mediated by enhanced host Treg Foxp3 expression. PMID:21423069

  10. Peptidoglycan synthesis and structure in Staphylococcus haemolyticus expressing increasing levels of resistance to glycopeptide antibiotics.

    PubMed Central

    Billot-Klein, D; Gutmann, L; Bryant, D; Bell, D; Van Heijenoort, J; Grewal, J; Shlaes, D M

    1996-01-01

    The structures of cytoplasmic peptidoglycan precursor and mature peptidoglycan of an isogenic series of Staphylococcus haemolyticus strains expressing increasing levels of resistance to the glycopeptide antibiotics teicoplanin and vancomycin (MICs, 8 to 32 and 4 to 16 microg/ml, respectively) were determined. High-performance liquid chromatography, mass spectrometry, amino acid analysis, digestion by R39 D,D-carboxypeptidase, and N-terminal amino acid sequencing were utilized. UDP-muramyl-tetrapeptide-D-lactate constituted 1.7% of total cytoplasmic peptidoglycan precursors in the most resistant strain. It is not clear if this amount of depsipeptide precursor can account for the levels of resistance achieved by this strain. Detailed structural analysis of mature peptidoglycan, examined for the first time for this species, revealed that the peptidoglycan of these strains, like that of other staphylococci, is highly cross-linked and is composed of a lysine muropeptide acceptor containing a substitution at its epsilon-amino position of a glycine-containing cross bridge to the D-Ala 4 of the donor, with disaccharide-pentapeptide frequently serving as an acceptor for transpeptidation. The predominant cross bridges were found to be COOH-Gly-Gly-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Gly-Gly-NH2. Liquid chromatography-mass spectrometry analysis of the peptidoglycan of resistant strains revealed polymeric muropeptides bearing cross bridges containing an additional serine in place of glycine (probable structures, COOH-Gly-Ser-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Ser-Gly-NH2). Muropeptides bearing an additional serine in their cross bridges are estimated to account for 13.6% of peptidoglycan analyzed from resistant strains of S. haemolyticus. A soluble glycopeptide target (L-Ala-gamma-D-iso-glutamyl-L-Lys-D-Ala-D-Ala) was able to more effectively compete for vancomycin when assayed in the presence of resistant cells than when assayed in the presence of susceptible cells

  11. Effects of testosterone on the expression levels of AMH, VEGF and HIF-1α in mouse granulosa cells

    PubMed Central

    Zhang, Ying; Wang, Shan-Feng; Zheng, Jin-Dan; Zhao, Chun-Bo; Zhang, Yi-Nan; Liu, Li-Li; Huang, Jian-Hua

    2016-01-01

    The present study aimed to investigate the effects of testosterone on mouse granulosa cell morphology, and the expression levels of anti-Müllerian hormone (AMH), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). Mouse granulosa cells were isolated and identified, and their morphology was examined using hematoxylin and eosin, F-actin, and follicle-stimulating hormone receptor staining. The mRNA expression levels of AMH, VEGF and HIF-1α were examined using reverse transcription-quantitative polymerase chain reaction, and their protein secretion levels were investigated using enzyme-linked immunosorbent assays. Testosterone treatment did not affect granulosa cell morphology; however, it significantly increased the mRNA expression levels of AMH and VEGF, and the protein secretion levels of AMH, VEGF and HIF-1α. These results suggested that testosterone was able to regulate the functions of granulosa cells by upregulating the expression levels of AMH, VEGF and HIF-1α. PMID:27446291

  12. High-level soluble expression of a bacterial N-acyl-d-glucosamine 2-epimerase in recombinant Escherichia coli.

    PubMed

    Klermund, Ludwig; Riederer, Amelie; Groher, Anna; Castiglione, Kathrin

    2015-07-01

    N-Acyl-d-glucosamine 2-epimerase (AGE) is an important enzyme for the biocatalytic synthesis of N-acetylneuraminic acid (Neu5Ac). Due to the wide range of biological applications of Neu5Ac and its derivatives, there has been great interest in its large-scale synthesis. Thus, suitable strategies for achieving high-level production of soluble AGE are needed. Several AGEs from various organisms have been recombinantly expressed in Escherichia coli. However, the soluble expression level was consistently low with an excessive formation of inclusion bodies. In this study, the effects of different solubility-enhancement tags, expression temperatures, chaperones and host strains on the soluble expression of the AGE from the freshwater cyanobacterium Anabaena variabilis ATCC 29413 (AvaAGE) were examined. The optimum combination of tag, expression temperature, co-expression of chaperones and host strain (His6-tag, 37°C, GroEL/GroES, E. coli BL21(DE3)) led to a 264-fold improvement of the volumetric epimerase activity, a measure of the soluble expression, compared to the starting conditions (His6-maltose-binding protein-tag, 20°C, without chaperones, E. coli BL21(DE3)). A maximum yield of 22.5mg isolated AvaAGE per liter shake flask culture was obtained. PMID:25804337

  13. Liver metal levels and expression of genes related to iron homeostasis in rhesus monkeys after inhalational manganese exposure

    PubMed Central

    Pettiglio, Michael A.; Herrera, Carolina; Foster, Melanie L.; Dorman, David C.; Bartnikas, Thomas B.

    2016-01-01

    Here we present data on liver metal levels and expression of genes related to iron homeostasis in rhesus monkeys after inhalational manganese exposure. Archived liver samples from rhesus monkeys exposed to 0 (n=6), 0.06 (n=6), 0.3 (n=4) and 1.5 (n=4) mg/m3 manganese inhalation for 65 days were obtained from a published study (“Tissue manganese concentrations in young male rhesus monkeys following subchronic manganese sulfate inhalation” [1]). Samples were analyzed by spectroscopy, immunoblotting and quantitative PCR to assess metal levels and gene expression. Liver manganese and iron levels were linearly correlated although only the intermediate manganese exposure level (0.3 mg Mn/m3) led to a statistically significant increase in liver iron levels. PMID:26958631

  14. Liver metal levels and expression of genes related to iron homeostasis in rhesus monkeys after inhalational manganese exposure.

    PubMed

    Pettiglio, Michael A; Herrera, Carolina; Foster, Melanie L; Dorman, David C; Bartnikas, Thomas B

    2016-03-01

    Here we present data on liver metal levels and expression of genes related to iron homeostasis in rhesus monkeys after inhalational manganese exposure. Archived liver samples from rhesus monkeys exposed to 0 (n=6), 0.06 (n=6), 0.3 (n=4) and 1.5 (n=4) mg/m(3) manganese inhalation for 65 days were obtained from a published study ("Tissue manganese concentrations in young male rhesus monkeys following subchronic manganese sulfate inhalation" [1]). Samples were analyzed by spectroscopy, immunoblotting and quantitative PCR to assess metal levels and gene expression. Liver manganese and iron levels were linearly correlated although only the intermediate manganese exposure level (0.3 mg Mn/m(3)) led to a statistically significant increase in liver iron levels. PMID:26958631

  15. New adipokines vaspin and omentin. Circulating levels and gene expression in adipose tissue from morbidly obese women

    PubMed Central

    2011-01-01

    Background Vaspin and omentin are recently described molecules that belong to the adipokine family and seem to be related to metabolic risk factors. The objectives of this study were twofold: to evaluate vaspin and omentin circulating levels and mRNA expression in subcutaneous and visceral adipose tissues in non-diabetic morbidly obese women; and to assess the relationship of vaspin and omentin with anthropometric and metabolic parameters, and other adipo/cytokines. Design We analysed vaspin and omentin circulating levels in 71 women of European descent (40 morbidly obese [BMI ≥ 40 kg/m2] and 31 lean [BMI ≤ 25]). We assessed vaspin and omentin gene expression in paired samples of visceral and subcutaneous abdominal adipose tissue from 46 women: 40 morbidly obese and 6 lean. We determined serum vaspin and plasma omentin levels with an Enzyme-Linked Immunosorbent Assay and adipose tissue mRNA expression by real time RT-PCR. Results Serum vaspin levels in the morbidly obese were not significantly different from those in controls. They correlated inversely with levels of lipocalin 2 and interleukin 6. Vaspin mRNA expression was significantly higher in the morbidly obese, in both subcutaneous and visceral adipose tissue. Plasma omentin levels were significantly lower in the morbidly obese and they correlated inversely with glucidic metabolism parameters. Omentin circulating levels, then, correlated inversely with the metabolic syndrome (MS). Omentin expression in visceral adipose tissue was significantly lower in morbidly obese women than in controls. Conclusions The present study indicates that vaspin may have a compensatory role in the underlying inflammation of obesity. Decreased omentin circulating levels have a close association with MS in morbidly obese women. PMID:21526992

  16. Inverse Relationship of the CMKLR1 Relative Expression and Chemerin Serum Levels in Obesity with Dysmetabolic Phenotype and Insulin Resistance

    PubMed Central

    Corona-Meraz, Fernanda-Isadora; Navarro-Hernández, Rosa-Elena; Ruíz-Quezada, Sandra-Luz; Madrigal-Ruíz, Perla-Monserrat; Castro-Albarrán, Jorge; Chavarría-Ávila, Efraín; Guzmán-Ornelas, Milton-Omar; Gómez-Bañuelos, Eduardo; Petri, Marcelo-Herón; Ramírez-Cedano, Joel-Isidro; Aguilar-Aldrete, María-Elena; Ríos-Ibarra, Clara; Vázquez-Del Mercado, Mónica

    2016-01-01

    Background. In obesity there is a subclinical chronic low-grade inflammatory response where insulin resistance (IR) may develop. Chemerin is secreted in white adipose tissue and promotes low-grade inflammatory process, where it expressed CMKLR1 receptor. The role of chemerin and CMKLR1 in inflammatory process secondary to obesity is not defined yet. Methods. Cross-sectional study with 134 individuals classified as with and without obesity by body mass index (BMI) and IR. Body fat storage measurements and metabolic and inflammatory markers were measured by routine methods. Soluble chemerin and basal levels of insulin by ELISA and relative expression of CMKLR1 were evaluated with qPCR and 2−ΔΔCT method. Results. Differences (P < 0.05) were observed between obesity and lean individuals in body fat storage measurements and metabolic-inflammatory markers. Both CMKLR1 expression and chemerin levels were increased in obesity without IR. Soluble chemerin levels correlate with adiposity and metabolic markers (r = 8.8% to 38.5%), P < 0.05. Conclusion. The increment of CMKLR1 expression was associated with insulin production. Increased serum levels of chemerin in obesity were observed, favoring a dysmetabolic response. The results observed in this study suggest that both chemerin and CMKLR1 have opposite expression in the context of low-grade inflammatory response manifested in the development of IR. PMID:27239101

  17. Examining how p16(INK4a) expression levels are linked to handgrip strength in the elderly.

    PubMed

    Kao, Tung-Wei; Chen, Wei-Liang; Han, Der-Sheng; Huang, Ying-Hsin; Chen, Chi-Ling; Yang, Wei-Shiung

    2016-01-01

    Although many studies have shown that p16(INK4a) is more highly expressed in the human body during senescence, studies on its relevance to handgrip strength among old adults, are relatively sparse. We enrolled 205 community-dwelling old adults aged 65 years and older without specific medical conditions. Handgrip strength of the dominant hand was measured. Low handgrip strength was defined as the lowest quartile of handgrip strength among the participants. RNA was extracted from peripheral white blood cells. Use quantitative polymerase chain reaction to estimate the p16(INK4a) mRNA expression level. The average handgrip strength was 25.22 ± 8.98 kg, and gender difference was observed. In the linear regression model, the p16(INK4a) mRNA expression level was significantly negatively associated with handgrip strength in men but not in women. The β coefficient, representing the change of handgrip strength for each increment in the p16(INK4a) mRNA expression level, was -0.208 (p = 0.024) among old men. The negative association remained after additional covariates adjustment. In the multiple logistic regression model among old men, the odds ratio (OR) of low handgrip strength was 1.246 (p = 0.032). In this study, we observed the p16(INK4a) mRNA expression level was negative associated with handgrip strength among community-dwelling old men. PMID:27549351

  18. Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

    PubMed

    Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

    2016-06-01

    The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms. PMID:27543868

  19. Examining how p16INK4a expression levels are linked to handgrip strength in the elderly

    PubMed Central

    Kao, Tung-Wei; Chen, Wei-Liang; Han, Der- Sheng; Huang, Ying-Hsin; Chen, Chi-Ling; Yang, Wei-Shiung

    2016-01-01

    Although many studies have shown that p16INK4a is more highly expressed in the human body during senescence, studies on its relevance to handgrip strength among old adults, are relatively sparse. We enrolled 205 community-dwelling old adults aged 65 years and older without specific medical conditions. Handgrip strength of the dominant hand was measured. Low handgrip strength was defined as the lowest quartile of handgrip strength among the participants. RNA was extracted from peripheral white blood cells. Use quantitative polymerase chain reaction to estimate the p16INK4a mRNA expression level. The average handgrip strength was 25.22 ± 8.98 kg, and gender difference was observed. In the linear regression model, the p16INK4a mRNA expression level was significantly negatively associated with handgrip strength in men but not in women. The β coefficient, representing the change of handgrip strength for each increment in the p16INK4a mRNA expression level, was −0.208 (p = 0.024) among old men. The negative association remained after additional covariates adjustment. In the multiple logistic regression model among old men, the odds ratio (OR) of low handgrip strength was 1.246 (p = 0.032). In this study, we observed the p16INK4a mRNA expression level was negative associated with handgrip strength among community-dwelling old men. PMID:27549351

  20. Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs

    PubMed Central

    Ma, Zhiyu; Su, Juan; Guo, Tingting; Jin, Mengmeng; Li, Xiang; Lei, Zhihai; Hou, Yuanlong; Li, Xiaoliang; Jia, Cuicui; Zhang, Zheng; Ahmed, Ejlal

    2016-01-01

    Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs. PMID:27010315

  1. Morphology, sex steroid level and gene expression analysis in gonadal sex reversal of triploid female (XXX) rainbow trout (Oncorhynchus mykiss).

    PubMed

    Xu, Gefeng; Huang, Tianqing; Jin, Xian; Cui, Cunhe; Li, Depeng; Sun, Cong; Han, Ying; Mu, Zhenbo

    2016-02-01

    In non-mammalian vertebrates, estrogens and expressions of cyp19a1 and foxl2 play critical roles in maintaining ovary differentiation and development, while dmrt1 and sox9 are male-specific genes in testicular differentiation and are highly conserved. In order to deeply understand the morphological change, sex steroids level and molecular mechanism of triploid female gonadal reversal in rainbow trout, we studied the ovary morphology, tendency of estradiol-17β (E2) and testosterone (T) levels and the relative expressions of dmrt1, cyp19a1, sox9 and foxl2 in juvenile and adult fish. Our results demonstrated that the development of triploid female gonads in rainbow trout went through arrested development, oocytes dedifferentiation, ovary reconstruction and sex reversal finally. During early gonadal development (154-334 days post-fertilization), the expressions of foxl2 and cyp19a1 increased linearly, while expressions of dmrt1 and sox9 were extremely suppressed, and E2 level was higher, while T level was lower. During the mid-to-late period of triploid female gonadal development (574-964 days post-fertilization), the expressions of dmrt1 and sox9 remained high and were very close to the quantity of diploid male genes, and T levels were even reaching diploid male plasma concentrations, while expressions of cyp19a1 and foxl2 were decreased, leading to decrease in E2 level. We realized that the development model of rainbow trout triploid female gonads was extremely rare, and the regulatory mechanism was very special. Genes involved in gonadal development and endogenous estrogens are pivotal factors in fish natural sex reversal. PMID:26373423

  2. Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs.

    PubMed

    Ma, Zhiyu; Su, Juan; Guo, Tingting; Jin, Mengmeng; Li, Xiang; Lei, Zhihai; Hou, Yuanlong; Li, Xiaoliang; Jia, Cuicui; Zhang, Zheng; Ahmed, Ejlal

    2016-01-01

    Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs. PMID:27010315

  3. The Expression Levels of XLF and Mutant P53 Are Inversely Correlated in Head and Neck Cancer Cells

    PubMed Central

    Feng, Sizhe; Rabii, Ramin; Liang, Guobiao; Song, Chenxi; Chen, Wei; Guo, Mian; Wei, Xuezhong; Messadi, Diana; Hu, Shen

    2016-01-01

    XRCC4-like factor (XLF), also known as Cernunnos, is a protein encoded by the human NHEJ1 gene and an important repair factor for DNA double-strand breaks. In this study, we have found that XLF is over-expressed in HPV(+) versus HPV(-) head and neck squamous cell carcinoma (HNSCC) and significantly down-regulated in the HNSCC cell lines expressing high level of mutant p53 protein versus those cell lines harboring wild-type TP53 gene with low p53 protein expression. We have also demonstrated that Werner syndrome protein (WRN), a member of the NHEJ repair pathway, binds to both mutant p53 protein and NHEJ1 gene promoter, and siRNA knockdown of WRN leads to the inhibition of XLF expression in the HNSCC cells. Collectively, these findings suggest that WRN and p53 are involved in the regulation of XLF expression and the activity of WRN might be affected by mutant p53 protein in the HNSCC cells with aberrant TP53 gene mutations, due to the interaction of mutant p53 with WRN. As a result, the expression of XLF in these cancer cells is significantly suppressed. Our study also suggests that XLF is over-expressed in HPV(+) HNSCC with low expression of wild type p53, and might serve as a potential biomarker for HPV(+) HNSCC. Further studies are warranted to investigate the mechanisms underlying the interactive role of WRN and XLF in NHEJ repair pathway. PMID:27471552

  4. The Expression Levels of XLF and Mutant P53 Are Inversely Correlated in Head and Neck Cancer Cells.

    PubMed

    Feng, Sizhe; Rabii, Ramin; Liang, Guobiao; Song, Chenxi; Chen, Wei; Guo, Mian; Wei, Xuezhong; Messadi, Diana; Hu, Shen

    2016-01-01

    XRCC4-like factor (XLF), also known as Cernunnos, is a protein encoded by the human NHEJ1 gene and an important repair factor for DNA double-strand breaks. In this study, we have found that XLF is over-expressed in HPV(+) versus HPV(-) head and neck squamous cell carcinoma (HNSCC) and significantly down-regulated in the HNSCC cell lines expressing high level of mutant p53 protein versus those cell lines harboring wild-type TP53 gene with low p53 protein expression. We have also demonstrated that Werner syndrome protein (WRN), a member of the NHEJ repair pathway, binds to both mutant p53 protein and NHEJ1 gene promoter, and siRNA knockdown of WRN leads to the inhibition of XLF expression in the HNSCC cells. Collectively, these findings suggest that WRN and p53 are involved in the regulation of XLF expression and the activity of WRN might be affected by mutant p53 protein in the HNSCC cells with aberrant TP53 gene mutations, due to the interaction of mutant p53 with WRN. As a result, the expression of XLF in these cancer cells is significantly suppressed. Our study also suggests that XLF is over-expressed in HPV(+) HNSCC with low expression of wild type p53, and might serve as a potential biomarker for HPV(+) HNSCC. Further studies are warranted to investigate the mechanisms underlying the interactive role of WRN and XLF in NHEJ repair pathway. PMID:27471552

  5. High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.

    PubMed

    Nie, Yao; Yan, Wei; Xu, Yan; Chen, Wen Bo; Mu, Xiao Qing; Wang, Xinye; Xiao, Rong

    2013-01-01

    Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3)/pET-20b(+)-pul and E. coli BL21(DE3)/pET-22b(+)-pul, were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3)/pET-20b(+)-pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3)/pET-22b(+)-pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3)/pET-22b(+)-pul. When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The successful

  6. High-Level Expression of Bacillus naganoensis Pullulanase from Recombinant Escherichia coli with Auto-Induction: Effect of lac Operator

    PubMed Central

    Xu, Yan; Chen, Wen Bo; Mu, Xiao Qing; Wang, Xinye; Xiao, Rong

    2013-01-01

    Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3)/pET-20b(+)-pul and E. coli BL21(DE3)/pET-22b(+)-pul, were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3)/pET-20b(+)-pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3)/pET-22b(+)-pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3)/pET-22b(+)-pul. When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The successful

  7. A distinct ERCC1 haplotype is associated with mRNA expression levels in prostate cancer patients.

    PubMed

    Woelfelschneider, Andreas; Popanda, Odilia; Lilla, Carmen; Linseisen, Jakob; Mayer, Claudia; Celebi, Oktay; Debus, Jürgen; Bartsch, Helmut; Chang-Claude, Jenny; Schmezer, Peter

    2008-09-01

    Both genetic variants and messenger RNA (mRNA) expression of DNA repair and tumor suppressor genes have been investigated as molecular markers for therapy outcome. However, the phenotypic impact of genetic variants often remained unclear, thus the rationale of their use in risk prediction may be limited. We therefore analyzed genetic variants together with anthropometric and lifestyle factors to see how these affect mRNA levels of ERCC1, MDM2 and TP53 in primary blood lymphocytes. mRNA expression was measured in 376 prostate cancer patients by quantitative real-time polymerase chain reaction after reverse transcription, and ERCC1 rs11615 T>C, ERCC1 rs3212986 C>A, MDM2 rs2279744 T>G and TP53 rs17878362 (p53PIN3) polymorphisms were determined. Considerable interindividual differences in mRNA expression were found (coefficients of variation: ERCC1, 45%; MDM2, 43% and TP53, 35%). ERCC1 expression was positively correlated with plasma levels of beta-carotene (P = 0.03) and negatively correlated with canthaxanthin (P = 0.02) and lutein (P = 0.02). Overall, the polymorphisms affected mRNA expression only weakly. Carriers of a distinct ERCC1 haplotype (CC) showed, however, significantly lower expression values than non-carriers (P = 0.001). Applying logistic regression, we found that CC haplotype carriers had a 1.69-fold increased odds ratio (95% confidence interval: 1.06-2.71) for reduced ERCC1 mRNA levels. This low ERCC1 expression might be associated with reduced DNA repair and better therapy response. In summary, the association we have found between ERCC1 genotype and mRNA expression supports recent clinical observations that genetic variation in ERCC1 can affect treatment outcome and prognosis. Our study further revealed a modulating effect by nutritional factors. PMID:18332046

  8. Low-level laser therapy enhances the expression of osteogenic factors during bone repair in rats.

    PubMed

    Tim, Carla Roberta; Pinto, Karina Nogueira Zambone; Rossi, Bruno Rafael Orsini; Fernandes, Kelly; Matsumoto, Mariza Akemi; Parizotto, Nivaldo Antonio; Rennó, Ana Claudia Muniz

    2014-01-01

    The aim of this study was to evaluate the effects of low-level laser therapy (LLLT) on bone formation, immunoexpression of osteogenic factors, and biomechanical properties in a tibial bone defect model in rats. Sixty male Wistar rats were distributed into bone defect control group (CG) and laser irradiated group (LG). Animals were euthanized on days 15, 30, and 45 post-injury. The histological and morphometric analysis showed that the treated animals presented no inflammatory infiltrate and a better tissue organization at 15 and 30 days postsurgery. Also, a higher amount of newly formed bone was observed at 15 days postsurgery. No statistically significant difference was observed in cyclooxygenase-2 immunoexpression among the groups at 15, 30, and 45 days in the immunohistochemical analysis. Considering RUNX-2, the immunoexpression was statistically higher in the LG compared to the CG at 45 days. BMP-9 immunoexpression was significantly higher in the LG in comparison to CG at day 30. However, there was no expressivity for this immunomarker, both in the CG and LG, at the day 45 postsurgery. No statistically significant difference was observed in the receptor activator of nuclear factor kappa-B ligand immunoexpression among the groups in all periods evaluated. No statistically significant difference among the groups was observed in the maximal load in any period of time. Our findings indicate that laser therapy improved bone healing by accelerating the development of newly formed bone and activating the osteogenic factors on tibial defects, but the biomechanical properties in LG were not improved. PMID:23515631

  9. Integration of mRNA expression profile, copy number alterations, and microRNA expression levels in breast cancer to improve grade definition.

    PubMed

    Cava, Claudia; Bertoli, Gloria; Ripamonti, Marilena; Mauri, Giancarlo; Zoppis, Italo; Della Rosa, Pasquale Anthony; Gilardi, Maria Carla; Castiglioni, Isabella

    2014-01-01

    Defining the aggressiveness and growth rate of a malignant cell population is a key step in the clinical approach to treating tumor disease. The correct grading of breast cancer (BC) is a fundamental part in determining the appropriate treatment. Biological variables can make it difficult to elucidate the mechanisms underlying BC development. To identify potential markers that can be used for BC classification, we analyzed mRNAs expression profiles, gene copy numbers, microRNAs expression and their association with tumor grade in BC microarray-derived datasets. From mRNA expression results, we found that grade 2 BC is most likely a mixture of grade 1 and grade 3 that have been misclassified, being described by the gene signature of either grade 1 or grade 3. We assessed the potential of the new approach of integrating mRNA expression profile, copy number alterations, and microRNA expression levels to select a limited number of genomic BC biomarkers. The combination of mRNA profile analysis and copy number data with microRNA expression levels led to the identification of two gene signatures of 42 and 4 altered genes (FOXM1, KPNA4, H2AFV and DDX19A) respectively, the latter obtained through a meta-analytical procedure. The 42-based gene signature identifies 4 classes of up- or down-regulated microRNAs (17 microRNAs) and of their 17 target mRNA, and the 4-based genes signature identified 4 microRNAs (Hsa-miR-320d, Hsa-miR-139-5p, Hsa-miR-567 and Hsa-let-7c). These results are discussed from a biological point of view with respect to pathological features of BC. Our identified mRNAs and microRNAs were validated as prognostic factors of BC disease progression, and could potentially facilitate the implementation of assays for laboratory validation, due to their reduced number. PMID:24866763

  10. The Utility of Shallow RNA-Seq for Documenting Differential Gene Expression in Genes with High and Low Levels of Expression

    PubMed Central

    Plachetzki, David C.; Jasper, W. Cameron

    2013-01-01

    The sequencing depth necessary for documenting differential gene expression using RNA-Seq has been little explored outside of model systems. In particular, the depth required to analyze large-scale patterns of differential transcription factor expression is not known. The goal of the present study is to explore the effectiveness of shallow (relatively low read depth) RNA-Seq. We focus on two tissues in the honey bee: the sting gland and the digestive tract. The sting gland is an experimentally well-understood tissue that we use to benchmark the utility of this approach. We use the digestive tract to test the results obtained with the sting gland, and to conduct RNA-Seq between tissue types. Using a list of experimentally verified genes conferring tissue-specific functions in the sting gland, we show that relatively little read depth is necessary to identify them. We argue that this result should be broadly applicable, since genes important for tissue-specific functions often have robust expression patterns, and because we obtained similar results in our analysis of the digestive tract. Furthermore, we demonstrate that the differential expression of transcription factors, which are transcribed at low levels compared to other genes, can nevertheless often be determined using shallow RNA-Seq. Overall, we find over 150 differentially expressed transcription factors in our tissues at a read depth of only 12 million. This work shows the utility of low-depth sequencing for identifying genes important for tissue-specific functions. It also verifies the often-held belief that transcription factors show low levels of expression, while demonstrating that, in spite of this, they are frequently amenable to shallow RNA-Seq. Our findings should be of benefit to researchers using RNA-Seq in many different biological systems. PMID:24358338

  11. SOURCES OF VARIATION IN BASELINE GENE EXPRESSION LEVELS FROM TOXICOGENOMIC STUDY CONTROL ANIMALS ACROSS MULTIPLE LABORATORIES

    EPA Science Inventory

    Variations in study design are typical for toxicogenomic studies, but their impact on gene expression in control animals has not been well characterized. A dataset of control animal microarray expression data was assembled by a working group of the Health and Environmental Scienc...

  12. A Phenomenological Exploration of the Experiences of Master's Level Counselor Trainees in Expressive Arts Group Supervision

    ERIC Educational Resources Information Center

    Rossi, Martha Howe

    2010-01-01

    Expressive arts group supervision is the use of music, stories, movement, poetry or prose, role-play or psychodrama, art, guided imagery, or play to help trainees develop reflective skills (Wilkins, 1995), express thoughts and feelings (Knill, Levine & Levine, 2005; Lahad, 2000), develop new perspectives (Gladding, 2005), increase communication…

  13. Reversing the reduced level of endometrial GLUT4 expression in polycystic ovary syndrome: a mechanistic study of metformin action

    PubMed Central

    Li, Xin; Cui, Peng; Jiang, Hong-Yuan; Guo, Yan-Rong; Pishdari, Bano; Hu, Min; Feng, Yi; Billig, Håkan; Shao, Ruijin

    2015-01-01

    Conflicting results have been reported regarding whether or not insulin-regulated glucose transporter 4 (GLUT4) is expressed in human and rodent endometria. There is an inverse relationship between androgen levels and insulin-dependent glucose metabolism in women. Hyperandrogenemia, hyperinsulinemia, and insulin resistance are believed to contribute to endometrial abnormalities in women with polycystic ovary syndrome (PCOS). However, it has been unclear in previous studies if endometrial GLUT4 expression is regulated by androgen-dependent androgen receptors (ARs) and/or the insulin receptor/Akt/mTOR signaling network. In this study, we demonstrate that GLUT4 is expressed in normal endometrial cells (mainly in the epithelial cells) and is down-regulated under conditions of hyperandrogenemia in tissues from PCOS patients and in a 5α-dihydrotestosterone-induced PCOS-like rat model. Western blot analysis revealed reduced endometrial GLUT4 expression and increased AR expression in PCOS patients. However, the reduced GLUT4 level was not always associated with an increase in AR in PCOS patients when comparing non-hyperplasia with hyperplasia. Using a human tissue culture system, we investigated the molecular basis by which GLUT4 regulation in endometrial hyperplasia tissues is affected by metformin in PCOS patients. We show that specific endogenous organic cation transporter isoforms are regulated by metformin, and this suggests a direct effect of metformin on endometrial hyperplasia. Moreover, we demonstrate that metformin induces GLUT4 expression and inhibits AR expression and blocks insulin receptor/PI3K/Akt/mTOR signaling in the same hyperplasia human tissues. These findings indicate that changes in endometrial GLUT4 expression in PCOS patients involve the androgen-dependent alteration of AR expression and changes in the insulin receptor/PI3K/Akt/mTOR signaling network. PMID:26045896

  14. Reversing the reduced level of endometrial GLUT4 expression in polycystic ovary syndrome: a mechanistic study of metformin action.

    PubMed

    Li, Xin; Cui, Peng; Jiang, Hong-Yuan; Guo, Yan-Rong; Pishdari, Bano; Hu, Min; Feng, Yi; Billig, Håkan; Shao, Ruijin

    2015-01-01

    Conflicting results have been reported regarding whether or not insulin-regulated glucose transporter 4 (GLUT4) is expressed in human and rodent endometria. There is an inverse relationship between androgen levels and insulin-dependent glucose metabolism in women. Hyperandrogenemia, hyperinsulinemia, and insulin resistance are believed to contribute to endometrial abnormalities in women with polycystic ovary syndrome (PCOS). However, it has been unclear in previous studies if endometrial GLUT4 expression is regulated by androgen-dependent androgen receptors (ARs) and/or the insulin receptor/Akt/mTOR signaling network. In this study, we demonstrate that GLUT4 is expressed in normal endometrial cells (mainly in the epithelial cells) and is down-regulated under conditions of hyperandrogenemia in tissues from PCOS patients and in a 5α-dihydrotestosterone-induced PCOS-like rat model. Western blot analysis revealed reduced endometrial GLUT4 expression and increased AR expression in PCOS patients. However, the reduced GLUT4 level was not always associated with an increase in AR in PCOS patients when comparing non-hyperplasia with hyperplasia. Using a human tissue culture system, we investigated the molecular basis by which GLUT4 regulation in endometrial hyperplasia tissues is affected by metformin in PCOS patients. We show that specific endogenous organic cation transporter isoforms are regulated by metformin, and this suggests a direct effect of metformin on endometrial hyperplasia. Moreover, we demonstrate that metformin induces GLUT4 expression and inhibits AR expression and blocks insulin receptor/PI3K/Akt/mTOR signaling in the same hyperplasia human tissues. These findings indicate that changes in endometrial GLUT4 expression in PCOS patients involve the androgen-dependent alteration of AR expression and changes in the insulin receptor/PI3K/Akt/mTOR signaling network. PMID:26045896

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

    PubMed

    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

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

    PubMed Central

    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

  17. Expression of the hereditary hemochromatosis protein HFE increases ferritin levels by inhibiting iron export in HT29 cells.

    PubMed

    Davies, Paige S; Enns, Caroline A

    2004-06-11

    Iron is essential for life in almost all organisms and, in mammals, is absorbed through the villus cells of the duodenum. Using a human colonic carcinoma cell line that has many duodenal characteristics, HT29, we show that genes involved in intestinal iron transport are endogenously expressed. When stably transfected to express the hereditary hemochromatosis protein HFE these cells have increased ferritin levels. We demonstrate that this is not due to an effect on the transferrin (TF)-mediated iron uptake pathway but rather due to inhibition of iron efflux from the cell. The effect of HFE was independent of its interaction with TF receptor 1 as indicated by similar results using both the wild type HFE and the W81A mutant that binds TF receptor 1 with greatly reduced affinity. HFE expression did not affect the mRNA levels of most of the genes involved in iron absorption that were tested; however, it did correspond to a decrease in hephaestin message levels. These results point to a role for HFE in inhibition of iron efflux in HT29 cells. This is a distinct role from that in HeLa and human embryonic kidney 293 cells where HFE has been shown to inhibit TF-mediated iron uptake resulting in decreased ferritin levels. Such a distinction suggests a multifunctional role for HFE that is dependent upon expression levels of proteins involved in iron transport. PMID:15044462

  18. Differential viral levels and immune gene expression in three stocks of Apis mellifera induced by different numbers of Varroa destructor.

    PubMed

    Khongphinitbunjong, Kitiphong; de Guzman, Lilia I; Tarver, Matthew R; Rinderer, Thomas E; Chen, Yanping; Chantawannakul, Panuwan

    2015-01-01

    The viral levels and immune responses of Italian honey bees (IHB), Russian honey bees (RHB) and an outcross of Varroa Sensitive Hygienic bees (POL) deliberately infested with one or two foundress Varroa were compared. We found that the Deformed wing virus (DWV) level in IHB inoculated with one or two foundress Varroa increased to about 10(3) or 10(5) fold the levels of their uninfested brood. In contrast, POL (10(2) or 10(4) fold) and RHB (10(2) or l0(4) fold) supported a lower increase in DWV levels. The feeding of different stages of Varroa nymphs did not increase DWV levels of their pupal hosts. Analyses of their corresponding Varroa mites showed the same trends: two foundress Varroa yielded higher DWV levels than one foundress, and the addition of nymphs did not increase viral levels. Using the same pupae examined for the presence of viruses, 16 out of 24 genes evaluated showed significant differential mRNA expression levels among the three honey bee stocks. However, only four genes (Defensin, Dscam, PPOact and spaetzle), which were expressed at similar levels in uninfested pupae, were altered by the number of feeding foundress Varroa and levels of DWV regardless of stocks. This research provides the first evidence that immune response profiles of different honey bee stocks are induced by Varroa parasitism. PMID:25456452

  19. Expression System for High Levels of GAG Lyase Gene Expression and Study of the hepA Upstream Region in Flavobacterium heparinum

    PubMed Central

    Blain, Françoise; Tkalec, A. Lydia; Shao, Zhongqi; Poulin, Catherine; Pedneault, Marc; Gu, Kangfu; Eggimann, Bernhard; Zimmermann, Joe; Su, Hongsheng

    2002-01-01

    A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts. PMID:12029040

  20. Expression system for high levels of GAG lyase gene expression and study of the hepA upstream region in Flavobacterium heparinum.

    PubMed

    Blain, Françoise; Tkalec, A Lydia; Shao, Zhongqi; Poulin, Catherine; Pedneault, Marc; Gu, Kangfu; Eggimann, Bernhard; Zimmermann, Joe; Su, Hongsheng

    2002-06-01

    A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts. PMID:12029040

  1. Loss of Pnn expression attenuates expression levels of SR family splicing factors and modulates alternative pre-mRNA splicing in vivo

    SciTech Connect

    Chiu Yali; Ouyang Pin . E-mail: ouyang@mail.cgu.edu.tw

    2006-03-10

    SR and SR-related proteins have been implicated as trans-acting factors that play an important role in splice selection and are involved at specific stages of spliceosome formation. A well-established property of SR protein splicing factors is their ability to influence selection of alternative splice sites in a concentration-dependent manner. Identification of molecules that regulate SR family protein expression is therefore of vital importance in RNA biology. Here we report that depletion of Pnn expression, a SR-related protein with functions involved in pre-mRNA splicing and mRNA export, induces reduced expression of a subset of cellular proteins, especially that of SR family proteins, including SC35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, Mdm2, and ki67. Knocking down Pnn expression was achieved in vitro by siRNA transfection. Expression levels of SR and SR-related proteins in Pnn-depleted cells as compared to those in control cells were evaluated by immunofluorescent staining and Western blot with specific antibodies. In addition, we also demonstrate that loss of Pnn expression could modulate splice site selection of model reporter gene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role in the control of the cellular amount of family SR proteins through down-regulation of its own expression, thereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function.

  2. The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings.

    PubMed

    Wu, Chung-Shen; Kuo, Wei-Tin; Chang, Chia-Yu; Kuo, Jun-Yi; Tsai, Yi-Ting; Yu, Su-May; Wu, Hsi-Ten; Chen, Peng-Wen

    2014-05-01

    Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O2 deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8% of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8% of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia. PMID:24445591

  3. Circulating promyelocytes and low levels of CD16 expression on polymorphonuclear leukocytes accompany early-onset periodontitis.

    PubMed Central

    Nemoto, E; Nakamura, M; Shoji, S; Horiuchi, H

    1997-01-01

    Early-onset periodontitis (EOP) is characterized by rapidly progressive alveolar bone loss, chemotactic defects of neutrophils, and significant familial aggregation. We found immature myeloid lineage cells, defined as promyelocytes, in the peripheral blood in patients with EOP. A hematological examination of peripheral blood cells showed normal reference values regarding cell proportions. Flow cytometry revealed significantly lower expression of CD16, a glycosylphosphatidylinositol (GPI)-anchored protein, on peripheral neutrophils in patients compared with those in age- and sex-matched healthy controls, whereas the levels of CD11a and CD11b expression were similar. The chemotactic response of neutrophils was lower toward not only formyl-methionyl-leucyl-phenylalanine but also complement fragment C5a than that of healthy controls. The expression of another GPI-anchored protein, CD14, was equally expressed by controls and patients. Therefore, the low level of CD16 expression was not due to the incomplete synthesis of the GPI anchor. GPI anchors of CD16 on neutrophils from controls and patients were both partially resistant to phosphatidylinositol-specific phospholipase C. The presence of promyelocytes in peripheral blood, low expression of CD16, and low chemotactic response of neutrophils suggest that patients with EOP have an abnormal maturation system in myeloid lineage cells in the bone marrow, which may be associated with the onset and course of EOP. PMID:9284170

  4. Changes of thyroid hormone levels and related gene expression in zebrafish on early life stage exposure to triadimefon.

    PubMed

    Liu, Shaoying; Chang, Juhua; Zhao, Ying; Zhu, Guonian

    2011-11-01

    In this study, zebrafish was exposed to triadimefon. Thyroid hormones levels and the expression of related genes in the hypothalamic-pituitary-thyroid (HPT) axis, including thyroid-stimulating hormone (TSH-beta), deiodinases (dio1 and dio2) and the thyroid hormone receptor (thraa and thrb) were evaluated. After triadimefon exposure, increased T4 can be explained by increased thyroid-stimulating hormone (TSH-beta). The conversion of T4 to T3 (deiodinase type I-dio1) was decreased, which reduced the T3 level. Thyroid hormone receptor beta (thrb) mRNA levels were significantly down-regulated, possibly as a response to the decreased T3 levels. The overall results indicated that triadimefon exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by triadimefon could occur at several steps in the synthesis, regulation, and action of thyroid hormones. PMID:22004968

  5. Prostacyclin receptor expression on platelets of humans with type 2 diabetes is inversely correlated with hemoglobin A1c levels.

    PubMed

    Knebel, Stephanie M; Sprague, Randy S; Stephenson, Alan H

    2015-01-01

    Inappropriate platelet aggregation can result in thrombosis and tissue ischemia. When compared to healthy human platelets, those of humans with type 2 diabetes (DM2) exhibit increased aggregation when stimulated. Activation of the platelet prostacyclin receptor (IPR) results in cAMP accumulation and inhibition of platelet aggregation. We hypothesized that DM2 platelets express decreased IPR when compared to platelets of healthy humans, resulting in decreased IPR agonist-induced cAMP accumulation. We measured IPR expression with radioligand binding of [(3)H]-iloprost, a stable prostacyclin analog, and with Western blotting of the IPR protein. Iloprost-stimulated platelet cAMP levels were used to identify the functional response to IPR activation. IPR binding, expression of the IPR protein and the levels of cAMP in platelets incubated with iloprost were significantly decreased in DM2 platelets when compared to platelets of healthy humans. IPR expression decreased in platelets as glycemic control of the subjects worsened, as indicated by increased hemoglobin A1c levels. Taken together, these findings suggest that reduced IPR expression in DM2 platelets may contribute to platelet hyperactivity in humans with type 2 diabetes. PMID:25617843

  6. Validation of reference genes for real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang.

    PubMed

    Zhao, Wenjing; Li, Yan; Gao, Pengfei; Sun, Zhihong; Sun, Tiansong; Zhang, Heping

    2011-09-01

    Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments under all the different experimental conditions tested. The systematic validation of candidate reference genes is important for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang. PMID:21104423

  7. Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers

    PubMed Central

    Went, P; Vasei, M; Bubendorf, L; Terracciano, L; Tornillo, L; Riede, U; Kononen, J; Simon, R; Sauter, G; Baeuerle, P A

    2006-01-01

    Epithelial cell adhesion molecule (Ep-CAM; CD326) is used as a target by many immunotherapeutic approaches, but little data are available about Ep-CAM expression in major human malignancies with respect to level, frequency, tumour stage, grade, histologic tumour type and impact on survival. We analysed by immunohistochemical staining tissue microarrays with 4046 primary human carcinoma samples from colon, stomach, prostate and lung cancers for both frequency and intensity of Ep-CAM expression under highly standardised conditions. A total of 3360 samples were analysable. High-level Ep-CAM expression was observed in 97.7% (n=1186) of colon, 90.7% of gastric (n=473), and 87.2% of prostate cancers (n=414), and in 63.9% of lung cancers (n=1287). No detectable Ep-CAM staining was found with only 0.4% of colon, 2.5% of gastric, 1.9% of prostate cancers, and 13.5% of lung cancers. The only significant correlation of Ep-CAM expression with tumour grading was observed in colon cancer where high-level Ep-CAM expression on grade 3 tumours was down to 92.1% (P<0.0001). Adenosquamous and squamous carcinomas of the lung had a lower percentage of high-level Ep-CAM expression compared to adenocarcinomas with 35.4 and 53.6%, respectively, and with 45.5 and 17.3% of tumours being Ep-CAM negative. With the exception of moderately differentiated colon carcinoma, where patients not expressing Ep-CAM on their tumours showed an inferior survival (P=0.0014), correlation of Ep-CAM expression with survival did not reach statistical significance for any of the other cancer indications and subgroups. In conclusion, the data strongly support the notion that Ep-CAM is a prime target for immunotherapies in major human malignancies. This is because the most common human cancers show (i) a low frequency of Ep-CAM-negative tumours, (ii) a high frequency of Ep-CAM expression on cells of a given tumour, and (iii) for most cancers, an insignificant influence of tumour staging, grading and histology on

  8. Expression Levels of PPARγ and CYP-19 in Polycystic Ovarian Syndrome Primary Granulosa Cells: Influence of ω-3 Fatty Acid

    PubMed Central

    Zaree, Mina; Shahnazi, Vahideh; Fayezi, Shabnam; Darabi, Maryam; Mehrzad-Sadaghiani, Mahzad; Darabi, Masoud; Khani, Sajjad; Nouri, Mohammad

    2015-01-01

    Background The omega-3 fatty acid (ω-3 fatty acid) such as eicosapentaenoic acid (EPA) is currently used in the clinic as a nutritional supplement in the treatment of poly- cystic ovarian syndrome (PCOS). The present study was designed to investigate the ef- fect of EPA on the expression levels of peroxisome proliferator-activated receptor gamma (PPARγ) and cytochrome P450 aromatase (encoded by the CYP-19) in primary cultured granulosa cells (GC) from patients undergoing in vitro fertilization (IVF), and also to compare these effects with those in GC of PCOS patients. Materials and Methods In this experimental study, human GC were isolated, pri- mary cultured in vitro, exposed to a range of concentrations of the EPA and in- vestigated with respect to gene expression levels of PPARγ and CYP-19 using real time-polymerase chain reaction (PCR). The participants (n=30) were the patients admitted to the IVF Center in February-March 2013 at Alzahra Hospital, Tabriz, Iran, who were divided into two groups as PCOS (n=15) and non-PCOS (n=15) women (controls). Results All doses of the EPA significantly induced PPARγ mRNA gene expression level as compared to the control recombinant follicle stimulating hormone (rFSH) alone condi- tion. High doses of EPA in the presence of rFSH produced a stimulatory effect on expres- sion level of PPARγ (2.15-fold, P=0.001) and a suppressive effect (0.56-fold, P=0.01) on the expression level of CYP-19, only in the PCOS GC. Conclusion EPA and FSH signaling pathway affect differentially on the gene ex- pression levels of PPARγ and CYP-19 in PCOS GC. Altered FSH-induced PPARγ activity in PCOS GC may modulate the CYP-19 gene expression in response to EPA, and possibly modulates the subsequent steroidogenesis of these cells. PMID:26246878

  9. Variation of expression levels of seven housekeeping genes at different life-history stages in Porphyra yezoensis.

    PubMed

    Wu, Xiaojie; Huang, Aiyou; Xu, Meiling; Wang, Chao; Jia, Zhaojun; Wang, Guangce; Niu, Jianfeng

    2013-01-01

    In order to identify the optimal internal control for relative real-time PCR when studying target gene expression in the red alga Porphyra yezoensis, we quantified the expression of seven housekeeping genes (18S ribosomal RNA, 30S ribosomal protein S8, Polyubiquitin-2, Glyceraldehyde-3-phosphate dehydrogenase, Elongation factor 1-alpha, Beta-tubulin and Actin 3) at different life-history stages. Absolute quantification was done by normalization to total RNA quantity and by normalization to genomic DNA quantity. We used these two normalization approaches, comparing the differences of expression levels of all candidate housekeeping genes between any two generations and across three life-history stages (filamentous sporophytes, leafy gametophytes and conchospores). We found GAPDH had the best stability in all cases and we recommend that GAPDH be considered as a potential internal control for gene expression studies at different life-history stages in P. yezoensis. PMID:23637763

  10. An internal ribosome entry site (IRES) mutant library for tuning expression level of multiple genes in mammalian cells.

    PubMed

    Koh, Esther Y C; Ho, Steven C L; Mariati; Song, Zhiwei; Bi, Xuezhi; Bardor, Muriel; Yang, Yuansheng

    2013-01-01

    A set of mutated Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) elements with varying strengths is generated by mutating the translation initiation codons of 10(th), 11(th), and 12(th) AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells. PMID:24349195

  11. An Internal Ribosome Entry Site (IRES) Mutant Library for Tuning Expression Level of Multiple Genes in Mammalian Cells

    PubMed Central

    Koh, Esther Y. C.; Ho, Steven C. L.; Mariati; Song, Zhiwei; Bi, Xuezhi; Bardor, Muriel; Yang, Yuansheng

    2013-01-01

    A set of mutated Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) elements with varying strengths is generated by mutating the translation initiation codons of 10th, 11th, and 12th AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells. PMID:24349195

  12. Co-Cultures of Pseudomonas aeruginosa and Roseobacter denitrificans Reveal Shifts in Gene Expression Levels Compared to Solo Cultures

    PubMed Central

    Conway, Crystal A.; Esiobu, Nwadiuto; Lopez, Jose V.

    2012-01-01

    Consistent biosynthesis of desired secondary metabolites (SMs) from pure microbial cultures is often unreliable. In a proof-of-principle study to induce SM gene expression and production, we describe mixed “co-culturing” conditions and monitoring of messages via quantitative real-time PCR (qPCR). Gene expression of model bacterial strains (Pseudomonas aeruginosa PAO1 and Roseobacter denitrificans Och114) was analyzed in pure solo and mixed cocultures to infer the effects of interspecies interactions on gene expression in vitro, Two P. aeruginosa genes (PhzH coding for portions of the phenazine antibiotic pathway leading to pyocyanin (PCN) and the RhdA gene for thiosulfate: cyanide sulfurtransferase (Rhodanese)) and two R. denitrificans genes (BetaLact for metallo-beta-lactamase and the DMSP gene for dimethylpropiothetin dethiomethylase) were assessed for differential expression. Results showed that R. denitrificans DMSP and BetaLact gene expression became elevated in a mixed culture. In contrast, P. aeruginosa co-cultures with R. denitrificans or a third species did not increase target gene expression above control levels. This paper provides insight for better control of target SM gene expression in vitro and bypass complex genetic engineering manipulations. PMID:22566756

  13. Serum levels of microRNA-133b and microRNA-206 expression predict prognosis in patients with osteosarcoma

    PubMed Central

    Zhang, Chun; Yao, Cong; Li, Haopeng; Wang, Guoyu; He, Xijing

    2014-01-01

    The aim of the present study was to investigate whether the aberrant expression of microRNA (miR)-133b and miR-206 can be used as potential prognostic markers of human osteosarcoma. Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis was performed to detect the expression levels of miR-133b and miR-206 in 100 pairs of osteosarcoma tissues and matched noncancerous bone tissues, and serum samples from 100 patients with osteosarcoma as well as in serum samples from 100 healthy controls. As a result, expression levels of miR-133b and miR-206 were both significantly decreased in osteosarcoma tissues and patients’ sera (both P<0.001). Then, the downregulation of miR-133b and miR-206 both more frequently occurred in osteosarcoma patients with high tumor grade (both P=0.01), positive metastasis (both P<0.001) and recurrence (both P<0.001). Moreover, the patients with low miR-133b expression and low miR-206 expression both had shorter overall survival (OS, both P<0.001) and disease-free survival (DFS, both P<0.001) than those with high expressions. Of note, the OS and DFS of patients with combined low expression of miR-133b and miR-206 (miR-133b-low/miR-206-low) were the shortest (both P<0.001). Furthermore, low miR-133b expression, low miR-206 expression and conjoined expression of miR-133b/miR-206 were all independent prognostic factors for OS and DFS of osteosarcoma patients. Collectively, the aberrant expression of miR-133b and miR-206 may be implicated in tumorigenesis and tumor progression of osteosarcoma. More interestingly, detection of serum miR-133b and miR-206 expression could be further developed as novel, non-invasive and efficient markers for prognosis in patients with osteosarcomas. PMID:25120799

  14. Dietary Selenium Levels Affect Selenoprotein Expression and Support the Interferon-γ and IL-6 Immune Response Pathways in Mice

    PubMed Central

    Tsuji, Petra A.; Carlson, Bradley A.; Anderson, Christine B.; Seifried, Harold E.; Hatfield, Dolph L.; Howard, Michael T.

    2015-01-01

    Selenium is an essential element that is required to support a number of cellular functions and biochemical pathways. The objective of this study was to examine the effects of reduced dietary selenium levels on gene expression to assess changes in expression of non-selenoprotein genes that may contribute to the physiological consequences of selenium deficiency. Mice were fed diets that were either deficient in selenium or supplemented with selenium in the form of sodium selenite for six weeks. Differences in liver mRNA expression and translation were measured using a combination of ribosome profiling, RNA-Seq, microarrays, and qPCR. Expression levels and translation of mRNAs encoding stress-related selenoproteins were shown to be up-regulated by increased selenium status, as were genes involved in inflammation and response to interferon-γ. Changes in serum cytokine levels were measured which confirmed that interferon-γ, as well as IL-6, were increased in selenium adequate mice. Finally, microarray and qPCR analysis of lung tissue demonstrated that the selenium effects on immune function are not limited to liver. These data are consistent with previous reports indicating that adequate selenium levels can support beneficial immune responses, and further identify the IL-6 and interferon-γ pathways as being responsive to dietary selenium intake. PMID:26258789

  15. High-level soluble expression of bioactive porcine myostatin propeptide in E. coli.

    PubMed

    Haq, Wing Yeung; Kang, Sang Kee; Lee, Sang Beum; Kang, Hee Chul; Choi, Yun Jaie; Lee, Chin Nyean; Kim, Yong Soo

    2013-10-01

    Myostatin (MSTN) is a potent negative regulator of skeletal muscle mass. The activity of MSTN is suppressed by MSTN propeptide (MSTNPro), the N-terminal part of unprocessed MSTN that is cleaved off during posttranslational MSTN processing. Easy availability of MSTNPro would help to investigate the potential of the protein as an agent to enhance muscle growth in agricultural animal species. Thus, this study was designed to produce bioactive wild-type porcine MSTN propeptide (pMSTNProW) and its mutated form at the BMP-1/TLD proteolytic cleavage site (pMSTNProM) in Escherichia coli. The pMSTNProW and pMSTNProM genes were separately cloned into pMAL-c5X vector downstream of the maltose-binding protein (MBP) gene and were transformed and expressed in soluble forms in E. coli. For each milliliter of cell culture, about 40 μg of soluble MBP-pMSTNProW and MBP-pMSTNProM proteins were purified by amylose resin affinity chromatography. Further purification by anion exchange chromatography of the affinity-purified fractions yielded about 10 μg/mL culture of MBP-pMSTNProW and MBP-pMSTNProM proteins. Factor Xa protease cleaved the fusion partner MBP from MBP-pMSTNPro proteins, and approximately 4.2 μg of pMSTNProW and pMSTNProM proteins were purified per milliliter of culture. MBP-pMSTNProM was resistant to digestion by BMP-1 metalloproteinase, while MBP-pMSTNProW was cleaved into two fragments by BMP-1. Both MBP-pMSTNProW and MBP-pMSTNProM demonstrated their MSTN binding affinities in a pulldown assay. In an in vitro gene reporter assay, both proteins inhibited MSTN bioactivity without a significant difference in their inhibitory capacities, indicating that the cell culture-based gene reporter assay has limitation in detecting the true in vivo biological potencies of mutant forms of MSTNPro proteins at the BMP-1/TLD cleavage site. Current results show that a high-level production of bioactive porcine MSTNpro is possible in E. coli, and it remains to be investigated

  16. Effect of cadmium on the expression levels of interleukin-1α and interleukin-10 cytokines in human lung cells

    PubMed Central

    ODEWUMI, CAROLINE; LATINWO, LEKAN M.; SINCLAIR, ANDRE; BADISA, VEERA L.D.; ABDULLAH, AHKINYALA; BADISA, RAMESH B.

    2015-01-01

    Cadmium is an environmentally hazardous metal, which causes toxicity in humans. Inhalation of cigarette smoke and industrial fumes containing cadmium are sources of cadmium exposure. It is responsible for the malfunction of various organs, leading to disease particularly in the lungs, liver and kidneys. In the present study, the effect of cadmium chloride (CdCl2) on cell viability, and the expression levels of interleukin (IL)-1α and IL-10 cytokines at various concentrations and incubation durations were assessed in MRC-9 human normal lung and A549 human lung cancer cells to elucidate the mechanism of cadmium toxicity. Cell viability was measured using a crystal violet dye binding assay. The expression levels of the cytokines were measured by cytokine specific enzyme-linked immunosorbent assay kits. The viability assay results revealed higher sensitivity of the A549 lung cancer cells to CdCl2 compared with the normal MRC-9 lung cells. In the normal MRC-9 lung cells, higher expression levels of the cytokines were observed at the lowest CdCl2 concentration at a shorter exposure time compared with the lung cancer cells. Higher levels of the cytokines were observed in the A549 lung cancer cells at all other times and concentrations compared with the MRC-9 cells, indicating higher levels of inflammation. The cytokine levels were reduced at higher CdCl2 concentrations and longer exposure durations, demonstrating the toxic effect of cadmium. The results indicated that CdCl2 affected the expression levels of the cytokines and led to cytotoxicity in human lung cells, and suggested that compounds which reduce inflammation may prevent cadmium toxicity. PMID:26397147

  17. Prepartal dietary energy level affects peripartal bovine blood neutrophil metabolic, antioxidant, and inflammatory gene expression.

    PubMed

    Zhou, Z; Bu, D P; Vailati Riboni, M; Khan, M J; Graugnard, D E; Luo, J; Cardoso, F C; Loor, J J

    2015-08-01

    During the dry period, cows can easily overconsume higher-grain diets, a scenario that could impair immune function during the peripartal period. Objectives were to investigate the effects of energy overfeeding on expression profile of genes associated with inflammation, lipid metabolism, and neutrophil function, in 12 multiparous Holstein cows (n=6/dietary group) fed control [CON, 1.34 Mcal/kg of dry matter (DM)] or higher-energy (HE, 1.62 Mcal/kg of DM) diets during the last 45 d of pregnancy. Blood was collected to evaluate 43 genes in polymorphonuclear neutrophil leukocytes (PMNL) isolated at -14, 7, and 14 d relative to parturition. We detected greater expression of inflammatory-related cytokines (IL1B, STAT3, NFKB1) and eicosanoid synthesis (ALOX5AP and PLA2G4A) in HE cows than in CON cows. Around parturition, all cows had a close balance in mRNA expression of the pro-inflammatory IL1B and the anti-inflammatory IL10, with greater expression of both in cows fed HE than CON. The expression of CCL2, LEPR, TLR4, IL6, and LTC4S was undetectable. Cows in the HE group had greater expression of genes involved in PMNL adhesion, motility, migration, and phagocytosis, which was similar to expression of genes related to the pro-inflammatory cytokine. This response suggests that HE cows experienced a chronic state of inflammation. The greater expression of G6PD in HE cows could have been associated with the greater plasma insulin, which would have diverted glucose to other tissues. Cows fed the HE diet also had greater expression of transcription factors involved in metabolism of long-chain fatty acids (PPARD, RXRA), suggesting that immune cells might be predisposed to use endogenous ligands such as nonesterified fatty acids available in the circulation when glucose is in high demand for milk synthesis. The lower overall expression of SLC2A1 postpartum than prepartum supports this suggestion. Targeting interleukin-1β signaling might be of value in terms of controlling

  18. Penicillin-Binding Protein 5 Sequence Alteration and Levels of plp5 mRNA Expression in Clinical Isolates of Enterococcus faecium with Different Levels of Ampicillin Resistance.

    PubMed

    Belhaj, Mondher; Boutiba-Ben Boubaker, Ilhem; Slim, Amin

    2016-04-01

    Eighty-two nonduplicated ampicillin-resistant Enterococcus faecium (AREF) isolates from clinical infections at the Charles Nicolle Hospital of Tunisia were investigated. They were collected from January 2001 to December 2009. Genetic relationship between them was studied using pulsed-field gel electrophoresis. The amino acid sequence difference variations of the C-terminal part of penicillin-binding protein 5 (PBP5) versus levels of expressed mRNA were investigated by polymerase chain reaction (PCR), sequencing, and real-time PCR quantification of (PBP5), respectively. No β-lactamase activity was detected and none of our strains showed resistance to glycopeptides, which retain their therapeutic efficiency against enterococcal infections in our hospital. Pattern analysis of the strains revealed six main clones disseminating in different wards. Sequence data revealed the existence of 19 different plp5 alleles with a difference in 16 amino acid positions spanning from residue 414 to 632. Each allele presented at least five amino acid substitutions (His-470→Gln, Asn-496→Lys, Ala-499→Thr, Glu-525→Asp, and Glu-629→Val). No correlation between amino acid sequence polymorphism of PBP5 and levels of ampicillin resistance was detected. The levels of plp5 mRNA expression varied between strains and did not always correlate with levels of ampicillin resistance in clinical AREF. PMID:26618475

  19. Enhanced expression of glucose-regulated protein 78 correlates with malondialdehyde levels during the formation of liver cirrhosis in rats

    PubMed Central

    ZHANG, YUN; ZHANG, HUIYING; ZHAO, ZHONGFU; LV, MINLI; JIA, JIANTAO; ZHANG, LILI; TIAN, XIAOXIA; CHEN, YUNXIA; LI, BAOHONG; LIU, MINGSHE; HAN, DEWU; JI, CHENG

    2015-01-01

    The aim of the present study was to explore the role of glucose-regulated protein 78 (GRP78) in the development of liver cirrhosis promoted by intestinal endotoxemia in rats. Fifty-one male Wistar rats were randomly divided into the liver cirrhosis 4-week, 6-week and 8-week groups and the normal control group at each time point. Liver cirrhosis was induced by employing multiple pathogenic factors in the rats. Blood and liver tissues were collected. The levels of alanine aminotransferase (ALT), homocysteine, endotoxin and tumor necrosis factor-α (TNF-α) in the plasma, and TNF-α, malondialdehyde (MDA) and procollagen type III peptide (PIIIP) in the liver tissues were determined. The mRNA and protein expression levels of GRP78 in the liver were detected using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Morphological changes were observed through hematoxylin and eosin and van Gieson staining of the liver. Liver cirrhosis caused marked histopathological changes to the livers of the rats. Following significant increases in the levels of ALT, homocysteine, endotoxin and TNF-α in the plasma, and TNF-α, MDA and PIIIP in the liver tissues of all experimental groups with the progression of liver cirrhosis, the mRNA and protein expression levels of GRP78 also gradually increased. In addition, correlation analysis indicated that the enhanced expression of GRP78 correlated with the MDA levels of the rats during the formation of liver cirrhosis. PMID:26668603

  20. Effect of High Fat Diets on Body Mass, Oleylethanolamide Plasma Levels and Oxytocin Expression in Growing Rats.

    PubMed

    Sospedra, Isabel; Moral, Raquel; Escrich, Raquel; Solanas, Montserrat; Vela, Elena; Escrich, Eduard

    2015-06-01

    Obesity prevalence in developed countries has promoted the need to identify the mechanisms involved in control of feeding and energy balance. We have tested the hypothesis that different fats present in diet composition may contribute in body weight gain and body indexes by regulation of oxytocin gene (oxt) expression in hypothalamus and Oleylethanolamide (OEA) levels in plasma. Sprague-Dawley rats were fed two high fat diets, based on corn (HCO) and extra virgin olive oil (HOO) and results were compared to a low fat diet (LF). LC-MS/MS analysis showed an increasing trend of OEA plasma levels in HOO group, although no significant differences were found. However, body weight gain of LF and HOO were similar and significantly lower than HCO. HCO rats also had higher Lee index than HOO. Rats fed HOO diet showed higher levels of hypothalamic oxt mRNA expression, which could indicate that oxytocin may be modulated by dietary lipids. PMID:25976631

  1. Relationship between circadian oscillations of Rev-erb{alpha} expression and intracellular levels of its ligand, heme

    SciTech Connect

    Rogers, Pamela M.; Ying Ling; Burris, Thomas P.

    2008-04-18

    The nuclear hormone receptors, REV-ERB{alpha} [NR1D1] and REV-ERB{beta} [NR1D1], were recently demonstrated to be receptors for the porphyrin, heme. Heme regulates the ability of these receptors to repress transcription of their target genes via modulation of the affinity of the receptor's ligand binding domain for the corepressor, NCoR. The REV-ERBs function as critical components of the mammalian clock and their expression oscillates in a circadian manner. Here, we show that in NIH3T3 cells intracellular heme levels also oscillate in a circadian fashion. These data are the first to show the temporal relationship of intracellular heme levels to the expression of its receptor, Rev-erb{alpha}, and suggest that the rapid oscillations in heme levels may an important component regulating REV-ERB transcriptional activity.

  2. Vitamin D receptor expression levels determine the severity and complexity of disease progression among leprosy reaction patients

    PubMed Central

    Mandal, D.; Reja, A.H.H.; Biswas, N.; Bhattacharyya, P.; Patra, P.K.; Bhattacharya, B.

    2015-01-01

    We studied the roles of vitamin D and its receptor, VDR, in the progression of leprosy. The majority of individuals with leprosy from Kolkata, India, with a type 1 or type 2 reaction have low levels of vitamin D3 in serum samples. Interestingly, individuals with a type 2 reaction associated with neuritis/erythema nodosum leprosum had very low VDR mRNA expression levels, ranging from 5% to 10%, compared to that of healthy control subjects; these patients also had a high bacilli index, ranging from 3+ to 5+. This is the first report to indicate that VDR expression levels may determine the complexity and severity of the progression of leprosy. PMID:26106480

  3. Vitamin D receptor expression levels determine the severity and complexity of disease progression among leprosy reaction patients.

    PubMed

    Mandal, D; Reja, A H H; Biswas, N; Bhattacharyya, P; Patra, P K; Bhattacharya, B

    2015-07-01

    We studied the roles of vitamin D and its receptor, VDR, in the progression of leprosy. The majority of individuals with leprosy from Kolkata, India, with a type 1 or type 2 reaction have low levels of vitamin D3 in serum samples. Interestingly, individuals with a type 2 reaction associated with neuritis/erythema nodosum leprosum had very low VDR mRNA expression levels, ranging from 5% to 10%, compared to that of healthy control subjects; these patients also had a high bacilli index, ranging from 3+ to 5+. This is the first report to indicate that VDR expression levels may determine the complexity and severity of the progression of leprosy. PMID:26106480

  4. Characteristics of carboxylesterase genes and their expression-level between acaricide-susceptible and resistant Tetranychus cinnabarinus (Boisduval).

    PubMed

    Wei, Peng; Shi, Li; Shen, Guangmao; Xu, Zhifeng; Liu, Jialu; Pan, Yu; He, Lin

    2016-07-01

    Carboxylesterases (CarEs) play important roles in metabolism and detoxification of dietary and environmental xenobiotics in insects and mites. On the basis of the Tetranychuscinnabarinus transcriptome dataset, 23 CarE genes (6 genes are full sequence and 17 genes are partial sequence) were identified. Synergist bioassay showed that CarEs were involved in acaricide detoxification and resistance in fenpropathrin- (FeR) and cyflumetofen-resistant (CyR) strains. In order to further reveal the relationship between CarE gene's expression and acaricide-resistance in T. cinnabarinus, we profiled their expression in susceptible (SS) and resistant strains (FeR, and CyR). There were 8 and 4 over-expressed carboxylesterase genes in FeR and CyR, respectively, from which the over-expressions were detected at mRNA level, but not DNA level. Pesticide induction experiment elucidated that 4 of 8 and 2 of 4 up-regulated genes were inducible with significance in FeR and CyR strains, respectively, but they could not be induced in SS strain, which indicated that these genes became more enhanced and effective to withstand the pesticides' stress in resistant T. cinnabarinus. Most expression-changed and all inducible genes possess the Abhydrolase_3 motif, which is a catalytic domain for hydrolyzing. As a whole, these findings in current study provide clues for further elucidating the function and regulation mechanism of these carboxylesterase genes in T. cinnabarinus' resistance formation. PMID:27265830

  5. Expression of vaspin in the joint and the levels in the serum and synovial fluid of patients with osteoarthritis

    PubMed Central

    Bao, Jia-Peng; Jiang, Li-Feng; Chen, Wei-Ping; Hu, Peng-Fei; Wu, Li-Dong

    2014-01-01

    The aim of this study was to determine the expression of vaspin in the joint and investigate the distribution between paired serum and synovial fluid (SF) in osteoarthritis (OA) patients, and serum in healthy controls. The gene expression of vaspin was measured by quantitative real-time polymerase chain reaction (qPCR) in the OA joint tissues. The vaspin protein expression in the cartilage, synovium and osteophyte from OA patients who required total knee replacement (TKR) were detected by immunohistochemistry (IHC). Levels of vaspin in serum and SF were analyzed by enzyme-linked immunosorbent assay (ELISA), including 26 OA patients and 23 healthy controls. All the joint tissues including cartilage, synovium, meniscus, infrapatellar fat pad and osteophyte from OA patients expressed vaspin messenger RNA (mRNA), and the expression of vaspin protein was observed in OA cartilage, synovium and osteophyte. Furthermore, serum vaspin was reduced in OA patients compared to healthy controls, and serum vaspin levels from OA patients exceed those in the paired SF. Serum or SF vaspin were not related to age, gender, or body mass index (BMI). These results suggest that vaspin may be involved in the pathophysiology of OA and may have local effects in the joint during the process of OA. PMID:25419381

  6. Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

    PubMed Central

    Wiencierz, Anne Maria; Kernbach, Manuel; Ecklebe, Josephine; Monnerat, Gustavo; Tomiuk, Stefan; Raulf, Alexandra; Christalla, Peter; Malan, Daniela; Hesse, Michael; Bosio, Andreas; Fleischmann, Bernd K.; Eckardt, Dominik

    2015-01-01

    Rationale Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers. Methods and Results In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis. Conclusion Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications. PMID:26618511

  7. MMBGX: a method for estimating expression at the isoform level and detecting differential splicing using whole-transcript Affymetrix arrays

    PubMed Central

    Turro, Ernest; Lewin, Alex; Rose, Anna; Dallman, Margaret J.; Richardson, Sylvia

    2010-01-01

    Affymetrix has recently developed whole-transcript GeneChips—‘Gene’ and ‘Exon’ arrays—which interrogate exons along the length of each gene. Although each probe on these arrays is intended to hybridize perfectly to only one transcriptional target, many probes match multiple transcripts located in different parts of the genome or alternative isoforms of the same gene. Existing statistical methods for estimating expression do not take this into account and are thus prone to producing inflated estimates. We propose a method, Multi-Mapping Bayesian Gene eXpression (MMBGX), which disaggregates the signal at ‘multi-match’ probes. When applied to Gene arrays, MMBGX removes the upward bias of gene-level expression estimates. When applied to Exon arrays, it can further disaggregate the signal between alternative transcripts of the same gene, providing expression estimates of individual splice variants. We demonstrate the performance of MMBGX on simulated data and a tissue mixture data set. We then show that MMBGX can estimate the expression of alternative isoforms within one experimental condition, confirming our results by RT-PCR. Finally, we show that our method for detecting differential splicing has a lower error rate than standard exon-level approaches on a previously validated colon cancer data set. PMID:19854940

  8. Boric acid increases the expression levels of human anion exchanger genes SLC4A2 and SLC4A3.

    PubMed

    Akbas, F; Aydin, Z

    2012-01-01

    Boron is an important micronutrient in plants and animals. The role of boron in living systems includes coordinated regulation of gene expression, growth and proliferation of higher plants and animals. There are several well-defined genes associated with boron transportation and tolerance in plants and these genes show close homology with human anion exchanger genes. Mutation of these genes also characterizes some genetic disorders. We investigated the toxic effects of boric acid on HEK293 cells and mRNA expression of anion exchanger (SLC4A1, SLC4A2 and SLC4A3) genes. Cytotoxicity of boric acid at different concentrations was tested by using the methylthiazolyldiphenyl-tetrazolium bromide assay. Gene expression profiles were examined using quantitative real-time PCR. In the HEK293 cells, the nontoxic upper concentration of boric acid was 250 μM; more than 500 μM caused cytotoxicity. The 250 μM boric acid concentration increased gene expression level of SLC4A2 up to 8.6-fold and SLC4A3 up to 2.6-fold, after 36-h incubation. There was no significant effect of boric acid on SLC4A1 mRNA expression levels. PMID:22576912

  9. Synergistic and antagonistic interplay between myostatin gene expression and physical activity levels on gene expression patterns in triceps Brachii muscles of C57/BL6 mice.

    PubMed

    Caetano-Anollés, Kelsey; Mishra, Sanjibita; Rodriguez-Zas, Sandra L

    2015-01-01

    Levels of myostatin expression and physical activity have both been associated with transcriptome dysregulation and skeletal muscle hypertrophy. The transcriptome of triceps brachii muscles from male C57/BL6 mice corresponding to two genotypes (wild-type and myostatin-reduced) under two conditions (high and low physical activity) was characterized using RNA-Seq. Synergistic and antagonistic interaction and ortholog modes of action of myostatin genotype and activity level on genes and gene pathways in this skeletal muscle were uncovered; 1,836, 238, and 399 genes exhibited significant (FDR-adjusted P-value < 0.005) activity-by-genotype interaction, genotype and activity effects, respectively. The most common differentially expressed profiles were (i) inactive myostatin-reduced relative to active and inactive wild-type, (ii) inactive myostatin-reduced and active wild-type, and (iii) inactive myostatin-reduced and inactive wild-type. Several remarkable genes and gene pathways were identified. The expression profile of nascent polypeptide-associated complex alpha subunit (Naca) supports a synergistic interaction between activity level and myostatin genotype, while Gremlin 2 (Grem2) displayed an antagonistic interaction. Comparison between activity levels revealed expression changes in genes encoding for structural proteins important for muscle function (including troponin, tropomyosin and myoglobin) and for fatty acid metabolism (some linked to diabetes and obesity, DNA-repair, stem cell renewal, and various forms of cancer). Conversely, comparison between genotype groups revealed changes in genes associated with G1-to-S-phase transition of the cell cycle of myoblasts and the expression of Grem2 proteins that modulate the cleavage of the myostatin propeptide. A number of myostatin-feedback regulated gene products that are primarily regulatory were uncovered, including microRNA impacting central functions and Piezo proteins that make cationic current

  10. Synergistic and Antagonistic Interplay between Myostatin Gene Expression and Physical Activity Levels on Gene Expression Patterns in Triceps Brachii Muscles of C57/BL6 Mice

    PubMed Central

    Caetano-Anollés, Kelsey; Mishra, Sanjibita; Rodriguez-Zas, Sandra L.

    2015-01-01

    Levels of myostatin expression and physical activity have both been associated with transcriptome dysregulation and skeletal muscle hypertrophy. The transcriptome of triceps brachii muscles from male C57/BL6 mice corresponding to two genotypes (wild-type and myostatin-reduced) under two conditions (high and low physical activity) was characterized using RNA-Seq. Synergistic and antagonistic interaction and ortholog modes of action of myostatin genotype and activity level on genes and gene pathways in this skeletal muscle were uncovered; 1,836, 238, and 399 genes exhibited significant (FDR-adjusted P-value < 0.005) activity-by-genotype interaction, genotype and activity effects, respectively. The most common differentially expressed profiles were (i) inactive myostatin-reduced relative to active and inactive wild-type, (ii) inactive myostatin-reduced and active wild-type, and (iii) inactive myostatin-reduced and inactive wild-type. Several remarkable genes and gene pathways were identified. The expression profile of nascent polypeptide-associated complex alpha subunit (Naca) supports a synergistic interaction between activity level and myostatin genotype, while Gremlin 2 (Grem2) displayed an antagonistic interaction. Comparison between activity levels revealed expression changes in genes encoding for structural proteins important for muscle function (including troponin, tropomyosin and myoglobin) and for fatty acid metabolism (some linked to diabetes and obesity, DNA-repair, stem cell renewal, and various forms of cancer). Conversely, comparison between genotype groups revealed changes in genes associated with G1-to-S-phase transition of the cell cycle of myoblasts and the expression of Grem2 proteins that modulate the cleavage of the myostatin propeptide. A number of myostatin-feedback regulated gene products that are primarily regulatory were uncovered, including microRNA impacting central functions and Piezo proteins that make cationic current

  11. Express

    Integrated Risk Information System (IRIS)

    Express ; CASRN 101200 - 48 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effect

  12. High expression levels of egfl7 correlate with low endothelial cell activation in peritumoral vessels of human breast cancer

    PubMed Central

    Pannier, Diane; Philippin-Lauridant, Géraldine; Baranzelli, Marie-Christine; Bertin, Delphine; Bogart, Emilie; Delprat, Victor; Villain, Gaëlle; Mattot, Virginie; Bonneterre, Jacques; Soncin, Fabrice

    2016-01-01

    Tumor blood vessels participate in the immune response against cancer cells and we previously used pre-clinical models to demonstrate that egfl7 (VE-statin) promotes tumor cell evasion from the immune system by repressing endothelial cell activation, preventing immune cells from entering the tumor mass. In the present study, the expression levels of egfl7 and that of ICAM-1 as a marker of endothelium activation, were evaluated in peritumoral vessels of human breast cancer samples. Breast cancer samples (174 invasive and 30 in situ) from 204 patients treated in 2005 were immunostained for CD31, ICAM-1 and stained for egfl7 using in situ hybridization. The expression levels of ICAM-1 and egfl7 were assessed in peritumoral areas using semi-quantitative scales. There was a strong and significant inverse correlation between the expression of ICAM-1 and that of egfl7 in CD31+ blood vessels. When the ICAM-1 score increased, the egfl7 score reduced significantly (P=0.004), and vice-versa (Cuzick's test for trend across ordered groups). In order to determine which gene influenced the other gene between egfl7 and ICAM-1, the expression levels of either gene were modulated in endothelial cells. Egfl7 regulated ICAM-1 expression while ICAM-1 had no effects on egfl7 expression in the same conditions. Altogether, these results provide further results that egfl7 serves a regulatory role in endothelial cell activation in relation to immune infiltration and that it is a potential therapeutic target to consider for improving anticancer immunotherapies. PMID:27446447

  13. High levels of expression of the Iron-Sulfur Proteins Phthalate Dioxygenase and Phthalate Dioxygenase Reductase in Escherichia coli

    PubMed Central

    Jaganaman, Sunil; Pinto, Alex; Tarasev, Michael; Ballou, David P.

    2007-01-01

    Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II) - mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at ∼30 mg of crude PDO and ∼1 mg of crude PDR per liter of cell culture when grown with phthalate as the main carbon source. A high level expression system in Escherichia coli was developed for PDO and PDR. Optimization relative to Escherichia coli cell line, growth parameters, time of induction, media composition, and iron-sulfur additives resulted in yields of about 1 g/L for PDO and about 0.2 g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20 hours) lag after the induction. The specific activity of purified PDO did not depend on the pH of the cell culture when harvested. However, when the pH of the culture reached 8.5-9, a large fraction of the PDR that was expressed lacked its ferredoxin domain, presumably because of proteolysis. Termination of growth while the pH of the cell culture was < 8 decreased the fraction of proteolyzed enzyme, whereas yields of the unclipped PDR were only marginally lower. Overall, changes in pH of the cell culture were found to be an excellent indicator of the overall level of native protein expression. Its monitoring allowed the real time tracking of the protein expression and made it possible to tailor the expression times to achieve a combination of high quality and high yield of protein. PMID:17049880

  14. HLA reduces KIR expression level and frequency in a humanized mouse model1

    PubMed Central

    van Bergen, Jeroen; Thompson, Allan; Retière, Christelle; van Pel, Melissa; Salvatori, Daniela; Lemonnier, François; Raulet, David; Trowsdale, John; Koning, Frits

    2014-01-01

    Many human Natural Killer (NK) cells are prevented from killing autologous cells by virtue of inhibitory Killer cell Immunoglobulin-like Receptors (KIR) binding `self' HLA class I molecules. Individual NK cells stably express a selected set of KIR, but it is currently disputed whether the fraction of NK cells expressing a particular inhibitory KIR is influenced by the presence of the corresponding HLA ligand. This issue has been particularly hard to tackle in a statistically meaningful way due to the extreme polymorphism of the KIR and HLA loci, with widely varying affinities for individual KIR and HLA allele combinations. Here, we use a transgenic mouse model to investigate the effect of HLA on KIR repertoire and function. In this model system, a functional interaction between HLA-Cw3 and KIR2DL2 reduced both the surface expression of KIR2DL2 as well as the frequency of KIR2DL2+ cells. PMID:23390293

  15. [Bioinformatics analysis and expressed level of histone methyltransferase genes in Lonicera japonica].

    PubMed

    Qi, Lin-jie; Yuan, Yuan; Huang, Lu-qi; Long, Ping; Zha, Liang-ping; Wang, Yao-long

    2015-06-01

    Twenty-three histone methyltransferase genes were obtained from transcriptome dataset of Lonicera japonica. The nucleotide and proteins characteristics, subcellular localization, senior structural domains and conservative forecasting were analyzed. The result of phylogenetic tree showed that 23 histone methyltransferases were mainly divided into two groups: lysine methyltransferase and arginine methyltransferases. The result of gene expression showed that 23 histone methyltransferases showed preference in terms of interspecies and organs. They were more expressed in buds of L. japonica than in L. japonica var. chinensis and lower in leaves of L. japonica than in L. japonica var. chinensis. Eight genes were specific expressed in flower. These results provided basis for further understanding the function of histone methyltransferase and epigenetic regulation of active ingredients of L. japonica. PMID:26552158

  16. Effects of ultrasound on estradiol level, bone mineral density, bone biomechanics and matrix metalloproteinase-13 expression in ovariectomized rabbits

    PubMed Central

    XIA, LU; HE, HONGCHEN; GUO, HUA; QING, YUXI; HE, CHENG-QI

    2015-01-01

    The aim of the present study was to observe the effect of ultrasound (US) on estradiol level, bone mineral density (BMD), bone biomechanics and matrix metalloproteinase-13 (MMP-13) expression in ovariectomized (OVX) rabbits. A total of 28 virgin New Zealand white rabbits were randomly assigned into the following groups: Control (control group), ovariectomy (OVX group), ovariectomy with ultrasound therapy (US group) and ovariectomy with estrogen replacement therapy group (ERT group). At 8 weeks after ovariectomy, the US group received ultrasound treatment while the ERT group were orally treated with conjugated estrogens, and the control and OVX groups remained untreated. The estradiol level, BMD and bone biomechanics, cartilage histology and the MMP-13 expression were analyzed after the intervention. The results indicate that the US treatment increased estradiol level, BMD and bone biomechanical function. Furthermore, the US treatment appeared to improve the recovery of cartilage morphology and decreased the expression of MMP-13 in OVX models. Furthermore, the results suggest that 10 days of US therapy was sufficient to prevent the reduction of estradiol, BMD and bone biomechanical function, to protect osteoarthritis cartilage structure, and to reduce MMP-13 transcription and expression in OVX rabbits. Therefore, US treatment may be a potential treatment for postmenopausal osteoarthritis and osteoporosis. PMID:26622502

  17. Hepatocyte growth factor stimulates motility, chemotaxis and mitogenesis in ovarian carcinoma cells expressing high levels of c-met.

    PubMed

    Corps, A N; Sowter, H M; Smith, S K

    1997-09-26

    A proportion of ovarian carcinomas markedly overexpress the proto-oncogene c-met, which encodes the receptor for hepatocyte growth factor (HGF). HGF may either stimulate or inhibit the multiplication of its target cells, and may also promote motogenesis and morphogenesis. In this study, we established that the ovarian carcinoma-derived cell-line SK-OV-3 expressed about 20-fold higher levels of c-met protein than are expressed by a second line, CH1. This enabled us to test functional consequences of high-level expression of c-met in ovarian carcinoma cells. The addition of HGF to attached cultures of SK-OV-3 cells caused a change to a motile phenotype, that was evident after 4-6 hr and affected essentially all of the cells by 24 hr. When HGF was placed in the lower compartment of a migration chamber, it induced a 17-fold increase in the migration of SK-OV-3 cells to the lower surface of the filter. Finally, HGF stimulated the incorporation of [3H]-thymidine by cultures of SK-OV-3 cells incubated in medium containing either low (0.2%) or full (10%) FCS. None of these responses were obtained when HGF was added to CH1 cells. We conclude that high levels of c-met expression in ovarian cancer cells may lead to a range of responses to HGF that would promote tumour growth and dissemination. PMID:9334823

  18. Effects of low-level laser therapy on M1-related cytokine expression in monocytes via histone modification.

    PubMed

    Chen, Chia-Hsin; Wang, Chau-Zen; Wang, Yan-Hsiung; Liao, Wei-Ting; Chen, Yi-Jen; Kuo, Chang-Hung; Kuo, Hsuan-Fu; Hung, Chih-Hsing

    2014-01-01

    Low-level laser therapy (LLLT) has been used in the treatment of radiotherapy-induced oral mucositis and allergic rhinitis. However, the effects of LLLT on human monocyte polarization into M1 macrophages are unknown. To evaluate the effects of LLLT on M1-related cytokine and chemokine production and elucidate the mechanism, the human monocyte cell line THP-1 was treated with different doses of LLLT. The expression of M1-related cytokines and chemokines (CCL2, CXCL10, and TNF-α) was determined by ELISA and real-time PCR. LLLT-associated histone modifications were examined by chromatin immunoprecipitation (ChIP) assays. Mitochondrial involvement in the LLLT-induced M1-related cytokine expression was evaluated by quantitative real-time PCR. Flow cytometry was used to detect the cell surface markers for monocyte polarization. The results showed that LLLT (660 nm) significantly enhanced M1-related cytokine and chemokine expression in mRNA and protein levels. Mitochondrial copy number and mRNA levels of complex I-V protein were increased by LLLT (1 J/cm(2)). Activation of M1 polarization was concomitant with histone modification at TNF-α gene locus and IP-10 gene promoter area. This study indicates that LLLT (660 nm) enhanced M1-related cytokine and chemokine expression via mitochondrial biogenesis and histone modification, which may be a potent immune-enhancing agent for the treatment of allergic diseases. PMID:24692853

  19. Post-harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers.

    PubMed

    Larraya, Luis M; Fernández-San Millán, Alicia; Ancín, María; Farran, Inmaculada; Veramendi, Jon

    2015-09-01

    Plastid genetic engineering represents an attractive system for the production of foreign proteins in plants. Although high expression levels can be achieved in leaf chloroplasts, the results for non-photosynthetic plastids are generally discouraging. Here, we report the expression of two thioredoxin genes (trx f and trx m) from the potato plastid genome to study transgene expression in amyloplasts. As expected, the highest transgene expression was detected in the leaf (up to 4.2% of TSP). The Trx protein content in the tuber was approximately two to three orders of magnitude lower than in the leaf. However, we demonstrate that a simple post-harvest light treatment of microtubers developed in vitro or soil-grown tubers induces up to 55 times higher accumulation of the recombinant protein in just seven to ten days. After the applied treatment, the Trx f levels in microtubers and soil-grown tubers increased to 0.14% and 0.11% of TSP, respectively. Moreover, tubers stored for eight months maintained the capacity of increasing the foreign protein levels after the light treatment. Post-harvest cold induction (up to five times) at 4°C was also detected in microtubers. We conclude that plastid transformation and post-harvest light treatment could be an interesting approach for the production of foreign proteins in potato. PMID:26121393

  20. ACC2 Is Expressed at High Levels Human White Adipose and Has an Isoform with a Novel N-Terminus

    PubMed Central

    Raymond, Christopher K.; Garrett-Engele, Philip; Ohwaki, Kenji; Kan, Zhengyan; Kusunoki, Jun; Johnson, Jason M.

    2009-01-01

    Acetyl-CoA carboxylases ACC1 and ACC2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA, regulating fatty-acid synthesis and oxidation, and are potential targets for treatment of metabolic syndrome. Expression of ACC1 in rodent lipogenic tissues and ACC2 in rodent oxidative tissues, coupled with the predicted localization of ACC2 to the mitochondrial membrane, have suggested separate functional roles for ACC1 in lipogenesis and ACC2 in fatty acid oxidation. We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart. Human adipose, along with human liver, expresses more ACC2 than ACC1. Using RT-PCR, real-time PCR, and immunoprecipitation we report a novel isoform of ACC2 (ACC2.v2) that is expressed at significant levels in human adipose. The protein generated by this isoform has enzymatic activity, is endogenously expressed in adipose, and lacks the N-terminal sequence. Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis. The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors. PMID:19190759

  1. A structured sparse regression method for estimating isoform expression level from multi-sample RNA-seq data.

    PubMed

    Zhang, L; Liu, X J

    2016-01-01

    With the rapid development of next-generation high-throughput sequencing technology, RNA-seq has become a standard and important technique for transcriptome analysis. For multi-sample RNA-seq data, the existing expression estimation methods usually deal with each single-RNA-seq sample, and ignore that the read distributions are consistent across multiple samples. In the current study, we propose a structured sparse regression method, SSRSeq, to estimate isoform expression using multi-sample RNA-seq data. SSRSeq uses a non-parameter model to capture the general tendency of non-uniformity read distribution for all genes across multiple samples. Additionally, our method adds a structured sparse regularization, which not only incorporates the sparse specificity between a gene and its corresponding isoform expression levels, but also reduces the effects of noisy reads, especially for lowly expressed genes and isoforms. Four real datasets were used to evaluate our method on isoform expression estimation. Compared with other popular methods, SSRSeq reduced the variance between multiple samples, and produced more accurate isoform expression estimations, and thus more meaningful biological interpretations. PMID:27323111

  2. Olfactory discrimination varies in mice with different levels of α7-nicotinic acetylcholine receptor expression

    PubMed Central

    Hellier, Jennifer L.; Arevalo, Nicole L.; Blatner, Megan J.; Dang, An K.; Clevenger, Amy C.; Adams, Catherine E.; Restrepo, Diego

    2010-01-01

    Previous studies have shown that schizophrenics have decreased expression of α7-nicotinic acetylcholine (α7) receptors in the hippocampus and other brain regions, paranoid delusions, disorganized speech, deficits in auditory gating (i.e., inability to inhibit neuronal responses to repetitive auditory stimuli), and difficulties in odor discrimination and detection. Here we use mice with decreased α7 expression that also show a deficit in auditory gating to determine if these mice have similar deficits in olfaction. In the adult mouse olfactory bulb (OB), α7 expression localizes in the glomerular layer; however, the functional role of α7 is unknown. We show that inbred mouse strains (i.e., C3H and C57) with varying α7 expression (e.g., α7 wild-type [α7+/+], α7 heterozygous knock-out [α7+/−] and α7 homozygous knockout mice [α7−/−]) significantly differ in odor discrimination and detection of chemically related odorant pairs. Using [125I] α-bungarotoxin (α-BGT) autoradiography, α7 expression was measured in the OB. As previously demonstrated, α-BGT binding was localized to the glomerular layer. Significantly more expression of α7 was observed in C57 α7+/+ mice compared to C3H α7+/+ mice. Furthermore, C57 α7+/+ mice were able to detect a significantly lower concentration of an odor in a mixture compared to C3H α7+/+ mice. Both C57 and C3H α7+/+ mice discriminated between chemically related odorants sooner than α7+/− or α7−/− mice. These data suggest that α7-nicotinic-receptors contribute strongly to olfactory discrimination and detection in mice and may be one of the mechanisms producing olfactory dysfunction in schizophrenics. PMID:20713028

  3. Expression levels of IL-17 and TNF-α in degenerated lumbar intervertebral discs and their correlation

    PubMed Central

    LIU, XIAO-GANG; HOU, HONG-WEI; LIU, YI-LIN

    2016-01-01

    The present study aimed to investigate the expression and roles of interleukin (IL)-17 and tumor necrosis factor (TNF)-α in intervertebral disc degeneration (IDD) and to identify the association between the effects of IL-17 and TNF-α in IDD. This may increase understanding of the pathogenic mechanism underlying IDD, and aid the development of alternative therapies. The experimental group consisted of 40 samples of nucleus pulposus tissue obtained from the intervertebral discs (IVDs) of patients with IDD by surgical intervention, and was further divided into an annulus fibrosus disrupted group, comprising 18 patients in which the external annulus was ruptured, and an annulus fibrosus intact group comprising 22 patients. The control group consisted of 20 samples of nucleus pulposus tissue from the IVDs of patients with traumatic lumbar disc fractures. The mRNA and protein expression levels of IL-17 and TNF-α in the 50 tissue samples were detected by semi-quantitative reverse transcription polymerase chain reaction and immunohistochemical staining, respectively, and the results were statistically analyzed. The IL-17 and TNF-α protein and mRNA expression levels in the annulus fibrosus disrupted and annulus fibrosus intact groups were both higher compared with those in the control group. In addition, the expression levels of IL-17 and TNF-α in the annulus fibrosus disrupted group were significantly higher compared with those in the annulus fibrosus intact group (P<0.01). A positive correlation was identified between the mRNA and protein expression levels of IL-17 and TNF-α in the experimental group (r=0.957, P<0.01). IL-17 and TNF-α may therefore be involved in the progression of human IDD, and may have synergistic effects in the development of IDD. PMID:27284317

  4. High-level expression of HOXB13 is closely associated with tumor angiogenesis and poor prognosis of hepatocellular carcinoma

    PubMed Central

    Zhu, Jian-Yu; Sun, Qi-Kai; Wang, Wei; Jia, Wei-Dong

    2014-01-01

    Homeobox B13 (HOXB13) is generally considered as a crucial regulator of terminal cellular differentiation. More recently, the absent or aberrant expression of HOXB13 has been increasingly implicated in cancer development and metastasis. However, the expression of HOXB13 in hepatocellular carcinoma (HCC) and its correlation with tumor angiogenesis and prognosis still remain unclear. The aim of the study was to evaluate the expression of HOXB13 in patients with HCC and explore the relationship of HOXB13 expression with clinicopathologic factors, tumor angiogenesis and prognosis. Immunohistochemistry was performed to determine the expression of HOXB13 in HCC and corresponding paracarcinomatous tissues from 72 patients. Vascular endothelial growth factor (VEGF) and CD31 were only examined in tissues of HCC patients mentioned above. The results showed that HOXB13 expression was significantly (P <0.001) higher in HCC (69.4%) than that in surrounding non-tumor tissues (26.4%), positively correlated with tumor VEGF (P <0.001) and microvessel density (MVD) (P = 0.013). Besides, it was associated with tumor capsula (P <0.001), vascular invasion (P <0.001), Edmondson grade (P <0.001), AFP (P = 0.007) and TNM stage (P <0.001). Univariate analysis showed poorer overall survival (OS) rate and disease free survival (DFS) rate in patients expressing higher levels of HOXB13. HOXB13 was also found to be an independent poor prognostic factor of OS and DFS in multivariate analysis. Taken together, our results suggest that increased HOXB13 expression is associated with tumor angiogenesis and progression in HCC and may function as a promising biomarker for unfavorable prognosis of HCC. PMID:25031711

  5. Diagnostic investigations of DKK-1 and PDCD5 expression levels as independent prognostic markers of human chondrosarcoma.

    PubMed

    Zarea, Mojtaba; Mohammadian Bajgiran, Amirhossein; Sedaghati, Farnoush; Hatami, Negin; Taheriazam, Afshin; Yahaghi, Emad; Shakeri, Mohammadreza

    2016-07-01

    In this study, we investigated the expression levels of Dickkopf-1 (DKK-1) and programmed cell death 5 (PDCD5) by using quantitative real-time PCR and immunohistochemistry in patients with chondrosarcoma. The DKK-1 mRNA levels were significantly higher in chondrosarcoma when compared with the corresponding nontumor tissues (mean ± SD: 4.23 ± 1.54; 1.54 ± 0.87; P = 0.001). PDCD5 mRNA levels were remarkably deceased in tumor tissues when compared with corresponding nontumor tissues (mean ± SD: 1.94 ± 0.73; 5.42 ± 1.73; P = 0.001). The high and moderate DKK-1 expressions were observed for 60% of chondrosarcoma samples in comparison with 27.5% of corresponding nontumor tissues (P  =  0.001). Moreover, low expression of PDCD5 was found in 67.5% of the tumor tissues when compared with the nontumor tissues (32.5%; P = 0.002). The results of this study showed that high DKK-1 expression levels were strongly related to MSTS stage (P = 0.011) and the advancement of histological grade (P < 0.001). Furthermore, the PDCD5 expression levels were correlated with histological grade (P < 0.001), MSTS stage (P = 0.016), and distant metastasis (P = 0.001). Kaplan-Meier survival and log-rank survival showed that patients with high DKK-1 levels and low PDCD5 levels were correlated with shorter overall survival (log-rank test P < 0.001). PDCD5 levels, histological grade, and tumor stage were independent predictors of overall survival. In conclusion, DKK-1 and PDCD5 can be independent predictors of overall survival in patients suffering from chondrosarcoma. © 2016 IUBMB Life, 68(7):597-601, 2016. PMID:27255549

  6. Changes in endogenous gene transcript and protein levels in maize plants expressing the soybean ferritin transgene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic agricultural crops with increased nutritive value present prospects for contributing to public health. However, their acceptance is poor in many countries due to the perception that genetic modification may cause unintended effects on expression of native genes in the host plant. Here, w...

  7. Expression of a foreign Rubisco small subunit in tobacco with reduced levels of the native protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cDNA, ArRbcS3, for the small subunit of Rubisco from Amaranthus retroflexus (pigweed) was expressed in tobacco (Nicotiana tabacum) under the control of a strong leaf-specific Lhcb promoter. The coding region of the ArRbcS3 was fused to the plastid targeting sequence of the native tobacco rbcS to...

  8. Characteristics of High-Risk College Student Drinkers Expressing High and Low Levels of Distress

    ERIC Educational Resources Information Center

    Graceffo, James M.; Hayes, Jeffrey A.; Chun-Kennedy, Caitlin; Locke, Benjamin D.

    2012-01-01

    The aim of this study was to identify variables that reliably differentiated between 2 groups of students who reported binge drinking at the same rate (6 to more than 10 times within the previous 2 weeks) but who exhibited different distress associated with their behavior. Results indicated that students who received an external expression of…

  9. Protein expression levels in the medullary visceral zone of rats with subarachnoid hemorrhage.

    PubMed

    Sun, L H; Xing, L F; Zhang, G H; Pan, S Y

    2015-01-01

    We investigated protein expression in the medullary visceral zone (MVZ) of rats with multiple-organ dysfunction syndrome (MODS) caused by subarachnoid hemorrhage (SAH) to discuss the possible regulatory mechanism of the MVZ in the course of SAH-induced MODS. A SAH-induced MODS model was established in rats by injecting arterial blood into the Willis' circle. Protein expression in the MVZ was analyzed by immunohistochemistry assay. Protein expression in the MVZ peaked 24-36 h after SAH, and was significantly higher than in the control and sham operation groups. Organs at each time point exhibited inflammatory injuries to varying degrees after SAH, which reached a maximum at 24-36 h. Incidences of systemic inflammatory response syndrome and MODS were 100 and 71.67%, respectively, after SAH. There is a consistency between MVZ protein expression and inflammatory changes in each organ after SAH. This prompts the suggestion that the MVZ may be one of the direct regulative centers in SAH-induced MODS, and may be involved in the functional regulation of the surrounding organs after SAH. PMID:26345815

  10. Sources of variation in baseline gene expression levels from toxicogenomics study control animals

    EPA Science Inventory

    The use of gene expression profiling in both clinical and laboratory settings would be enhanced by better characterization ofvariance due to individual, environmental, and technical factors. Meta-analysis ofmicroarray data from untreated or vehicle-treated animals within the con...

  11. Quantitative analysis of the mRNA expression levels of BCL2 and BAX genes in human osteoarthritis and normal articular cartilage: An investigation into their differential expression.

    PubMed

    Karaliotas, Georgios I; Mavridis, Konstantinos; Scorilas, Andreas; Babis, George C

    2015-09-01

    Osteoarthritis (OA) is primarily characterized by articular cartilage degeneration and chondrocyte loss. Although the role of apoptosis in cartilage pathobiology remains to be elucidated, the apoptotic B‑cell CLL/lymphoma 2 (BCL2) gene family is considered to be involved in OA. The purpose of the present study was to quantitatively analyze the mRNA expression profiles of the BCL2‑associated X protein (BAX) and BCL2 genes in human OA and in normal cartilage. Cartilage tissue samples were obtained from 78 patients undergoing total knee arthroplasty for OA (OA group) and orthopedic interventions for causes other than OA (control group). Total RNA was isolated from the cartilage tissue specimens and reverse transcribed into cDNA. A highly sensitive and specific reverse transcription quantitative polymerase chain reaction assay was developed for quantification of the mRNA levels of BAX and BCL2, using beta‑2 microglobulin as an endogenous control for normalization purposes. Gene expression analysis was performed using the comparative Ct (2(‑ΔΔCt)) method. The mRNA expression of BAX presented an increasing trend in the OA group compared with the control group, although without statistically significace (P=0.099). By contrast, the expression ratio of BCL2/BAX was found to be significantly decreased (2.76‑fold) in the OA group compared with the normal cartilage control group (P=0.022). A notable 4.6‑fold overexpression of median mRNA levels of BAX was also observed in patients with stage III OA compared with the control (P=0.034), while the BCL2/BAX ratio was markedly (2.5‑fold) decreased (P=0.024). A marked positive correlation was observed between the mRNA levels of BAX and BCL2 in the control group (r(s)=0.728; P<0.001), which was also present in the OA group, although to a lesser degree (r(s)=0.532; P<0.001). These results further implicate apoptosis in the pathogenesis of OA, through molecular mechanisms, which include the aberrant expression of the

  12. The expression level of HJURP has an independent prognostic impact and predicts the sensitivity to radiotherapy in breast cancer

    SciTech Connect

    Hu, Zhi; Huang, Ge; Sadanandam, Anguraj; Gu, Shenda; Lenburg, Marc E; Pai, Melody; Bayani, Nora; Blakely, Eleanor A; Gray, Joe W; Mao, Jian-Hua

    2010-06-25

    Introduction: HJURP (Holliday Junction Recognition Protein) is a newly discovered gene reported to function at centromeres and to interact with CENPA. However its role in tumor development remains largely unknown. The goal of this study was to investigate the clinical significance of HJURP in breast cancer and its correlation with radiotherapeutic outcome. Methods: We measured HJURP expression level in human breast cancer cell lines and primary breast cancers by Western blot and/or by Affymetrix Microarray; and determined its associations with clinical variables using standard statistical methods. Validation was performed with the use of published microarray data. We assessed cell growth and apoptosis of breast cancer cells after radiation using high-content image analysis. Results: HJURP was expressed at higher level in breast cancer than in normal breast tissue. HJURP mRNA levels were significantly associated with estrogen receptor (ER), progesterone receptor (PR), Scarff-Bloom-Richardson (SBR) grade, age and Ki67 proliferation indices, but not with pathologic stage, ERBB2, tumor size, or lymph node status. Higher HJURP mRNA levels significantly decreased disease-free and overall survival. HJURP mRNA levels predicted the prognosis better than Ki67 proliferation indices. In a multivariate Cox proportional-hazard regression, including clinical variables as covariates, HJURP mRNA levels remained an independent prognostic factor for disease-free and overall survival. In addition HJURP mRNA levels were an independent prognostic factor over molecular subtypes (normal like, luminal, Erbb2 and basal). Poor clinical outcomes among patients with high HJURP expression werevalidated in five additional breast cancer cohorts. Furthermore, the patients with high HJURP levels were much more sensitive to radiotherapy. In vitro studies in breast cancer cell lines showed that cells with high HJURP levels were more sensitive to radiation treatment and had a higher rate of apoptosis

  13. Inconformity of CXCL3 plasma level and placenta expression in preeclampsia and its effect on trophoblast viability and invasion.

    PubMed

    Gui, Shunping; Ni, Shanshan; Jia, Jin; Gong, Yunhui; Gao, Linbo; Zhang, Lin; Zhou, Rong

    2014-01-01

    As a member of the chemokine family, CXCL3 was previously known to participate in many pathophysiological events. However, whether CXCL3 stimulates trophoblast invasion as a key process of preeclampsia pathogenesis remains largely unknown. Therefore, the aim of this study was to investigate this hypothesis and determine the effect of CXCL3 on the first trimester trophoblast. Seventy gravidas were included in this study. ELISA was used to detect CXCL3 plasma levels on preeclampsia and normal pregnant groups. CXCL3 protein and mRNA levels were detected via Western blot and real-time quantitative PCR analysis after immunolocalized in human placenta. Moreover, the CXCL3 function in HTR-8/Svneo was analyzed via WST-1 assay, flow cytometry and invasion test. The plasma CXCL3 level in preeclampsia was significantly higher than that in normal pregnancy. CXCL3 expression was observed in the cytoplasm of placental trophoblasts and vascular endothelium in all groups without significant difference between maternal and fetal sides. In addition, placenta CXCL3 expression in severe preeclampsia was significantly lower than those in normal and mild PE groups. Moreover, exogenous CXCL3 can promote the proliferation and invasion of HTR-8/Svneo; however, its effect on apoptosis remains unclear. In summary, a significant abnormality of plasma CXCL3 level and placental CXCL3 expression was discovered in severe preeclampsia; CXCL3 had a function in trophoblast invasion, which indicated its participation in shallow implantation. Therefore CXCL3 might be involved in severe preeclampsia pathogenesis. PMID:25485631

  14. Gene expression patterns unveil a new level of molecular heterogeneity in colorectal cancer.

    PubMed

    Budinska, Eva; Popovici, Vlad; Tejpar, Sabine; D'Ario, Giovanni; Lapique, Nicolas; Sikora, Katarzyna Otylia; Di Narzo, Antonio Fabio; Yan, Pu; Hodgson, John Graeme; Weinrich, Scott; Bosman, Fred; Roth, Arnaud; Delorenzi, Mauro

    2013-09-01

    The recognition that colorectal cancer (CRC) is a heterogeneous disease in terms of clinical behaviour and response to therapy translates into an urgent need for robust molecular disease subclassifiers that can explain this heterogeneity beyond current parameters (MSI, KRAS, BRAF). Attempts to fill this gap are emerging. The Cancer Genome Atlas (TGCA) reported two main CRC groups, based on the incidence and spectrum of mutated genes, and another paper reported an EMT expression signature defined subgroup. We performed a prior free analysis of CRC heterogeneity on 1113 CRC gene expression profiles and confronted our findings to established molecular determinants and clinical, histopathological and survival data. Unsupervised clustering based on gene modules allowed us to distinguish at least five different gene expression CRC subtypes, which we call surface crypt-like, lower crypt-like, CIMP-H-like, mesenchymal and mixed. A gene set enrichment analysis combined with literature search of gene module members identified distinct biological motifs in different subtypes. The subtypes, which were not derived based on outcome, nonetheless showed differences in prognosis. Known gene copy number variations and mutations in key cancer-associated genes differed between subtypes, but the subtypes provided molecular information beyond that contained in these variables. Morphological features significantly differed between subtypes. The objective existence of the subtypes and their clinical and molecular characteristics were validated in an independent set of 720 CRC expression profiles. Our subtypes provide a novel perspective on the heterogeneity of CRC. The proposed subtypes should be further explored retrospectively on existing clinical trial datasets and, when sufficiently robust, be prospectively assessed for clinical relevance in terms of prognosis and treatment response predictive capacity. Original microarray data were uploaded to the ArrayExpress database (http

  15. Estimation of Gene Expression at Isoform Level from mRNA-Seq Data by Bayesian Hierarchical Modeling

    PubMed Central

    Bhattacharjee, M.; Gupta, Ravi; Davuluri, R. V.

    2012-01-01

    mRNA-Seq is a precise and highly reproducible technique for measurement of transcripts levels and yields sequence information of a transcriptome at a single nucleotide base-level thus enabling us to determine splice junctions and alternative splicing events with high confidence. Often analysis of mRNA-Seq data does not attempt to quantify the expressions at isoform level. In this paper our objective would be use the mRNA-Seq data to infer expression at isoform level, where splicing patterns of a gene is assumed to be known. A Bayesian latent variable based modeling framework is proposed here, where the parameterization enables us to infer at various levels. For example, expression variability of an isoform across different conditions; the model parameterization also allows us to carry out two-sample comparisons, e.g., using a Bayesian t-test, in addition simple presence or absence of an isoform can also be estimated by the use of the latent variables present in the model. In this paper we would carry out inference on isoform expression under different normalization techniques, since it has been recently shown that one of the most prominent sources of variation in differential call using mRNA-Seq data is the normalization method used. The statistical framework is developed for multiple isoforms and easily extends to reads mapping to multiple genes. This could be achieved by slight conceptual modifications in definitions of what we consider as a gene and what as an exon. Additionally proposed framework can be extended by appropriate modeling of the design matrix to infer about yet unknown novel transcripts. However such attempts should be made judiciously since the input date used in the proposed model does not use reads from splice junctions. PMID:23293650

  16. The Promoter Methylation Status and mRNA Expression Levels of CTCF and SIRT6 in Sporadic Breast Cancer

    PubMed Central

    Wang, Da; Zhang, Xuemei

    2014-01-01

    Promoter hypermethylation causes gene silencing and is thought to be an early event in carcinogenesis. This study was to detect promoter methylation status and mRNA expression levels of CCCTC-binding factor (CTCF) and sirtuin 6 (SIRT6), and to explore the relationship between methylation and mRNA expression in breast cancer patient samples. Promoter methylation analysis and expression profile analysis of two genes were performed by methylation-specific PCR, bisulfite sequencing PCR, and quantitative real-time PCR in cancer lesions and matched normal tissues. The promoter region of CTCF has not been hypermethylated in all patient samples. In contrast, methylation of SIRT6 gene was present in invasive cancers (93.5%) and matched normal tissues (96.8%) from 62 patients. Promoter hypermethylation of SIRT6 was also observed in ductal carcinoma in situ (three of three) and matched normal tissues (two of three). mRNA expression of CTCF and SIRT6 in invasive tumors showed a lower level than that in paired normal tissues (p=0.008 and p=0.030, respectively). The fold change values of CTCF expression were significantly lower in invasive ductal cancer lesions with Ki-67-positive status (p=0.042). In conclusion, our data showed that the methylation status of CTCF and SIRT6 promoter regions was not statistically different in cancer lesions compared with matched normal tissues. No significant association between promoter methylation status and expression profiles of CTCF and SIRT6 was found in invasive breast cancers. PMID:24842653

  17. Exercise-induced increase in IL-6 level enhances GLUT4 expression and insulin sensitivity in mouse skeletal muscle.

    PubMed

    Ikeda, Shin-Ichi; Tamura, Yoshifumi; Kakehi, Saori; Sanada, Hiromi; Kawamori, Ryuzo; Watada, Hirotaka

    2016-05-13

    A single bout of exercise is known to increase the insulin sensitivity of skeletal muscle; however, the underlying mechanism of this phenomenon is not fully understood. Because a single bout of exercise induces a transient increase in blood interleukin-6 (IL-6) level, we hypothesized that the enhancement of insulin sensitivity after a single bout of exercise in skeletal muscle is mediated at least in part through IL-6-dependent mechanisms. To test this hypothesis, C57BL6J mice were intravenously injected with normal IgG or an IL-6 neutralizing antibody before exercise. Twenty-four hours after a single bout of exercise, the plantaris muscle was harvested to measure insulin sensitivity and glucose transporter (GLUT)-4 expression levels by ex-vivo insulin-stimulated 2-deoxyglucose (2-DG) uptake and Western blotting, respectively. Compared with sedentary mice, mice that performed exercise showed enhanced IL-6 concentration, insulin-stimulated 2-DG uptake, and GLUT-4 expression in the plantaris muscle. The enhanced insulin sensitivity and GLUT4 expression were canceled by injection of the IL-6 neutralizing antibody before exercise. In addition, IL-6 injection increased GLUT4 expression, both in the plantaris muscle and the soleus muscle in C57BL6J mice. Furthermore, a short period of incubation with IL-6 increased GLUT4 expression in differentiated C2C12 myotubes. In summary, these results suggested that IL-6 increased GLUT4 expression in muscle and that this phenomenon may play a role in the post-exercise enhancement of insulin sensitivity in skeletal muscle. PMID:27040770

  18. Evaluation of discoidin domain receptor-2 (DDR2) expression level in normal, benign, and malignant human prostate tissues

    PubMed Central

    Azemikhah, Mitra; Ashtiani, Hamidreza Ahmadi; Aghaei, Mahmoud; Rastegar, Hosein

    2015-01-01

    Discoidin domain receptor (DDR) is a new member of the receptor tyrosine kinase family. There are two isoforms of discoidin domain receptor (DDR), DDR1 and DDR2. These receptors play a major role in the adhesion, motility and cell proliferation. Due to the important role of DDR2 in the development of tumor extension, this receptor is pivotal in the field of carcinogenesis. The aim of this study was to investigate the mRNA and protein expression of DDR2, in the malignant, benign prostatic hyperplasia (BPH) and normal tissues of patients with prostate cancer. In this study the gene and protein expression of DDR2 in adjacent normal (n=40), BPH (n=40), and malignant (n=40) prostate tissue were measured using real-time PCR and Western blotting. Then, the correlation of DDR2 gene and protein expression with prognostic factors such as age, tumor grade, tumor stage, lymph node involvement, and serum prostate-specific antigen (PSA) concentration were evaluated. The relative mRNA and protein expression level of DDR2 in malignant and benign prostate tissue was significantly higher than those of adjacent normal tissues (P<0.01). This expression was found to increase approximately 3.5 and 2.1 fold for mRNA and protein levels, respectively. Spearman test indicated a significant correlation between DDR2 mRNA and protein expression with prognostic factors such as tumor grade, stage, lymph node involvement, and serum PSA concentration. However, significant correlation with age was not observed. These findings suggest that DDR2 is a cancer-related gene associated with the aggressive progression of prostate cancer patients. PMID:26600862

  19. Evaluation of discoidin domain receptor-2 (DDR2) expression level in normal, benign, and malignant human prostate tissues.

    PubMed

    Azemikhah, Mitra; Ashtiani, Hamidreza Ahmadi; Aghaei, Mahmoud; Rastegar, Hosein

    2015-01-01

    Discoidin domain receptor (DDR) is a new member of the receptor tyrosine kinase family. There are two isoforms of discoidin domain receptor (DDR), DDR1 and DDR2. These receptors play a major role in the adhesion, motility and cell proliferation. Due to the important role of DDR2 in the development of tumor extension, this receptor is pivotal in the field of carcinogenesis. The aim of this study was to investigate the mRNA and protein expression of DDR2, in the malignant, benign prostatic hyperplasia (BPH) and normal tissues of patients with prostate cancer. In this study the gene and protein expression of DDR2 in adjacent normal (n=40), BPH (n=40), and malignant (n=40) prostate tissue were measured using real-time PCR and Western blotting. Then, the correlation of DDR2 gene and protein expression with prognostic factors such as age, tumor grade, tumor stage, lymph node involvement, and serum prostate-specific antigen (PSA) concentration were evaluated. The relative mRNA and protein expression level of DDR2 in malignant and benign prostate tissue was significantly higher than those of adjacent normal tissues (P<0.01). This expression was found to increase approximately 3.5 and 2.1 fold for mRNA and protein levels, respectively. Spearman test indicated a significant correlation between DDR2 mRNA and protein expression with prognostic factors such as tumor grade, stage, lymph node involvement, and serum PSA concentration. However, significant correlation with age was not observed. These findings suggest that DDR2 is a cancer-related gene associated with the aggressive progression of prostate cancer patients. PMID:26600862

  20. High-level expression and characterization of a novel serine protease in Pichia pastoris by multi-copy integration.

    PubMed

    Shu, Min; Shen, Wei; Yang, Shihui; Wang, Xiaojuan; Wang, Fei; Wang, Yaping; Ma, Lixin

    2016-10-01

    A novel serine protease from Trichoderma koningii (SPTK) was synthesized and expressed in Pichia pastoris. The recombinant SPTK was completely inhibited by phenyl methyl sulfonyl fluoride (PMSF), suggesting that SPTK belonged to the subgroup of serine proteases. The optimum pH and temperature for the recombinant SPTK reaction were 6.0 and 55°C, respectively. SPTK performed a tolerance to most organic solvents and metal ions, and the addition of Triton X-100 exhibited an activation of SPTK up to 243% of its initial activity but SDS strongly inhibited. Moreover, our study showed that a portion of SPTK was N-glycosylated during fermentation. The activity and thermal stability of the recombinant SPTK were improved after the removal of glycosylation, and the N-glycosylation of SPTK could be efficiently removed through co-culture with P. pastoris strains expressing Endo-β-N-acetylglucosaminidase H. We constructed expression vectors harboring from one to four repeats of Sptk-expressing cassettes via an in vitro BioBrick assembly approach. And the result of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the genome of P. pastoris through a single recombination event. These strains were used to study the correlation between the gene copy number and the expression level of SPTK. The results of qPCR and enzyme activity assays indicated that the copy number variation of Sptk gene generally had a positive effect on the expression level of SPTK, while an increase in integration of target gene did not guarantee its high expression. The maximum yield and specific activity of SPTK in P. pastoris were obtained from the recombinant yeast strain harboring two-copy tandem Sptk-expressing cassettes, the yield reached 0.48g/l after a 6-d induction using menthol in shake flasks and 3.2g/l in high-density fermentation with specific activity of 5200U/mg. In addition, the recombinant SPTK could efficiently degrade chicken

  1. Ecdysteroids regulate the levels of Molt-Inhibiting Hormone (MIH) expression in the blue crab, Callinectes sapidus.

    PubMed

    Techa, Sirinart; Chung, J Sook

    2015-01-01

    Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Little has been known of the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH transcripts using in vitro incubation study with ecdysteroids and in vivo RNAi in the blue crab, Callinectes sapidus. Our results show a specific expression of ecdysone receptor (EcR) in which EcR1 is the major isoform in eyestalk ganglia. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of eyestalk ganglia with ecdysteroids that mimic the intrinsic conditions of D0 stage: the concentration (75 ng/ml) and composition (ponasterone A and 20-hydroxyecdysone at a 3:1 (w:w) ratio). Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation during molting cycle, specifically how the molt cycle is repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at early premolt stage may act positively on the levels of MIH expression in the eyestalk ganglia. Subsequently, the increased MIH titers in the hemolymph at postmolt would inhibit the synthesis and release of ecdysteroids by Y-organs, resulting in re-setting the subsequent molt cycle. PMID:25849453

  2. Ecdysteroids Regulate the Levels of Molt-Inhibiting Hormone (MIH) Expression in the Blue Crab, Callinectes sapidus

    PubMed Central

    Techa, Sirinart; Chung, J. Sook

    2015-01-01

    Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Little has been known of the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH transcripts using in vitro incubation study with ecdysteroids and in vivo RNAi in the blue crab, Callinectes sapidus. Our results show a specific expression of ecdysone receptor (EcR) in which EcR1 is the major isoform in eyestalk ganglia. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of eyestalk ganglia with ecdysteroids that mimic the intrinsic conditions of D0 stage: the concentration (75 ng/ml) and composition (ponasterone A and 20-hydroxyecdysone at a 3:1 (w:w) ratio). Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation during molting cycle, specifically how the molt cycle is repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at early premolt stage may act positively on the levels of MIH expression in the eyestalk ganglia. Subsequently, the increased MIH titers in the hemolymph at postmolt would inhibit the synthesis and release of ecdysteroids by Y-organs, resulting in re-setting the subsequent molt cycle. PMID:25849453

  3. High levels of p53 protein expression do not correlate with p53 gene mutations in anaplastic large cell lymphoma.

    PubMed Central

    Cesarman, E.; Inghirami, G.; Chadburn, A.; Knowles, D. M.

    1993-01-01

    Strong immunohistochemical reactivity for p53 tumor suppressor gene product has been reported in a variety of different human malignancies including CD30- (Ki-1) positive anaplastic large cell lymphoma (ALCL). Although high levels of p53 protein have been interpreted as abnormal, rapidly proliferating benign and neoplastic lymphoid cells may have increased p53 expression in the absence of structural alterations. On the other hand, mutations in the p53 gene can lead to a lack of p53 protein production. Structural alterations of the p53 gene have not been documented in cases of ALCL and the mechanism for an abnormal pattern of p53 expression in these lymphomas has not been elucidated. Therefore, to determine whether an altered pattern of p53 expression correlates with mutations in the p53 locus in ALCL, we analyzed the expression of p53 protein immunohistochemically, compared it with the proliferation index using monoclonal antibody Ki-67, and assessed the presence of mutations in exons 5 though 9 of the p53 gene using a single-strand conformation polymorphism assay in a panel of 17 ALCLs. Furthermore, we studied the presence of allelic deletions of chromosome 17p by restriction fragment length polymorphism analysis. We found significant levels of p53 protein expression in 12 of the 15 cases studied, but identified mutations in only one of 17 cases. An allelic deletion in chromosome 17p was identified only in the one case containing a mutated p53 gene. Whereas the case containing structural alterations in the p53 gene did have strong p53 immunoreactivity, 11 cases that lacked p53 mutations in the regions examined also had significant levels of p53. Thus, our studies indicate that strong immunohistochemical reactivity for p53 is not a reliable indicator of the presence of structural alterations of p53 gene exons 5 through 9 in ALCL. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8103295

  4. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.

    PubMed Central

    Sadelain, M; Wang, C H; Antoniou, M; Grosveld, F; Mulligan, R C

    1995-01-01

    Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of

  5. Identification and cloning of a novel phosphatase expressed at high levels in differentiating growth plate chondrocytes.

    PubMed

    Houston, B; Seawright, E; Jefferies, D; Hoogland, E; Lester, D; Whitehead, C; Farquharson, C

    1999-01-11

    Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differential display to these populations we cloned a cDNA encoding a novel 268 amino acid protein (3X11A). 3X11A contains two peptide motifs that are conserved in a recently identified superfamily of phosphotransferases. It is likely that 3X11A is a phosphatase, but its substrate specificity remains uncertain. 3X11A expression is upregulated 5-fold during chondrocyte terminal differentiation and its expression is approximately 100-fold higher in hypertrophic chondrocytes than in non-chondrogenic tissues. This suggests that 3X11A participates in a biochemical pathway that is particularly active in differentiating chondrocytes. PMID:9990301

  6. Alterations in methylation and expression levels of imprinted genes H19 and Igf2 in the fetuses of diabetic mice.

    PubMed

    Shao, Wei-Juan; Tao, Ling-Yun; Gao, Cheng; Xie, Jian-Yun; Zhao, Ru-Qian

    2008-08-01

    The study aimed to reveal alterations in expression and methylation levels of the growth-related imprinted genes H19 and Igf2 in fetuses of diabetic mice. Diabetes was induced in female mice by intraperitoneal injection of streptozotocin. DNA and total RNA were extracted from fetuses obtained from diabetic and control dams on embryonic day (E) 14. Real-time RT-PCR analysis revealed that the mRNA expression of Igf2 in fetuses from diabetic mice was 0.65-fold of the control counterparts. Bisulfite genomic sequencing demonstrated that the methylation level of the H19-Igf2 imprint control region was 19.1% higher in diabetic fetuses than in those of control dams. In addition, the body weight of pups born to diabetic dams was 26.5% lower than that of the control group. The results indicate that maternal diabetes can affect fetal development by means of altered expression of imprinted genes. The modified genomic DNA methylation status of imprinting genes may account for the change in gene expression. PMID:18724775

  7. Codon Optimization Significantly Improves the Expression Level of α -Amylase Gene from Bacillus licheniformis in Pichia pastoris.

    PubMed

    Wang, Jian-Rong; Li, Yang-Yuan; Liu, Dan-Ni; Liu, Jing-Shan; Li, Peng; Chen, Li-Zhi; Xu, Shu-De

    2015-01-01

    α-Amylase as an important industrial enzyme has been widely used in starch processing, detergent, and paper industries. To improve expression efficiency of recombinant α-amylase from Bacillus licheniformis (B. licheniformis), the α-amylase gene from B. licheniformis was optimized according to the codon usage of Pichia pastoris (P. pastoris) and expressed in P. pastoris. Totally, the codons encoding 305 amino acids were optimized in which a total of 328 nucleotides were changed and the G+C content was increased from 47.6 to 49.2%. The recombinants were cultured in 96-deep-well microplates and screened by a new plate assay method. Compared with the wild-type gene, the optimized gene is expressed at a significantly higher level in P. pastoris after methanol induction for 168 h in 5- and 50-L bioreactor with the maximum activity of 8100 and 11000 U/mL, which was 2.31- and 2.62-fold higher than that by wild-type gene. The improved expression level makes the enzyme a good candidate for α-amylase production in industrial use. PMID:26171389

  8. High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris.

    PubMed

    Li, Yang-yuan; Zhong, Kai-xin; Hu, Ai-hong; Liu, Dan-ni; Chen, Li-zhi; Xu, Shu-de

    2015-04-01

    A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZαA. Recombinant Mxyn2p was functionally expressed in P. pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75°C, while recombinant protein encoded by the "wild type" xylanase gene xyn2, also expressed in Pichia, was 20°C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80°C for 10min. The effects of the optimal pH and temperature for higher expression levels in P. pastoris were also determined, 6.0 and 22°C, respectively. The maximum xylanase activity of Mxyn2p was 13,000nkat/mg (9.88g/l) in fed-batch cultivation after 168h induction with methanol in a 50l bioreactor. PMID:25434687

  9. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown.

    PubMed

    Pertea, Mihaela; Kim, Daehwan; Pertea, Geo M; Leek, Jeffrey T; Salzberg, Steven L

    2016-09-01

    High-throughput sequencing of mRNA (RNA-seq) has become the standard method for measuring and comparing the levels of gene expression in a wide variety of species and conditions. RNA-seq experiments generate very large, complex data sets that demand fast, accurate and flexible software to reduce the raw read data to comprehensible results. HISAT (hierarchical indexing for spliced alignment of transcripts), StringTie and Ballgown are free, open-source software tools for comprehensive analysis of RNA-seq experiments. Together, they allow scientists to align reads to a genome, assemble transcripts including novel splice variants, compute the abundance of these transcripts in each sample and compare experiments to identify differentially expressed genes and transcripts. This protocol describes all the steps necessary to process a large set of raw sequencing reads and create lists of gene transcripts, expression levels, and differentially expressed genes and transcripts. The protocol's execution time depends on the computing resources, but it typically takes under 45 min of computer time. HISAT, StringTie and Ballgown are available from http://ccb.jhu.edu/software.shtml. PMID:27560171

  10. Codon Optimization Significantly Improves the Expression Level of α-Amylase Gene from Bacillus licheniformis in Pichia pastoris

    PubMed Central

    Wang, Jian-Rong; Li, Yang-Yuan; Liu, Dan-Ni; Liu, Jing-Shan; Li, Peng; Chen, Li-Zhi; Xu, Shu-De

    2015-01-01

    α-Amylase as an important industrial enzyme has been widely used in starch processing, detergent, and paper industries. To improve expression efficiency of recombinant α-amylase from Bacillus licheniformis (B. licheniformis), the α-amylase gene from B. licheniformis was optimized according to the codon usage of Pichia pastoris (P. pastoris) and expressed in P. pastoris. Totally, the codons encoding 305 amino acids were optimized in which a total of 328 nucleotides were changed and the G+C content was increased from 47.6 to 49.2%. The recombinants were cultured in 96-deep-well microplates and screened by a new plate assay method. Compared with the wild-type gene, the optimized gene is expressed at a significantly higher level in P. pastoris after methanol induction for 168 h in 5- and 50-L bioreactor with the maximum activity of 8100 and 11000 U/mL, which was 2.31- and 2.62-fold higher than that by wild-type gene. The improved expression level makes the enzyme a good candidate for α-amylase production in industrial use. PMID:26171389

  11. Quantitative in situ hybridization for the study of gene expression at the regional and cellular levels.

    PubMed

    Le Moine, Catherine

    2003-08-01

    Quantitative in situ hybridization allows measurement of mRNA level modifications in a variety of experimental conditions. This analysis may be performed both at the regional anatomical and cellular levels by densitometry, neuronal counting and silver grain measurements. PMID:18428577

  12. Distinct Expression Levels of ALS, LIP, and SAP Genes in Candida tropicalis with Diverse Virulent Activities

    PubMed Central

    Yu, Shuanbao; Li, Wenge; Liu, Xiaoshu; Che, Jie; Wu, Yuan; Lu, Jinxing

    2016-01-01

    Candia tropicalis is an increasingly important human pathogen, causing nosocomial fungemia among patients with neutropenia or malignancy. However, limited research has been published concerning its pathogenicity. Based on the phenotypes of C. tropicalis in our previous study, we selected nine representative strains with different activities of virulence factors (adhesion, biofilm formation, secreted aspartic proteinases, and hemolysins), and one reference strain, ATCC750. The present study aimed to investigate the filamentation ability, the expression of virulence genes (ALST1-3, LIP1, LIP4, and SAPT1-4) and the cell damage of C. tropicalis strains with diverse virulences. C. tropicalis exhibited strain-dependent filamentation ability, which was positively correlated with biofilm formation. Reverse transcriptase PCR analysis showed that the ALST3 and SAPT3 genes had the highest expression in their corresponding genes for most C. tropicalis. The expressions of virulence genes, except ALST3 on polystyrene, were upregulated compared with growth in the planktonic and on human urinary bladder epithelial cell line (TCC-SUP) surface. Clustering analysis of virulence genes showed that isolates had a high biofilm forming ability on polystyrene formed a group. Lactate dehydrogenase assays showed that the cell damage induced by C. tropicalis markedly increased with longer infection time (24 and 48 h). Strain FXCT01, isolated from blood, caused the most serious cell damage; while ZRCT52, which had no filamentation ability, caused the least cell damage. Correlation analysis demonstrated significant correlation existed between adhesion on epithelial cells or the expression of ALST2-3 and cell damage. Overall, our results supported the view that adhesion and filamentation may play significant roles in the cell damage caused by C. tropicalis. PMID:27524980

  13. Distinct Expression Levels of ALS, LIP, and SAP Genes in Candida tropicalis with Diverse Virulent Activities.

    PubMed

    Yu, Shuanbao; Li, Wenge; Liu, Xiaoshu; Che, Jie; Wu, Yuan; Lu, Jinxing

    2016-01-01

    Candia tropicalis is an increasingly important human pathogen, causing nosocomial fungemia among patients with neutropenia or malignancy. However, limited research has been published concerning its pathogenicity. Based on the phenotypes of C. tropicalis in our previous study, we selected nine representative strains with different activities of virulence factors (adhesion, biofilm formation, secreted aspartic proteinases, and hemolysins), and one reference strain, ATCC750. The present study aimed to investigate the filamentation ability, the expression of virulence genes (ALST1-3, LIP1, LIP4, and SAPT1-4) and the cell damage of C. tropicalis strains with diverse virulences. C. tropicalis exhibited strain-dependent filamentation ability, which was positively correlated with biofilm formation. Reverse transcriptase PCR analysis showed that the ALST3 and SAPT3 genes had the highest expression in their corresponding genes for most C. tropicalis. The expressions of virulence genes, except ALST3 on polystyrene, were upregulated compared with growth in the planktonic and on human urinary bladder epithelial cell line (TCC-SUP) surface. Clustering analysis of virulence genes showed that isolates had a high biofilm forming ability on polystyrene formed a group. Lactate dehydrogenase assays showed that the cell damage induced by C. tropicalis markedly increased with longer infection time (24 and 48 h). Strain FXCT01, isolated from blood, caused the most serious cell damage; while ZRCT52, which had no filamentation ability, caused the least cell damage. Correlation analysis demonstrated significant correlation existed between adhesion on epithelial cells or the expression of ALST2-3 and cell damage. Overall, our results supported the view that adhesion and filamentation may play significant roles in the cell damage caused by C. tropicalis. PMID:27524980

  14. Vascular endothelial growth factor A protein level and gene expression in intracranial meningiomas with brain edema.

    PubMed

    Nassehi, Damoun; Dyrbye, Henrik; Andresen, Morten; Thomsen, Carsten; Juhler, Marianne; Laursen, Henning; Broholm, Helle

    2011-12-01

    Meningiomas are the second most common primary intracranial tumors in adults. Although meningiomas are mostly benign, more than 50% of patients with meningioma develop peritumoral brain edema (PTBE), which may be fatal because of increased intracranial pressure. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and angiogen. VEGF-A protein, which is identical to vascular permeability factor, is a regulator of angiogenesis. In this study, 101 patients with meningiomas, and possible co-factors to PTBE, such as meningioma subtypes and tumor location, were examined. Forty-three patients had primary, solitary, supratentorial meningiomas with PTBE. In these, correlations in PTBE, edema index, VEGF-A protein, VEGF gene expression, capillary length, and tumor water content were investigated. DNA-branched hybridization was used for measuring VEGF gene expression in tissue homogenates prepared from frozen tissue samples. The method for VEGF-A analysis resembled an ELISA assay, but was based on chemiluminescence. The edema index was positively correlated to VEGF-A protein (p = 0.014) and VEGF gene expression (p < 0.05). The capillary length in the meningiomas was positively correlated to the PTBE (p = 0.038). If VEGF is responsible for the formation of PTBE, the edema may be treated with the anti-VEGF drug Bevacizumab (Avastin), which has been shown to reduce PTBE in patients with glioblastoma multiforme. PMID:22085359

  15. Differential expression of growth factors at the cellular level in virus-infected brain.

    PubMed

    Prosniak, Mikhail; Zborek, Anna; Scott, Gwen S; Roy, Anirban; Phares, Timothy W; Koprowski, Hilary; Hooper, D Craig

    2003-05-27

    The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS. PMID:12736376

  16. Differential expression of growth factors at the cellular level in virus-infected brain

    PubMed Central

    Prosniak, Mikhail; Zborek, Anna; Scott, Gwen S.; Roy, Anirban; Phares, Timothy W.; Koprowski, Hilary; Hooper, D. Craig

    2003-01-01

    The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS. PMID:12736376

  17. Transcranial light affects plasma monoamine levels and expression of brain encephalopsin in the mouse.

    PubMed

    Flyktman, Antti; Mänttäri, Satu; Nissilä, Juuso; Timonen, Markku; Saarela, Seppo

    2015-05-15

    Encephalopsin (OPN3) belongs to the light-sensitive transmembrane receptor family mainly expressed in the brain and retina. It is believed that light affects mammalian circadian rhythmicity only through the retinohypothalamic tract, which transmits light information to the suprachiasmatic nucleus in the hypothalamus. However, it has been shown that light penetrates the skull. Here, we present the effect of transcranial light treatment on OPN3 expression and monoamine concentrations in mouse brain and other tissues. Mice were randomly assigned to control group, morning-light group and evening-light group, and animals were illuminated transcranially five times a week for 8 min for a total of 4 weeks. The concentrations of OPN3 and monoamines were analysed using western blotting and HPLC, respectively. We report that transcranial light treatment affects OPN3 expression in different brain areas and plasma/adrenal gland monoamine concentrations. In addition, when light was administered at a different time of the day, the response varied in different tissues. These results provide new information on the effects of light on transmitters mediating mammalian rhythmicity. PMID:25805701

  18. Expression level and DNA methylation status of Glutathione-S-transferase genes in normal murine prostate and TRAMP tumors

    PubMed Central

    Mavis, Cory K.; Kinney, Shannon R. Morey; Foster, Barbara A.; Karpf, Adam R.

    2010-01-01

    BACKGROUND Glutathione-S-transferase (Gst) genes are down-regulated in human prostate cancer, and GSTP1 silencing is mediated by promoter DNA hypermethylation in this malignancy. We examined Gst gene expression and Gst promoter DNA methylation in normal murine prostates and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors. METHODS Primary and metastatic tumors were obtained from TRAMP mice, and normal prostates were obtained from strain-matched WT mice (n=15/group). Quantitative real-time RT-PCR was used to measure GstA4, GstK1, GstM1, GstO1, and GstP1 mRNA expression, and Western blotting and immunohistochemical staining was used to measure GstM1 and GstP1 protein expression. MassARRAY Quantitative Methylation Analysis was used to measure DNA methylation of the 5’ CpG islands of GstA4, GstK1, GstM1, GstO1, and GstP1. TRAMP-C2 cells were treated with the epigenetic remodeling drugs decitabine and trichostatin A (TSA) alone and in combination, and Gst gene expression was measured. RESULTS Of the genes analyzed, GstM1 and GstP1 were expressed at highest levels in normal prostate. All five Gst genes showed greatly reduced expression in primary tumors compared to normal prostate, but not in tumor metastases. Gst promoter methylation was unchanged in TRAMP tumors compared to normal prostate. Combined decitabine + TSA treatment significantly enhanced the expression of 4/5 Gst genes in TRAMP-C2 cells. CONCLUSIONS Gst genes are extensively downregulated in primary but not metastatic TRAMP tumors. Promoter DNA hypermethylation does not appear to drive Gst gene repression in TRAMP primary tumors; however, pharmacological studies using TRAMP cells suggest the involvement of epigenetic mechanisms in Gst gene repression. PMID:19444856

  19. High-level expression of pseudolysin, the extracellular elastase of Pseudomonas aeruginosa, in Escherichia coli and its purification.

    PubMed

    Odunuga, Odutayo O; Adekoya, Olayiwola A; Sylte, Ingebrigt

    2015-09-01

    Pseudolysin is the extracellular elastase of Pseudomonas aeruginosa and belongs to the thermolysin-like family of metallopeptidases. Pseudolysin has been identified as a robust drug target and a biotechnologically important enzyme in the tanning industry. Previous attempts to purify active pseudolysin from P. aeruginosa or by expression in Escherichia coli yielded low quantities. Considerable expression and purification of secreted pseudolysin from Pichia pastoris has been reported but it is time-consuming and not cost-effective. We report the successful large-scale expression of pseudolysin in E. coli and purification of the correctly folded and active protein. The lasB gene that codes for the enzymatically active mature 33-kilodalton pseudolysin was expressed with a histidine tag under the control of the T7 promoter. Pseudolysin expressed highly in E. coli and was solubilized and purified in 8M urea by metal affinity chromatography. The protein was simultaneously further purified, refolded and buffer-exchanged on a preparative Superdex 200 column by a modified urea reverse-gradient size exclusion chromatography. Using this technique, precipitation of pseudolysin was completely eliminated. Refolded pseudolysin was found to be active as assessed by its ability to hydrolyze N-succinyl-ala-ala-ala-p-nitroanilide. The purification scheme yielded approximately 40 mg of pseudolysin per liter of expression culture and specific activity of 3.2U/mg of protein using N-succinyl-ala-ala-ala-p-nitroanilide as substrate. This approach provides a reproducible strategy for high-level expression and purification of active metallopeptidases and perhaps other inclusion body-forming and precipitation-prone proteins. PMID:25979480

  20. Effects of voluntary exercise and genetic selection for high activity levels on HSP72 expression in house mice.

    PubMed

    Belter, Jason G; Carey, Hannah V; Garland, Theodore

    2004-04-01

    We studied expression of heat shock protein 72 (HSP72) in female mice from four replicate lines that had been selectively bred for high voluntary wheel running (S) and from four random-bred control lines (C). Mice from generation 23 were sampled after 6 days of wheel access, and those from generation 14 were sampled after 8 wk of access to wheels either free to rotate or locked. Mice from S lines ran approximately 2.6 times as many revolutions per day as did those from C lines. Western blotting of tissues from generation 23 mice indicated that S mice had elevated HSP72 expression in triceps surae muscle, but levels in spleen, kidney, heart, and lung were similar in S and C mice. HSP72 expression in triceps surae from generation 14 mice was measured by ELISA and analyzed with a two-way analysis of covariance. The interaction between wheel type and line type (S vs. C) was statistically significant, and subsequent analyses indicated that S mice had significantly elevated HSP72 expression only when housed with free wheels. Mice with the previously described mini-muscle phenotype (Houle-Leroy P, Guderley H, Swallow JG, and Garland T Jr. Am J Physiol Regul Integr Comp Physiol 284: R433-R443, 2003) occurred in both generations and had elevated HSP72 expression in triceps surae. For the generation 23 sample, wheel running as a covariate had a significant negative association with HSP72 expression, and the effect of line type was still statistically significant. Therefore, the increased HSP72 expression of S mice is not a simple proximate effect of their increased wheel running. PMID:14672969

  1. MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels.

    PubMed

    Goedeke, Leigh; Rotllan, Noemi; Canfrán-Duque, Alberto; Aranda, Juan F; Ramírez, Cristina M; Araldi, Elisa; Lin, Chin-Sheng; Anderson, Norma N; Wagschal, Alexandre; de Cabo, Rafael; Horton, Jay D; Lasunción, Miguel A; Näär, Anders M; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-11-01

    The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL cholesterol (LDL-C). Whereas the transcriptional regulation of LDLR is well characterized, the post-transcriptional mechanisms that govern LDLR expression are just beginning to emerge. Here we develop a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen we identified and characterized miR-148a as a negative regulator of LDLR expression and activity and defined a sterol regulatory element-binding protein 1 (SREBP1)-mediated pathway through which miR-148a regulates LDL-C uptake. In mice, inhibition of miR-148a increased hepatic LDLR expression and decreased plasma LDL-C. Moreover, we found that miR-148a regulates hepatic expression of ATP-binding cassette, subfamily A, member 1 (ABCA1) and circulating high-density lipoprotein cholesterol (HDL-C) levels in vivo. These studies uncover a role for miR-148a as a key regulator of hepatic LDL-C clearance through direct modulation of LDLR expression and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate an elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease. PMID:26437365

  2. Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels.

    PubMed

    Roney, Ian J; Rudner, Adam D; Couture, Jean-François; Kærn, Mads

    2016-01-01

    Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount. PMID:27323850

  3. Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels

    PubMed Central

    Roney, Ian J.; Rudner, Adam D.; Couture, Jean-François; Kærn, Mads

    2016-01-01

    Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount. PMID:27323850

  4. Low Levels of the Reverse Transactivator Fail to Induce Target Transgene Expression in Vascular Smooth Muscle Cells

    PubMed Central

    Viceconte, Nikenza; McKenna, Tomás; Eriksson, Maria

    2014-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease with multiple features that are suggestive of premature aging. Most patients with HGPS carry a mutation on one of their copies of the LMNA gene. The LMNA gene encodes the lamin A and lamin C proteins, which are the major proteins of the nuclear lamina. The organs of the cardiovascular system are amongst those that are most severely affected in HGPS, undergoing a progressive depletion of vascular smooth muscle cells, and most children with HGPS die in their early teens from cardio-vascular disease and other complications from atherosclerosis. In this study, we developed a transgenic mouse model based on the tet-ON system to increase the understanding of the molecular mechanisms leading to the most lethal aspect of HGPS. To induce the expression of the most common HGPS mutation, LMNA c.1824C>T; p.G608G, in the vascular smooth muscle cells of the aortic arch and thoracic aorta, we used the previously described reverse tetracycline-controlled transactivator, sm22α-rtTA. However, the expression of the reverse sm22α-transactivator was barely detectable in the arteries, and this low level of expression was not sufficient to induce the expression of the target human lamin A minigene. The results from this study are important because they suggest caution during the use of previously functional transgenic animal models and emphasize the importance of assessing transgene expression over time. PMID:25090270

  5. High-level expression of Egr-1 and Egr-1–inducible genes in mouse and human atherosclerosis

    PubMed Central

    McCaffrey, Timothy A.; Fu, Chenzhong; Du, Baoheng; Eksinar, Sukru; Kent, K. Craig; Bush, Harry; Kreiger, Karl; Rosengart, Todd; Cybulsky, Myron I.; Silverman, Eric S.; Collins, Tucker

    2000-01-01

    To understand the mRNA transcript profile in the human atherosclerotic lesion, RNA was prepared from the fibrous cap versus adjacent media of 13 patients undergoing carotid endarterectomy. cDNA expression arrays bearing 588 known genes indicated that lesions express unexpectedly high levels of the early growth response gene, Egr-1 (NGFI-A), a zinc-finger transcription factor that modulates a cluster of stress-responsive genes including PDGF and TGF-β. Expression of Egr-1 was an average of 5-fold higher in the lesion than in the adjacent media, a result confirmed by RT-PCR, and many Egr-1–inducible genes were also strongly elevated in the lesion. Time-course analyses revealed that Egr-1 was not induced ex vivo. Immunocytochemistry indicated that Egr-1 was expressed prominently in the smooth muscle–actin positive cells, particularly in areas of macrophage infiltration, and in other cell types, including endothelial cells. Induction of atherosclerosis in LDL receptor-null mice by feeding them a high-fat diet resulted in a progressive increase in Egr-1 expression in the aorta. Thus, induction of Egr-1 by atherogenic factors may be a key step in coordinating the cellular events that result in vascular lesions. PMID:10712437

  6. Increased astrocyte expression of IL-6 or CCL2 in transgenic mice alters levels of hippocampal and cerebellar proteins

    PubMed Central

    Gruol, Donna L.; Vo, Khanh; Bray, Jennifer G.

    2014-01-01

    Emerging research has identified that neuroimmune factors are produced by cells of the central nervous system (CNS) and play critical roles as regulators of CNS function, directors of neurodevelopment and responders to pathological processes. A wide range of neuroimmune factors are produced by CNS cells, primarily the glial cells, but the role of specific neuroimmune factors and their glial cell sources in CNS biology and pathology have yet to be fully elucidated. We have used transgenic mice that express elevated levels of a specific neuroimmune factor, the cytokine IL-6 or the chemokine CCL2, through genetic modification of astrocyte expression to identify targets of astrocyte produced IL-6 or CCL2 at the protein level. We found that in non-transgenic mice constitutive expression of IL-6 and CCL2 occurs in the two CNS regions studied, the hippocampus and cerebellum, as measured by ELISA. In the CCL2 transgenic mice elevated levels of CCL2 were evident in the hippocampus and cerebellum, whereas in the IL-6 transgenic mice, elevated levels of IL-6 were only evident in the cerebellum. Western blot analysis of the cellular and synaptic proteins in the hippocampus and cerebellum of the transgenic mice showed that the elevated levels of CCL2 or IL-6 resulted in alterations in the levels of specific proteins and that these actions differed for the two neuroimmune factors and for the two brain regions. These results are consistent with cell specific profiles of action for IL-6 and CCL2, actions that may be an important aspect of their respective roles in CNS physiology and pathophysiology. PMID:25177271

  7. Correlation of plasma nitrite/nitrate levels and inducible nitric oxide gene expression among women with cervical abnormalities and cancer.

    PubMed

    Sowjanya, A Pavani; Rao, Meera; Vedantham, Haripriya; Kalpana, Basany; Poli, Usha Rani; Marks, Morgan A; Sujatha, M

    2016-01-30

    Cervical cancer is caused by infection with high risk human papillomavirus (HR-HPV). Inducible nitric oxide synthase (iNOS), a soluble factor involved in chronic inflammation, may modulate cervical cancer risk among HPV infected women. The aim of the study was to measure and correlate plasma nitrite/nitrate levels with tissue specific expression of iNOS mRNA among women with different grades of cervical lesions and cervical cancer. Tissue biopsy and plasma specimens were collected from 120 women with cervical neoplasia or cancer (ASCUS, LSIL, HSIL and invasive cancer) and 35 women without cervical abnormalities. Inducible nitric oxide synthase (iNOS) mRNA from biopsy and plasma nitrite/nitrate levels of the same study subjects were measured. Single nucleotide polymorphism (SNP) analysis was performed on the promoter region and Ser608Leu (rs2297518) in exon 16 of the iNOS gene. Differences in iNOS gene expression and plasma nitrite/nitrate levels were compared across disease stage using linear and logistic regression analysis. Compared to normal controls, women diagnosed with HSIL or invasive cancer had a significantly higher concentration of plasma nitrite/nitrate and a higher median fold-change in iNOS mRNA gene expression. Genotyping of the promoter region showed three different variations: A pentanucleotide repeat (CCTTT) n, -1026T > G (rs2779249) and a novel variant -1153T > A. These variants were associated with increased levels of plasma nitrite/nitrate across all disease stages. The higher expression of iNOS mRNA and plasma nitrite/nitrate among women with pre-cancerous lesions suggests a role for nitric oxide in the natural history of cervical cancer. PMID:26435258

  8. Quantification of phase I / II metabolizing enzyme gene expression and polycyclic aromatic hydrocarbon-DNA adduct levels in human prostate

    PubMed Central

    John, Kaarthik; Ragavan, Narasimhan; Pratt, M. Margaret; Singh, Paras B.; Al-Buheissi, Salah; Matanhelia, Shyam S.; Phillips, David H.; Poirier, Miriam C.; Martin, Francis L.

    2008-01-01

    BACKGROUND Studies of migrant populations suggest that dietary and/or environmental factors play a crucial role in the aetiology of prostatic adenocarcinoma (CaP). The human prostate consists of the peripheral zone (PZ), transition zone (TZ) and central zone (CZ); CaP occurs most often in the PZ. METHODS To investigate the notion that an underlying differential expression of phase I/II genes, and/or the presence of polycyclic aromatic hydrocarbon (PAH)-DNA adducts might explain the elevated PZ susceptibility, we examined prostate tissues (matched tissue sets consisting of PZ and TZ) from men undergoing radical retropubic prostatectomy for CaP (n=26) or cystoprostatectomy (n=1). Quantitative gene expression analysis was employed for cytochrome P450 (CYP) isoforms CYP1A1, CYP1B1 and CYP1A2, as well as N-acetyltransferase 1 and 2 (NAT1 and NAT2) and catechol-O-methyl transferase (COMT). RESULTS CYP1B1, NAT1 and COMT were expressed in all tissue sets; levels of CYP1B1 and NAT1 were consistently higher in the PZ compared to TZ. Immunohistochemistry confirmed the presence of CYP1B1 (nuclear-associated and primarily in basal epithelial cells) and NAT1. Tissue sections from 23 of these aforementioned 27 matched tissue sets were analyzed for PAH-DNA adduct levels using antiserum elicited against DNA modified with r7, t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE). PAH-DNA adduct levels were highest in glandular epithelial cells, but a comparison of PZ and TZ showed no significant differences. CONCLUSION Although expression of activating and/or detoxifying enzymes may be higher in the PZ, PAH-DNA adduct levels appear to be similar in both zones. Therefore, factors other than PAH-DNA adducts may be responsible for promotion of tumour formation in the human prostate. PMID:19143007

  9. Association between ADSL, GARS-AIRS-GART, DGAT1, and DECR1 expression levels and pork meat quality traits.

    PubMed

    Zhang, X D; Zhang, S J; Ding, Y Y; Feng, Y F; Zhu, H Y; Huang, L; Wu, T; Zhou, J; Yin, Z J

    2015-01-01

    In this study, meat quality traits were compared between Chinese lard- and European lean-type pigs. The association between expression of four genes (ADSL, GARS-AIRS-GART, DGAT1, and DECR1) and meat quality traits was also investigated. Meat quality traits were found to differ significantly between pig breeds. Meat color parameter values (a* and b*) and intramuscular fat content in Anqingliubai were significantly higher than those in Landrace (P < 0.01). Meat pH at 1 and 24 h following slaughter was significantly higher in Landrace than in Wei pigs, and meat inosine monophosphate (IMP) content was significantly higher in Landrace than in Wei and Anqingliubai pigs (both P < 0.01). Expression levels of ADSL, GARS-AIRS-GART, and DGAT1 were higher in longissimus lumborum muscle than in heart or liver tissues. ADSL and GARS-AIRS-GART expression levels were correlated with meat IMP content and pH levels. The results of this study will contribute to the understanding of meat quality traits in Chinese lard- and European lean-type pigs. PMID:26600543

  10. Earth Mover’s Distance (EMD): A True Metric for Comparing Biomarker Expression Levels in Cell Populations

    PubMed Central

    Orlova, Darya Y.; Zimmerman, Noah; Meehan, Stephen; Meehan, Connor; Waters, Jeffrey; Ghosn, Eliver E. B.; Filatenkov, Alexander; Kolyagin, Gleb A.; Gernez, Yael; Tsuda, Shanel; Moore, Wayne; Moss, Richard B.; Herzenberg, Leonore A.; Walther, Guenther

    2016-01-01

    Changes in the frequencies of cell subsets that (co)express characteristic biomarkers, or levels of the biomarkers on the subsets, are widely used as indices of drug response, disease prognosis, stem cell reconstitution, etc. However, although the currently available computational “gating” tools accurately reveal subset frequencies and marker expression levels, they fail to enable statistically reliable judgements as to whether these frequencies and expression levels differ significantly between/among subject groups. Here we introduce flow cytometry data analysis pipeline which includes the Earth Mover’s Distance (EMD) metric as solution to this problem. Well known as an informative quantitative measure of differences between distributions, we present three exemplary studies showing that EMD 1) reveals clinically-relevant shifts in two markers on blood basophils responding to an offending allergen; 2) shows that ablative tumor radiation induces significant changes in the murine colon cancer tumor microenvironment; and, 3) ranks immunological differences in mouse peritoneal cavity cells harvested from three genetically distinct mouse strains. PMID:27008164

  11. CD26 Expression on T Helper Populations and sCD26 Serum Levels in Patients with Rheumatoid Arthritis

    PubMed Central

    Cordero, Oscar J.; Varela-Calviño, Rubén; López-González, Tania; Calviño-Sampedro, Cristina; Viñuela, Juan E.; Mouriño, Coral; Hernández-Rodríguez, Íñigo; Rodríguez-López, Marina; Aspe de la Iglesia, Bruno; Pego, José María

    2015-01-01

    We studied dipeptidyl peptidase IV (DPP-IV, CD26) expression in different T helper cells and serum soluble DPP-IV/sCD26 levels in rheumatoid arthritis (RA) patients, correlated these with disease activity score (DAS), and examined how they were affected by different therapies, conventional or biological (anti-TNF, anti-CD20 and anti-IL6R or Ig-CTLA4). The percentage of CD4+CD45R0+CD26- cells was greatly reduced in patients (up to 50%) when compared with healthy subjects. Three other subsets of CD4 cells, including a CD26high Th1-associated population, changed variably with therapies. Data from these subsets (frequency and staining density) significantly correlated with DAS28 or DAS28 components but different in each group of patients undergoing the different therapies. Th17 and Th22 subsets were implicated in RA as independent CCR4+ and CCR4- populations each, with distinct CD26 expression, and were targeted with varying efficiency by each therapy. Serum DPP-IV activity rather than sCD26 levels was lower in RA patients compared to healthy donors. DPP-IV and sCD26 serum levels were found related to specific T cell subsets but not to disease activity. We conclude that, according to their CD26 expression, different cell subsets could serve to monitor RA course, and an uncharacterized T helper CD26- subset, not targeted by therapies, should be monitored for early diagnosis. PMID:26177310

  12. A Replication Study for Genome-Wide Gene Expression Levels in Two Layer Lines Elucidates Differentially Expressed Genes of Pathways Involved in Bone Remodeling and Immune Responsiveness

    PubMed Central

    Habig, Christin; Geffers, Robert; Distl, Ottmar

    2014-01-01

    The current replication study confirmed significant differences in gene expression profiles of the cerebrum among the two commercial layer lines Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB). Microarray analyses were performed for 30 LSL and another 30 LB laying hens kept in the small group housing system Eurovent German. A total of 14,103 microarray probe sets using customized Affymetrix ChiGene-1_0-st Arrays with 20,399 probe sets were differentially expressed among the two layer lines LSL and LB (FDR adjusted P-value <0.05). An at least 2-fold change in expression levels could be observed for 388 of these probe sets. In LSL, 214 of the 388 probe sets were down- and 174 were up-regulated and vice versa for the LB layer line. Among the 174 up-regulated probe sets in LSL, we identified 51 significantly enriched Gene ontology (GO) terms of the biological process category. A total of 63 enriched GO-terms could be identified for the 214 down-regulated probe sets of the layer line LSL. We identified nine genes significantly differentially expressed between the two layer lines in both microarray experiments. These genes play a crucial role in protection of neuronal cells from oxidative stress, bone mineral density and immune response among the two layer lines LSL and LB. Thus, the different regulation of these genes may significantly contribute to phenotypic trait differences among these layer lines. In conclusion, these novel findings provide a basis for further research to improve animal welfare in laying hens and these layer lines may be of general interest as an animal model. PMID:24922511

  13. Enhanced expression levels of aquaporin-1 and aquaporin-4 in A549 cells exposed to silicon dioxide.

    PubMed

    Hao, Xiaohui; Wang, Hongli; Liu, Wei; Liu, Shupeng; Peng, Zihe; Sun, Yue; Zhao, Jinyuan; Jiang, Qiujie; Liu, Heliang

    2016-09-01

    Aquaporins (AQPs), water channel proteins in the cell membranes of mammals, have been reported to be important in maintaining the water balance of the respiratory system. However, little is known regarding the role of AQP in occupational pulmonary diseases such as silicosis. The present study investigated the expression of AQP1 and AQP4 in the human A549 alveolar epithelial cell line stimulated by silica (SiO2). A549 cells were cultured and divided into four groups: Control, SiO2‑stimulated, AQP1 inhibitor and AQP4 inhibitor. The cells of the SiO2‑stimulated group were stimulated with SiO2 dispersed suspension (50 mg/ml). The cells of the inhibitor group were pretreated with mercury (II) chloride (HgCl2; a specific channel inhibitor of AQP1) and 2‑(nicotinamide)‑1,3,4‑thiadiazole (TGN‑020; a specific channel inhibitor of AQP4) and stimulated with SiO2. The mRNA expression levels of AQP1 and AQP4 were detected by reverse transcription‑quantitative polymerase chain reaction, and the protein expression levels of AQP1 and AQP4 were detected by western blotting and immunocytochemistry. Compared with the control group, the expression levels of AQP1 and AQP4 mRNA and protein in SiO2‑stimulated groups increased and subsequently decreased (AQP1 peaked at 2 h and AQP4 at 1h; both P<0.001 compared with control group). In the inhibitor group, expression levels were increased compared with controls; however, they were significantly decreased compared with the SiO2‑stimulated group at 2 h (AQP1; P<0.001) and 1 h (AQP4; P<0.001). The expression of AQP1 and AQP4 increased when exposed to SiO2, and this was inhibited by HgCl2 and TGN‑020, suggesting that AQP1 and AQP4 may contribute to A549 cell damage induced by SiO2. AQP1 and AQP4 may thus be involved in the initiation and development of silicosis. PMID:27431275

  14. Nucleophosmin protein expression level, but not threonine 198 phosphorylation, is essential in growth and proliferation

    PubMed Central

    Brady, SN; Maggi, LB; Winkeler, CL; Toso, EA; Gwinn, AS; Pelletier, CL; Weber, JD

    2010-01-01

    Nucleophosmin (NPM), an oligomeric phosphoprotein and nucleolar target of the ARF tumor suppressor, contributes to several critical cellular processes. Previous studies have shown that the human NPM’s phosphorylation by cyclin E–cyclin-dependent kinase 2 (cdk2) on threonine (Thr) 199 regulates its translocation from the centrosome during cell cycle progression. Given our previous finding that ARF directly binds NPM, impeding its transit to the cytoplasm and arresting cells before S-phase entry, we hypothesized that ARF might also inhibit NPM phosphorylation. However, ARF induction did not impair phosphorylation of the cdk2 target residue in murine NPM, Thr198. Furthermore, phosphorylation of Thr198 occurred throughout the cell cycle and was concomitant with increases in overall NPM expression. To investigate the cell’s presumed requirement for NPM-Thr198 phosphorylation in promoting the processes of growth and proliferation, we examined the effects of a non-phosphorylatable NPM mutant, T198A, in a clean cell system in which endogenous NPM had been removed by RNA interference. Here, we show that the T198A mutant is fully capable of executing NPM’s described roles in nucleocytoplasmic shuttling, ribosome export and cell cycle progression. Moreover, the proliferative defects observed with stable NPM knockdown were restored by mutant NPM-T198A expression. Thus, we demonstrate that the reduction in NPM protein expression blocks cellular growth and proliferation, whereas phosphorylation of NPM-Thr198 is not essential for NPM’s capacity to drive cell cycle progression and proliferation. PMID:19561638

  15. Moderate level of HER2 expression and its prognostic significance in breast cancer with intermediate grade.

    PubMed

    Ignatov, Tanja; Eggemann, Holm; Burger, Elke; Fettke, Franziska; Costa, Serban Dan; Ignatov, Atanas

    2015-06-01

    Overexpression of human epidermal growth factor receptor 2 (HER2) is an important prognostic and predictive marker of response to anti-HER2 therapy in breast cancer. Our goal was to analyze the prognostic significance of moderate expression of HER2 in breast cancer with intermediate differentiation grade. We performed a multicenter retrospective register study of 8494 patients with primary non-metastatic breast cancer admitted between 2000 and 2011 to eight Clinics in Saxony-Anhalt, federal state of Germany. Patients were divided into three groups according to their HER2 score: 4073 were classified as HER2 negative (HER2 0 and 1+), 822 HER2 moderate (HER2 2+/HER2), and 1238 HER2 positive (HER2 3+ or HER2 2+/HER2+). HER2-positive cases were excluded from analysis. Tumors with moderate HER2 (HER2 2+) expression demonstrated an aggressive behavior and worse patient survival compared with HER2 0 and 1+ status. HER2 2+ status was associated with shorter median overall survival (OS) (P < 0.0001) in breast cancer patients with an intermediate grade of differentiation. Comparing low-grade and high-grade tumors, HER2 moderate expression did not significantly influence patient survival. In multivariate analysis after adjustment for other prognostic factors HER2 2+ status remained an unfavorable prognostic factor for OS (HR 1.224, 95 % CI 1.059-1.415, P = 0.006) in breast cancer patients with an intermediate grade of differentiation. HER2 2+ status is an unfavorable prognostic factor regarding the OS of breast cancer patients with intermediate grade of differentiation and could be used to identify patients, who may benefit from adjuvant therapy. PMID:25926338

  16. High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc.

    PubMed

    Chen, W J; Moomaw, J F; Overton, L; Kost, T A; Casey, P J

    1993-05-01

    The mammalian enzyme protein farnesyltransferase is a heterodimeric protein that catalyzes the addition of a farnesyl isoprenoid to a cysteine in ras proteins. Since oncogenic forms of ras proteins require the farnesyl group for transforming activity, the structure and mechanism of this enzyme are important to define. However, such studies have been difficult to approach because of the low abundance of the enzyme in mammalian tissues and hence the problems of obtaining large quantities of the protein. We report here the co-expression of the two subunits of protein farnesyltransferase by Sf9 cells infected with a recombinant baculovirus containing the coding sequences of both polypeptides. This results in the production of milligram quantities of enzyme which can be readily purified by conventional chromatographic methods. The individual subunits of the enzyme can also be expressed in the Sf9 cells, but the ability to reconstitute active enzyme from extracts containing individual subunits is quite low. In contrast, the enzyme produced by co-expression of the two subunits is fully active and retains the properties of the mammalian form, including the specificity for the COOH-terminal amino acid of substrate proteins and the ability to bind short peptides encompassing the prenylation site of a ras protein. Furthermore, through atomic absorption analysis of the purified protein, we have confirmed the previous tentative assignment of protein farnesyltransferase as a zinc metalloenzyme by demonstrating that it contains an essentially stoichiometric amount of zinc. The ability to produce and purify milligram quantities of protein farnesyltransferase readily will allow detailed mechanistic and structural studies on this enzyme. PMID:8486655

  17. NIPBL expression levels in CdLS probands as a predictor of mutation type and phenotypic severity.

    PubMed

    Kaur, Maninder; Mehta, Devanshi; Noon, Sarah E; Deardorff, Matthew A; Zhang, Zhe; Krantz, Ian D

    2016-06-01

    Cornelia de Lange syndrome (CdLS) is a rare, genetically heterogeneous multisystem developmental disorder with a high degree of variability in its clinical presentation. Approximately 65% of probands harbor mutations in genes that encode core components (SMC1A, SMC3, and RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex, of which mutations in NIPBL are the most common. Cohesin plays a canonical role in sister chromatid cohesion during cell division and non-canonical roles in DNA repair, stem cell maintenance and differentiation, and regulation of gene expression. Disruption of the latter role seems to be the major contributor to the underlying molecular pathogenesis of CdLS. NIPBL is required for loading and unloading the cohesin complex onto chromosomes. The expression levels of NIPBL itself appear to be tightly regulated and highly evolutionarily conserved. Droplet digital PCR was used to quantify NIPBL mRNA expression levels with high precision from a cohort of 37 samples (NIPBL, SMC1A, SMC3, and HDAC8 mutation positive probands and negative control). Probands with severe forms of CdLS or severe mutation types were found to have lower levels of NIPBL in comparison to phenotypically milder patients and controls. Levels of NIPBL also correlated with the presence of mutations in different CdLS-causing genes. The data suggests that NIPBL levels are closely correlated with the severity of CdLS and with specific causative genes and types of mutations. ddPCR may provide a tool to assist in diagnostic approaches to CdLS, for genetic counseling and prognosis, and for monitoring potential therapeutic modalities in the future. © 2016 Wiley Periodicals, Inc. PMID:27125329

  18. GENDER DIFFERENCES IN BRAIN SUSCEPTIBILITY TO OXIDATIVE STRESS ARE MEDIATED BY LEVELS OF PARAOXONASE-2 (PON2) EXPRESSION

    PubMed Central

    Giordano, G.; Tait, L.; Furlong, C.E.; Cole, T.B.; Kavanagh, T.J.; Costa, L.G.

    2013-01-01

    Paraoxonase 2 (PON2), a member of a gene family that also includes PON1 and PON3, is expressed in most tissues, including the brain. In mouse brain, PON2 levels are highest in dopaminergic areas (e.g. striatum), and are higher in astrocytes than in neurons. PON2 is primarily located in mitochondria and exerts a potent antioxidant effect, protecting mouse CNS cells against oxidative stress. The aim of this study was to characterize PON2 expression and functions in the brains of male and female mice. Levels of PON2 (protein, mRNA, and lactonase activity) were higher in brain regions and cells of female mice. Astrocytes and neurons from male mice were significantly more sensitive (by 3–4-fold) to oxidative stress-induced toxicity than the same cells from female mice. Glutathione levels did not differ between genders. Importantly, no significant gender differences in susceptibility to the same oxidants were seen in cells from PON2−/− mice. Treatment with estradiol induced a time- and concentration-dependent increase in the levels of PON2 protein and mRNA in male (4.5-fold) and female (1.8-fold) astrocytes, which was dependent on activation of estrogen receptor alpha. In ovariectomized mice, PON2 protein and mRNA were decreased to male levels in brain regions and in liver. Estradiol protected astrocytes from wild-type mice against oxidative stress-induced neurotoxicity, but did not protect cells from PON2−/− mice. These results suggest that PON2 is a novel major intracellular factor that protects CNS cells against oxidative stress, and confers gender-dependent susceptibility to such stress. The lower expression of PON2 in males may have broad ramifications for susceptibility to diseases involving oxidative stress, including neurodegenerative diseases. PMID:23376469

  19. Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level

    SciTech Connect

    Eisenstein, R.S.; Rosen, J.M.

    1988-08-01

    The mechanism by which individual peptide and steroid hormones and cell-substratum interactions regulate milk protein gene expression has been studied in the COMMA-D mammary epithelial cell line. In the presence of insulin, hydrocortisone, and prolactin, growth of COMMA-D cells on floating collagen gels in comparison with that on a plastic substratum resulted in a 2.5- to 3-fold increase in the relative rate of ..beta..-casein gene transcription but a 37-fold increase in ..beta..-casein mRNA accumulation. In contrast, whey acidic protein gene transcription was constitutive in COMMA-D cells grown on either substratum, but its mRNA was unstable and little intact mature mRNA was detected. Culturing COMMA-D cells on collagen also promoted increased expression of other genes expressed in differentiated mammary epithelial cells, including those encoding ..cap alpha..- and ..gamma..-casein, transferrin, malic enzyme, and phosphoenolpyruvate carboxykinase but decreased the expression of actin and histone genes. Using COMMA-D cells, the authors defined further the role of individual hormones in influencing ..beta..-casein gene transcription. With insulin alone, a basal level of ..beta..-casein gene transcription was detected in COMMA-D cells grown on floating collagen gels. Addition of prolactin but not hydrocortisone resulted in a 2.5- to 3.0-fold increase in ..beta..-casein gene transcription, but both hormones were required to elicit the maximal 73-fold induction in mRNA accumulation. The posttranscriptional effect of hormones on casein mRNA accummulation preceded any detectable changes in the relative rate of transcription. Thus, regulation by both hormones and cell substratum of casein gene expression is exerted primarily at the post transcriptional level.

  20. Short-term sleep deprivation impairs spatial working memory and modulates expression levels of ionotropic glutamate receptor subunits in hippocampus.

    PubMed

    Xie, Meilan; Yan, Jie; He, Chao; Yang, Li; Tan, Gang; Li, Chao; Hu, Zhian; Wang, Jiali

    2015-06-01

    Hippocampus-dependent learning memory is sensitive to sleep deprivation (SD). Although the ionotropic glutamate receptors play a vital role in synaptic plasticity and learning and memory, however, whether the expression of these receptor subunits is modulated by sleep loss remains unclear. In the present study, western blotting was performed by probing with specific antibodies against the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1, GluA2, GluA3, and against the N-methyl-d-aspartate (NMDA) glutamate receptor subunits GluN1, GluN2A, GluN2B. In hippocampus, down regulation of surface GluA1 and GluN2A surface expression were observed in both SD groups. However, surface expression level of GluA2, GluA3, GluN1 and GluN2B was significantly up-regulated in 8h-SD rats when compared to the 4h-SD rats. In parallel with the complex changes in AMPA and NMDA receptor subunit expressions, we found the 8h-SD impaired rat spatial working memory in 30-s-delay T-maze task, whereas no impairment of spatial learning was observed in 4h-SD rats. These results indicate that sleep loss alters the relative expression levels of the AMPA and NMDA receptors, thus affects the synaptic strength and capacity for plasticity and partially contributes to spatial memory impairment. PMID:25732956

  1. Natural Functional SNPs in miR-155 Alter Its Expression Level, Blood Cell Counts, and Immune Responses.

    PubMed

    Li, Congcong; He, Huabin; Liu, An; Liu, Huazhen; Huang, Haibo; Zhao, Changzhi; Jing, Lu; Ni, Juan; Yin, Lilin; Hu, Suqin; Wu, Hui; Li, Xinyun; Zhao, Shuhong

    2016-01-01

    miR-155 has been confirmed to be a key factor in immune responses in humans and other mammals. Therefore, investigation of variations in miR-155 could be useful for understanding the differences in immunity between individuals. In this study, four SNPs in miR-155 were identified in mice (Mus musculus) and humans (Homo sapiens). In mice, the four SNPs were closely linked and formed two miR-155 haplotypes (A and B). Ten distinct types of blood parameters were associated with miR-155 expression under normal conditions. Additionally, 4 and 14 blood parameters were significantly different between these two genotypes under normal and lipopolysaccharide (LPS) stimulation conditions, respectively. Moreover, the expression levels of miR-155, the inflammatory response to LPS stimulation, and the lethal ratio following Salmonella typhimurium infection were significantly increased in mice harboring the AA genotype. Further, two SNPs, one in the loop region and the other near the 3' terminal of pre-miR-155, were confirmed to be responsible for the differential expression of miR-155 in mice. Interestingly, two additional SNPs, one in the loop region and the other in the middle of miR-155*, modulated the function of miR-155 in humans. Predictions of secondary RNA structure using RNAfold showed that these SNPs affected the structure of miR-155 in both mice and humans. Our results provide novel evidence of the natural functional SNPs of miR-155 in both mice and humans, which may affect the expression levels of mature miR-155 by modulating its secondary structure. The SNPs of human miR-155 may be considered as causal mutations for some immune-related diseases in the clinic. The two genotypes of mice could be used as natural models for studying the mechanisms of immune diseases caused by abnormal expression of miR-155 in humans. PMID:27532002

  2. High levels of glucose-6-phosphatase gene and protein expression reflect an adaptive response in proliferating liver and diabetes.

    PubMed Central

    Haber, B A; Chin, S; Chuang, E; Buikhuisen, W; Naji, A; Taub, R

    1995-01-01

    The regenerating liver after partial hepatectomy is one of the few physiologic models of cellular proliferation in the adult animal. During hepatic regeneration, the animal is able to maintain metabolic homeostasis despite the acute loss of two thirds of hepatic tissue. In examining the molecular mechanisms regulating hepatic regeneration, we isolated novel immediate-early genes that are rapidly induced as the remnant liver undergoes the transition from its normal quiescent state into the G1 phase of the cell cycle. One of the most rapidly and highly induced genes which we initially termed RL-1, encodes rat glucose-6-phosphatase (rG6Pase). G6Pase mRNA peaks at 30 min and 36-48 h after hepatectomy correlating with the first and second rounds of cell division. This finding is compatible with studies that showed that G6Pase enzyme activity increases during liver regeneration. However, the increase in G6Pase mRNA is much more dramatic, indicating that it is a more sensitive indicator of this regulation. G6Pase gene expression peaks in the perinatal time period in the liver and remains elevated during the first month of life. The expression of the G6Pase gene is also dramatically elevated in BB diabetic rats, again higher than the enzyme elevation, and its relative induction after partial hepatectomy is blunted in these animals. Insulin treatment of partially hepatectomized diabetic animals downregulates the expression of G6Pase mRNA. Using specific antibodies against G6Pase, we detect a 36-kD G6Pase protein, and its level is elevated in regenerating and diabetic livers. The pattern of G6Pase mRNA expression appears to reflect similar changes in insulin and glucagon levels which accompany diabetes and hepatic proliferation. The elevation of G6Pase expression in these conditions is indicative of its importance as a regulator of glucose homeostasis in normal and abnormal physiologic states. Images PMID:7860767

  3. Natural Functional SNPs in miR-155 Alter Its Expression Level, Blood Cell Counts, and Immune Responses

    PubMed Central

    Li, Congcong; He, Huabin; Liu, An; Liu, Huazhen; Huang, Haibo; Zhao, Changzhi; Jing, Lu; Ni, Juan; Yin, Lilin; Hu, Suqin; Wu, Hui; Li, Xinyun; Zhao, Shuhong

    2016-01-01

    miR-155 has been confirmed to be a key factor in immune responses in humans and other mammals. Therefore, investigation of variations in miR-155 could be useful for understanding the differences in immunity between individuals. In this study, four SNPs in miR-155 were identified in mice (Mus musculus) and humans (Homo sapiens). In mice, the four SNPs were closely linked and formed two miR-155 haplotypes (A and B). Ten distinct types of blood parameters were associated with miR-155 expression under normal conditions. Additionally, 4 and 14 blood parameters were significantly different between these two genotypes under normal and lipopolysaccharide (LPS) stimulation conditions, respectively. Moreover, the expression levels of miR-155, the inflammatory response to LPS stimulation, and the lethal ratio following Salmonella typhimurium infection were significantly increased in mice harboring the AA genotype. Further, two SNPs, one in the loop region and the other near the 3′ terminal of pre-miR-155, were confirmed to be responsible for the differential expression of miR-155 in mice. Interestingly, two additional SNPs, one in the loop region and the other in the middle of miR-155*, modulated the function of miR-155 in humans. Predictions of secondary RNA structure using RNAfold showed that these SNPs affected the structure of miR-155 in both mice and humans. Our results provide novel evidence of the natural functional SNPs of miR-155 in both mice and humans, which may affect the expression levels of mature miR-155 by modulating its secondary structure. The SNPs of human miR-155 may be considered as causal mutations for some immune-related diseases in the clinic. The two genotypes of mice could be used as natural models for studying the mechanisms of immune diseases caused by abnormal expression of miR-155 in humans. PMID:27532002

  4. Leptin receptor gene expression and number in the brain are regulated by leptin level and nutritional status.

    PubMed

    Mitchell, Sharon E; Nogueiras, Ruben; Morris, Amanda; Tovar, Sulay; Grant, Christine; Cruickshank, Morven; Rayner, D Vernon; Dieguez, Carlos; Williams, Lynda M

    2009-07-15

    Hormone potency depends on receptor availability, regulated via gene expression and receptor trafficking. To ascertain how central leptin receptors are regulated, the effects of leptin challenge, high-fat diet, fasting and refeeding were measured on leptin receptor number and gene expression. These were measured using quantitative (125)I-labelled leptin in vitro autoradiography and in situ hybridisation, respectively. Ob-R (all forms of leptin receptor) expression in the choroid plexus (CP) was unchanged by high-fat diet or leptin challenge, whereas fasting increased but refeeding failed to decrease expression. (125)I-labelled leptin binding to the CP was increased by fasting and returned to basal levels on refeeding. (125)I-Labelled leptin was reduced by leptin challenge and increased by high-fat feeding. Ob-Rb (signalling form) in the arcuate (ARC) and ventromedial (VMH) nuclei was increased after fasting and decreased by refeeding. Leptin challenge increased Ob-Rb expression in the ARC, but not after high-fat feeding. In general, changes in gene expression in the ARC and VMH appeared to be largely due to changes in area rather than density of labelling, indicating that the number of cells expressing Ob-Rb was the parameter that contributed most to these changes. Leptin stimulation of suppressor of cytokine signalling 3 (SOCS3), a marker of stimulation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway, was unchanged after high-fat diet. Thus, early loss of leptin sensitivity after high-fat feeding is unrelated to down-regulation of leptin receptor expression or number and does not involve the JAK/STAT pathway. The effect of leptin to decrease (125)I-labelled leptin binding and the loss of ability of leptin to up-regulate Ob-Rb expression in the ARC after high-fat feeding offer potential mechanisms for the development of leptin insensitivity in response to both hyperleptinaemia and high-fat diet. PMID:19491239

  5. Genome-level identification, gene expression, and comparative analysis of porcine ß-defensin genes

    PubMed Central

    2012-01-01

    Background Beta-defensins (β-defensins) are innate immune peptides with evolutionary conservation across a wide range of species and has been suggested to play important roles in innate immune reactions against pathogens. However, the complete β-defensin repertoire in the pig has not been fully addressed. Result A BLAST analysis was performed against the available pig genomic sequence in the NCBI database to identify β-defensin-related sequences using previously reported β-defensin sequences of pigs, humans, and cattle. The porcine β-defensin gene clusters were mapped to chromosomes 7, 14, 15 and 17. The gene expression analysis of 17 newly annotated porcine β-defensin genes across 15 tissues using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) showed differences in their tissue distribution, with the kidney and testis having the largest pBD expression repertoire. We also analyzed single nucleotide polymorphisms (SNPs) in the mature peptide region of pBD genes from 35 pigs of 7 breeds. We found 8 cSNPs in 7 pBDs. Conclusion We identified 29 porcine β-defensin (pBD) gene-like sequences, including 17 unreported pBDs in the porcine genome. Comparative analysis of β-defensin genes in the pig genome with those in human and cattle genomes showed structural conservation of β-defensin syntenic regions among these species. PMID:23150902

  6. Effect of HPV on tumor expression levels of the most commonly used markers in HNSCC.

    PubMed

    Polanska, Hana; Heger, Zbynek; Gumulec, Jaromir; Raudenska, Martina; Svobodova, Marketa; Balvan, Jan; Fojtu, Michaela; Binkova, Hana; Horakova, Zuzana; Kostrica, Rom; Adam, Vojtech; Kizek, Rene; Masarik, Michal

    2016-06-01

    Approximately 90 % of head and neck cancers are squamous cell carcinomas (HNSCC), and the overall 5-year survival rate is not higher than 50 %. There is much evidence that human papillomavirus (HPV) infection may influence the expression of commonly studied HNSCC markers. Our study was focused on the possible HPV-specificity of molecular markers that could be key players in important steps of cancerogenesis (MKI67, EGF, EGFR, BCL-2, BAX, FOS, JUN, TP53, MT1A, MT2A, VEGFA, FLT1, MMP2, MMP9, and POU5F). qRT-PCR analysis of these selected genes was performed on 74 biopsy samples of tumors from patients with histologically verified HNSCC (22 HPV-, 52 HPV+). Kaplan-Meier analysis was done to determine the relevance of these selected markers for HNSCC prognosis. In conclusion, our study confirms the impact of HPV infection on commonly studied HNSCC markers MT2A, MMP9, FLT1, VEGFA, and POU5F that were more highly expressed in HPV-negative HNSCC patients and also shows the relevance of studied markers in HPV-positive and HPV-negative HNSCC patients. PMID:26666815

  7. Reduction of EGF receptor levels in human tumor cells transfected with an antisense RNA expression vector

    SciTech Connect

    Yamada, Hirotomo; Koizumi, Shinji; Kimura, Masami ); Shimizu, Nobuyoshi )

    1989-09-01

    An expression vector was constructed from part of pSV2neo with the 3{prime}-ClaI fragment of the epidermal growth factor (EGF) receptor cDNA inserted in an inverted orientation downstream from the human metallothionein (MT) IIa promoter. The human squamous carcinoma cell line NA, which overproduces EGF receptor, was transfected with this vector and selected for resistance to the neomycin derivative G418. One of the stable transfectants had a 90% reduction cell-surface EGF receptor in response to ZnSO{sub 4}. The nascent EGF receptor peptide was also decreased with concurrent induction of MT mRNA. These data suggest that the antisense transcript regulated by the MT promoter inhibits the expression of the endogenous EGF receptor genes. Although no transcripts from the antisense gene were detected, the results indicate that transfection with the antisense vector provides a technique by which to modulate the number of EGF receptors on the cell surface of squamous cell carcinomas.

  8. Analysis of genes that influence sheep follicular development by different nutrition levels during the luteal phase using expression profiling.

    PubMed

    Luo, F; Jia, R; Ying, S; Wang, Z; Wang, F

    2016-06-01

    Nutrition is an important factor that regulates reproductive performance of sheep and affects follicle development. However, the correlation between nutrition and follicle development is poorly understood at the molecular level. To study its possible molecular mechanisms, we performed expression profiling of granulosa cells isolated from sheep that were fed different levels of nutrition levels during the luteal phase. To do this, ewes received a maintenance diet (M), and their estrus was synchronized by intravaginal progestogen sponges for 12 days. Ewes were randomly divided into the short-term dietary-restricted group (R; 0.5 × M) and the nutrient-supplemented group (S; 1.5 × M). RNA samples were extracted from granulosa cells. Transcriptome libraries from each group were constructed by Illumina sequencing. Among 18 468 detected genes, 170 genes were significantly differentially expressed, of which 140 genes were upregulated and 30 genes were downregulated in group S relative to group R. These genes could be candidates regulating follicular development in sheep. Gene Ontology, KEGG and clustering analyses were performed. Genes related to oocyte meiosis, such as ADCY7, were upregulated. We identified two important groups of related genes that were upregulated with improved nutrition: one group comprising the genes PTGS2, UCP2 and steroidogenic acute regulatory protein and the other group comprising interleukin-1A and interleukin-1B. The genes within each group showed similar expression patterns. Additionally, all five genes are involved in the reproduction process. Quantitative real-time PCR was performed to validate the results of expression profiling. These data in our study are an abundant genomic resource to expand the understanding of the molecular and cellular events underlying follicle development. PMID:26970339

  9. The trifunctional antibody ertumaxomab destroys tumor cells that express low levels of human epidermal growth factor receptor 2.

    PubMed

    Jäger, Michael; Schoberth, Alexandra; Ruf, Peter; Hess, Jürgen; Lindhofer, Horst

    2009-05-15

    Human epidermal growth factor receptor 2 (HER2/neu) is an important target for the treatment of the breast cancers in which it is overexpressed. However, no approved anti-HER2/neu therapy is available for the majority of breast cancer patients, who express HER2/neu at low levels (with scores of 1+ or 2+/fluorescence in situ hybridization-negative). The trifunctional antibody ertumaxomab targets HER2/neu, CD3, and activating Fcgamma receptors. In presence of ertumaxomab, tri-cell complexes consisting of tumor cells, T cells, and accessory cells form to cause tumor cell lysis. In a phase I trial with metastatic breast cancer patients, ertumaxomab could be applied safely and resulted in radiographically confirmed clinical responses. In this study, we compare ertumaxomab- and trastuzumab-mediated killing of cancer cell lines that express HER2/neu at low and high levels. Under optimal conditions for trastuzumab-mediated destruction of HER2/neu-overexpressing cells, only ertumaxomab was able to mediate the elimination of tumor cell lines that express HER2/neu at low levels (1+). Ertumaxomab-mediated activity was accompanied by a Th1-based cytokine release, a unique mode of action of trifunctional antibodies. Competitive binding studies with trastuzumab and 520C9 mapped the binding site of ertumaxomab to the extracellular regions II and III of the HER2/neu ectodomain. This site is distinct from the binding site of trastuzumab, so that HER2/neu-expressing tumor cells can be eliminated by ertumaxomab in the presence of high amounts of trastuzumab. The ability of ertumaxomab to induce cytotoxicity against various tumor cell lines, including those with low HER2/neu antigen density, may provide a novel therapeutic option for breast cancer patients who are not eligible for trastuzumab treatment. PMID:19435924

  10. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

    PubMed Central

    de Moraes, Vanessa C S; Bernardinelli, Emanuele; Zocal, Nathalia; Fernandez, Jhonathan A; Nofziger, Charity; Castilho, Arthur M; Sartorato, Edi L; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

  11. Global miRNA expression and correlation with mRNA levels in primary human bone cells

    PubMed Central

    Laxman, Navya; Rubin, Carl-Johan; Mallmin, Hans; Nilsson, Olle; Pastinen, Tomi; Grundberg, Elin; Kindmark, Andreas

    2015-01-01

    MicroRNAs (miRNAs) are important post-transcriptional regulators that have recently introduced an additional level of intricacy to our understanding of gene regulation. The aim of this study was to investigate miRNA–mRNA interactions that may be relevant for bone metabolism by assessing correlations and interindividual variability in miRNA levels as well as global correlations between miRNA and mRNA levels in a large cohort of primary human osteoblasts (HOBs) obtained during orthopedic surgery in otherwise healthy individuals. We identified differential expression (DE) of 24 miRNAs, and found 9 miRNAs exhibiting DE between males and females. We identified hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b and their target genes as important modulators of bone metabolism. Further, we used an integrated analysis of global miRNA–mRNA correlations, mRNA-expression profiling, DE, bioinformatics analysis, and functional studies to identify novel target genes for miRNAs with the potential to regulate osteoblast differentiation and extracellular matrix production. Functional studies by overexpression and knockdown of miRNAs showed that, the differentially expressed miRNAs hsa-miR-29b, hsa-miR-30c2, and hsa-miR-125b target genes highly relevant to bone metabolism, e.g., collagen, type I, α1 (COL1A1), osteonectin (SPARC), Runt-related transcription factor 2 (RUNX2), osteocalcin (BGLAP), and frizzled-related protein (FRZB). These miRNAs orchestrate the activities of key regulators of osteoblast differentiation and extracellular matrix proteins by their convergent action on target genes and pathways to control the skeletal gene expression. PMID:26078267

  12. Extracellular glutamate level and NMDA receptor subunit expression in mouse olfactory bulb following nanoparticle-rich diesel exhaust exposure.

    PubMed

    Win-Shwe, Tin-Tin; Mitsushima, Dai; Yamamoto, Shoji; Fujitani, Yuji; Funabashi, Toshiya; Hirano, Seishiro; Fujimaki, Hidekazu

    2009-08-01

    In this present study, we aimed to investigate the extracellular glutamate level and memory function-related gene expression in the mouse olfactory bulb after exposure of the animals to nanoparticle-rich diesel exhaust (NRDE) with or without bacterial cell wall component. Lipoteichoic acid (LTA), a cell wall component derived from Staphylococcus aureus, was used to induce systemic inflammation. Male BALB/c mice were exposed to clean air (particle concentration, 4.58 microg/m(3)) or NRDE (148.86 microg/m(3)) 5 h per day on 5 consecutive days of the week for 4 wk with or without weekly intraperitoneal injection of LTA. We examined the extracellular glutamate levels in the olfactory bulb using in vivo microdialysis and high-performance liquid chromatography assay. Then, we collected the olfactory bulb to examine the expression of N-methyl-D-aspartate (NMDA) receptor subunits (NR1, NR2A, and NR2B) and calcium/calmodulin-dependent protein kinase (CaMK) IV and cyclic AMP response element binding protein (CREB)-1 using real-time reverse-transcription polymerase chain reaction (RT-PCR). NRDE and/or LTA caused significantly increased extracellular glutamate levels in the olfactory bulb of mice. Moreover, the exposure of mice to NRDE upregulates NR1, NR2A, NR2B, and CaMKIV mRNAs in the olfactory bulb, while LTA upregulates only NR2B and CREB1 mRNAs. These findings suggest that NRDE and LTA cause glutamate-induced neurotoxicity separately and accompanied by changes in the expression of NMDA receptor subunits and related kinase and transcription factor in the mouse olfactory bulb. This is the first study to show the correlation between glutamate toxicity and memory function-related gene expressions in the mouse olfactory bulb following exposure to NRDE. PMID:19653804

  13. High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

    PubMed Central

    Allen, G C; Hall, G; Michalowski, S; Newman, W; Spiker, S; Weissinger, A K; Thompson, W F

    1996-01-01

    We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold. In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies. PMID:8672887

  14. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment.

    PubMed

    Sustarsic, Elahu G; Junnila, Riia K; Kopchick, John J

    2013-11-01

    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 this data, GH could be a new therapeutic target in melanoma. PMID:24134847

  15. The level of nitric oxide regulates lipocalin-2 expression under inflammatory condition in RINm5F beta-cells.

    PubMed

    Chang, Seo-Yoon; Kim, Dong-Bin; Ko, Seung-Hyun; Jang, Hyun-Jong; Jo, Yang-Hyeok; Kim, Myung-Jun

    2016-07-15

    We previously reported that proinflammatory cytokines (interleukin-1β and interferon-γ) induced the expression of lipocalin-2 (LCN-2) together with inducible nitric oxide synthase (iNOS) in RINm5F beta-cells. Therefore, we examined the effect of nitric oxide (NO) on LCN-2 expression in cytokines-treated RINm5F beta-cells. Additionally, we observed the effect of LCN-2 on cell viability. First, we found the existence of LCN-2 receptor and the internalization of exogenous recombinant LCN-2 peptide in RINm5F and INS-1 beta-cells. Next, the effects of NO on LCN-2 expression were evaluated. Aminoguanidine, an iNOS inhibitor and iNOS gene silencing significantly inhibited cytokines-induced LCN-2 expression while sodium nitroprusside (SNP), an NO donor potentiated it. Luciferase reporter assay showed that transcription factor NF-κB was not involved in LCN-2 expression. Both LCN-2 mRNA and protein stability assays were conducted. SNP did not affect LCN-2 mRNA stability, however, it significantly reduced LCN-2 protein degradation. The LCN-2 protein degradation was significantly attenuated by MG132, a proteasome inhibitor. Finally, the effect of LCN-2 on cell viability was evaluated. LCN-2 peptide treatment and LCN-2 overexpression significantly reduced cell viability. FACS analysis showed that LCN-2 induced the apoptosis of the cells. Collectively, NO level affects LCN-2 expression via regulation of LCN-2 protein stability under inflammatory condition and LCN-2 may reduce beta-cell viability by promoting apoptosis. PMID:27233602

  16. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

    PubMed Central

    Sustarsic, Elahu G.; Junnila, Riia K.; Kopchick, John J.

    2013-01-01

    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 this data, GH could be a new therapeutic target in melanoma. PMID:24134847

  17. Expression levels of estrogen receptor α mRNA in peripheral blood cells are an independent biomarker for postmenopausal osteoporosis☆

    PubMed Central

    Chou, Chi-Wen; Chiang, Tsay-I; Chang, I-Chang; Huang, Chung-Hung; Cheng, Ya-Wen

    2016-01-01

    Background The up- and down-regulation of the osteoclastogenesis response depends on the estrogen/estrogen receptor (ER) signaling pathway. Previous reports have shown that the promoter hypermethylation and gene polymorphism of ERα are risks for menopausal osteoporosis. No previous study has evaluated the expression levels of ERα mRNA in menopausal osteoporosis using human subjects. We hypothesized that ERα mRNA expression may show less resistance to postmenopausal osteoporosis. Methods In this study, we enrolled 107 women older than 45 years without menstruation and classified them into control, osteopenia, and osteoporosis groups depending on their T-scores. The ERα mRNA levels in peripheral blood cells (PBCs) were analyzed via quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), and estrogen in the serum was detected via ELISA. Results ERα mRNA levels in PBCs had a negative correlation with age and a positive correlation with estrogen and BAP in the osteopenia and osteoporosis groups, but not in the control group. Additionally, multivariate analysis showed that older age (> 55 years), and low ERα mRNA levels in PBLs (≦ 250.39 copies/μg DNA) were associated with an approximately 9.188-, and 31.25-fold risk of osteoporosis. Conclusion We conclude that ERα mRNA levels in PBLs could be used as an independent risk factor for postmenopausal osteoporosis. General significance Our findings suggested that ERα mRNA levels in PBLs may be more important than age and serum estrogen levels. PMID:27051599

  18. B-chromosome effects on Hsp70 gene expression does not occur at transcriptional level in the grasshopper Eyprepocnemis plorans.

    PubMed

    Navarro-Domínguez, Beatriz; Cabrero, Josefa; Camacho, Juan Pedro M; López-León, María Dolores

    2016-10-01

    As intragenomic parasites, B chromosomes can elicit stress in the host genome, thus inducing a response for host adaptation to this kind of continuous parasitism. In the grasshopper Eyprepocnemis plorans, B-chromosome presence has been previously associated with a decrease in the amount of the heat-shock protein 70 (HSP70). To investigate whether this effect is already apparent at transcriptional level, we analyze the expression levels of the Hsp70 gene in gonads and somatic tissues of males and females with and without B chromosomes from two populations, where the predominant B chromosome variants (B2 and B24) exhibit different levels of parasitism, by means of quantitative real-time PCR (qPCR) on complementary DNA (cDNA). The results revealed the absence of significant differences for Hsp70 transcripts associated with B-chromosome presence in virtually all samples. This indicates that the decrease in HSP70 protein levels, formerly reported in this species, may not be a consequence of transcriptional down-regulation of Hsp70 genes, but the result of post-transcriptional regulation. These results will help to design future studies oriented to identifying factors modulating Hsp70 expression, and will also contribute to uncover the biological role of B chromosomes in eukaryotic genomes. PMID:27334602

  19. Coffee Polyphenols Change the Expression of STAT5B and ATF-2 Modifying Cyclin D1 Levels in Cancer Cells

    PubMed Central

    Oleaga, Carlota; Ciudad, Carlos J.; Noé, Véronique; Izquierdo-Pulido, Maria

    2012-01-01

    Background. Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown. Objective. To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC) or caffeic acid (CA) using whole-genome microarrays. Results. ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were downregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells. Conclusions. Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2. PMID:22919439

  20. Effect of floral cluster pruning on anthocyanin levels and anthocyanain-related gene expression in 'Houman' grape.

    PubMed

    Zhang, Lei; Xu, Yan-Shuai; Jia, Yue; Wang, Ji-Yuan; Yuan, Yue; Yu, Yang; Tao, Jian-Min

    2016-01-01

    Lateral floral clusters were removed from the main axis of the floral clusters of 'Houman' grape plants, leaving only 3-5-cm-long region of flowers at the end of the central axis. The floral clusters were pruned at 7 days prior to flowering. The effect of the pruning on fruit quality was assessed by determining the composition and levels of anthocyanins in the fruit and anthocyanin-related gene expression. Results indicated that floral cluster pruning significantly improved the quality of the fruit by increasing berry size, fruit weight and the total content of soluble solids. Floral cluster pruning also decreased the level of titratable acidity. Sixteen different anthocyanins were detected in fruit of the pruned clusters, while only 15 were detected in fruit from unpruned clusters. The level of anthocyanins was also significantly higher in fruit of the pruned clusters than in the unpruned clusters. Anthocyanin-related gene expression was also significantly upregulated to a higher level in fruit from pruned floral clusters as compared with unpruned clusters. The upregulation was closely associated with increases in anthocyanin biosynthesis. PMID:27555920

  1. Effect of floral cluster pruning on anthocyanin levels and anthocyanain-related gene expression in ‘Houman’ grape

    PubMed Central

    Zhang, Lei; Xu, Yan-shuai; Jia, Yue; Wang, Ji-yuan; Yuan, Yue; Yu, Yang; Tao, Jian-min

    2016-01-01

    Lateral floral clusters were removed from the main axis of the floral clusters of ‘Houman’ grape plants, leaving only 3–5-cm-long region of flowers at the end of the central axis. The floral clusters were pruned at 7 days prior to flowering. The effect of the pruning on fruit quality was assessed by determining the composition and levels of anthocyanins in the fruit and anthocyanin-related gene expression. Results indicated that floral cluster pruning significantly improved the quality of the fruit by increasing berry size, fruit weight and the total content of soluble solids. Floral cluster pruning also decreased the level of titratable acidity. Sixteen different anthocyanins were detected in fruit of the pruned clusters, while only 15 were detected in fruit from unpruned clusters. The level of anthocyanins was also significantly higher in fruit of the pruned clusters than in the unpruned clusters. Anthocyanin-related gene expression was also significantly upregulated to a higher level in fruit from pruned floral clusters as compared with unpruned clusters. The upregulation was closely associated with increases in anthocyanin biosynthesis. PMID:27555920

  2. The impact of reference gene selection in quantification of gene expression levels in guinea pig cervical tissues and cells

    PubMed Central

    2013-01-01

    Background Accurate measurements of mRNA expression levels in tissues or cells are crucially dependent on the use of relevant reference genes for normalization of data. In this study we used quantitative real-time PCR and two Excel-based applets (geNorm and BestKeeper) to determine the best reference genes for quantification of target gene mRNA in a complex tissue organ such as the guinea pig cervix. Results Gene expression studies were conducted in cervical epithelium and stroma during pregnancy and parturition and in cultures of primary cells from this tissue. Among 15 reference gene candidates examined, both geNorm and BestKeeper found CLF1 and CLTC to be the most stable in cervical stroma and cervical epithelium, ACTB and PPIB in primary stroma cells, and CLTC and PPIB in primary epithelial cells. The order of stability among the remaining candidate genes was not in such an agreement. Commonly used reference such as GAPDH and B2M demonstrated lower stability. Determination of pairwise variation values for reference gene combinations using geNorm revealed that the geometric mean of the two most stable genes provides sufficient normalization in most cases. However, for cervical stroma tissue in which many reference gene candidates displayed low stability, inclusion of three reference genes in the geometric mean may improve accuracy of target gene expression level analyses. Using the top ranked reference genes we examined the expression levels of target gene PTGS2 in cervical tissue and cultured cervical cells. We compared the results with PTGS2 expression normalized to the least stable gene and found significant differences in gene expression, up to 10-fold in some samples, emphasizing the importance of appropriately selecting reference genes. Conclusions We recommend using the geometric mean of CFL1 and CLTC for normalization of qPCR studies in guinea pig cervical tissue studies, ACTB and PPIB in primary stroma cells and CLTC and PPIB in primary epithelial cells

  3. High-level Expression and Purification of Active Human FGF-2 in Escherichia coli by Codon and Culture Condition Optimization

    PubMed Central

    Soleyman, Mohammad Reza; Khalili, Mostafa; Khansarinejad, Behzad; Baazm, Maryam

    2016-01-01

    Background: Basic fibroblast growth factor (bFGF) is a member of a highly conserved superfamily of proteins that are involved in cell proliferation, differentiation, and migration. Objectives: The objective of this study was to overexpress and purify the high-level active human bFGF in Escherichia coli (E. coli). Materials and Methods: This experimental study was conducted in the Islamic Republic of Iran. After codon optimization and gene synthesis, the optimized FGF-2 gene was subcloned into plasmid pET-32a. pET32-FGF-2 was transformed into E. coli BL21 for expression. The cultivation parameters were optimized to produce a high yield of FGF-2. Results: The optimal conditions were determined as follows: cultivation at 37°C in TB medium, with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), followed by post-induction expression for 6 h. Under the abovementioned conditions, the expression volumetric productivity of FGF-2 reached 1.48 g/L. Conclusions: A fusion tag from the pET32 expression plasmid permits the recovery of the recombinant fusion FGF-2 from E. coli, without affecting its biological activity. PMID:27175305

  4. High levels of functional endopeptidase 24.11 (CD10) activity on human thymocytes: preferential expression on immature subsets.

    PubMed Central

    Mari, B; Breittmayer, J P; Guerin, S; Belhacene, N; Peyron, J F; Deckert, M; Rossi, B; Auberger, P

    1994-01-01

    Although it is now well established that cells of the immune system express most of the exopeptidases described so far, little information is available concerning the identification and the characterization of the peptidases associated with the surface of human thymocytes. In the present study we have focused on CD10 expression on thymocytes using both FACS and enzymatic analysis. Unfractionated intact human thymocytes were shown to express significant levels of CD10-specific enzymatic activity, as assessed by the hydrolysis of the neutral endopeptidase (NEP) substrate Suc-Ala-Ala-Phe-pNA and of D-Ala2-Leu-enkephalin, a typical NEP substrate. CD10 activity was abolished by specific NEP inhibitors, including thiorphan, retrothiorphan and phosphoramidon. Moreover, high performance liquid chromatography (HPLC) analysis showed that intact thymocytes and purified NEP hydrolysed thymopentin, a thymic factor known to induce the maturation of prothymocytes into thymocytes. Finally, CD 10/NEP was preferentially associated with CD3- CD3low and immature CD4- CD8- thymocytes. The data demonstrate for the first time that human thymocytes express functional NEP and suggest a role for this enzyme in the maturation of human thymocytes. PMID:7959879

  5. Inflammatory modulating effects of low level laser therapy on iNOS expression by means of bioluminescence imaging

    NASA Astrophysics Data System (ADS)

    Moriyama, Yumi; Moriyama, Eduardo H.; Blackmore, Kristina; Akens, Margarete K.; Lilge, Lothar

    2005-09-01

    This study investigates the efficacy of low level laser therapy (LLLT) in modulating inducible nitric oxide synthase (iNOS) expression as molecular marker of the inflammation signaling pathway. LLLT was mediated by different therapeutic wavelengths using transgenic animals with the luciferase gene under control of the iNOS gene expression. Inflammation in 30 transgenic mice (iNOS-luc mice, from FVB strain) was induced by intra-articular injection of Zymosan-A in both knee joints. Four experimental groups were treated with one of four different wavelengths (λ=635, 785, 808 and 905nm) and one not laser-irradiated control group. Laser treatment (25 mW cm-2, 5 J cm-2) was applied to the knees 15 minutes after inflammation induction. Measurements of iNOS expression were performed at multiple times (0, 3, 5, 7, 9 and 24h) post-LLLT by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The responsivity of BLI was sufficient to demonstrate a significant increase in bioluminescence signals after laser irradiation of 635nm when compared to non-irradiated animals and the other LLLT treated groups, showing the wavelength-dependence of LLLT on iNOS expression during the acute inflammatory process.

  6. Expression levels of seven candidate genes in human peripheral blood mononuclear cells and their association with preeclampsia

    PubMed Central

    Garza-Veloz, I.; Carrillo-Sanchez, K.; Martinez-Gaytan, V.; Cortes-Flores, R.; Ochoa-Torres, M. A.; Guerrero, G. G.; Rodriguez-Sanchez, I. P.; Cancela-Murrieta, C. O.; Zamudio-Osuna, M.; Badillo-Almaraz, J. I.; Castruita-De la Rosa, C.

    2014-01-01

    Objective To evaluate the peripheral blood mononuclear cell (PBMC) expression levels of hemeoxygenase 1 (HMOX-1), superoxide dismutase 1 (SOD-1), vascular endothelial growth factor A (VEGF-A), transforming growth factor beta 1 (TGF-β1), interleukin (IL)-6, IL-15 and AdipoQ genes to study their association with preeclampsia (PE). Methods A total of 177 pregnant women were recruited: 108 cases and 69 controls. Quantification of gene expression was measured by quantitative real-time polymerase chain reaction (PCR) using TaqMan probes. Results Underexpression of VEGF-A and TGF-β1 was a constant in most of the cases (80.91% and 76.36%, respectively) and their expression was associated with onset and/or severity of disease (p values < 0.05). IL-6, IL-15 and AdipoQ, showed low or no expression in PBMC samples evaluated. Conclusion PBMC underexpression of VEGF-A and TGF-β1 is a hallmark of PE in the study population. PMID:24295154

  7. Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes.

    PubMed

    Dekker, Rob J; van Thienen, Johannes V; Rohlena, Jakub; de Jager, Saskia C; Elderkamp, Yvonne W; Seppen, Jurgen; de Vries, Carlie J M; Biessen, Erik A L; van Berkel, Theo J C; Pannekoek, Hans; Horrevoets, Anton J G

    2005-08-01

    Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than approximately 5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow. PMID:16049344

  8. The Level of AdpA Directly Affects Expression of Developmental Genes in Streptomyces coelicolor ▿ †

    PubMed Central

    Wolański, Marcin; Donczew, Rafał; Kois-Ostrowska, Agnieszka; Masiewicz, Paweł; Jakimowicz, Dagmara; Zakrzewska-Czerwińska, Jolanta

    2011-01-01

    AdpA is a key regulator of morphological differentiation in Streptomyces. In contrast to Streptomyces griseus, relatively little is known about AdpA protein functions in Streptomyces coelicolor. Here, we report for the first time the translation accumulation profile of the S. coelicolor adpA (adpASc) gene; the level of S. coelicolor AdpA (AdpASc) increased, reaching a maximum in the early stage of aerial mycelium formation (after 36 h), and remained relatively stable for the next several hours (48 to 60 h), and then the signal intensity decreased considerably. AdpASc specifically binds the adpASc promoter region in vitro and in vivo, suggesting that its expression is autoregulated; surprisingly, in contrast to S. griseus, the protein presumably acts as a transcriptional activator. We also demonstrate a direct influence of AdpASc on the expression of several genes whose products play key roles in the differentiation of S. coelicolor: STI, a protease inhibitor; RamR, an atypical response regulator that itself activates expression of the genes for a small modified peptide that is required for aerial growth; and ClpP1, an ATP-dependent protease. The diverse influence of AdpASc protein on the expression of the analyzed genes presumably results mainly from different affinities of AdpASc protein to individual promoters. PMID:21926228

  9. Vitamin E levels in soybean (Glycine max (L.) Merr.) expressing a p-hydroxyphenylpyruvate gene from oat (Avena sativa L.).

    PubMed

    Kramer, Catherine M; Launis, Karen L; Traber, Maret G; Ward, Dennis P

    2014-04-16

    The enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) is ubiquitous in plants and functions in the tyrosine catabolic pathway, resulting in the formation of homogentisate. Homogentisate is the aromatic precursor of all plastoquinones and tocochromanols, including tocopherols and tocotrienols. Soybean (Glycine max (L.) Merr.) has been genetically modified to express the gene avhppd-03 that encodes the protein AvHPPD-03 derived from oat (Avena sativa L.). The AvHPPD-03 isozyme has an inherent reduced binding affinity for mesotrione, a herbicide that inhibits the wild-type soybean HPPD enzyme. Expression of avhppd-03 in soybean plants confers a mesotrione-tolerant phenotype. Seeds from three different avhppd-03-expressing soybean events were quantitatively assessed for content of eight vitamin E isoforms. Although increased levels of two tocopherol isoforms were identified for each of the three soybean events, they were within, or not substantially different from, the ranges of these isoforms found in nontransgenic soybean varieties. The increases of these tocopherols in the avhppd-03-expressing soybean events may have a slight benefit with regard to vitamin E nutrition but, given the commercial processing of soybeans, are unlikely to have a material impact on human nutrition with regard to vitamin E concentrations in soybean oil. PMID:24684596

  10. High-level expression of a manganese superoxide dismutase (PoMn-SOD) from Pleurotus ostreatus in Pichia pastoris.

    PubMed

    Yin, Chaomin; Zhao, Wenxia; Zheng, Liesheng; Chen, Liguo; Tan, Qi; Shang, Xiaodong; Ma, Aimin

    2014-09-01

    The full-length cDNA of Pleurotus ostreatus superoxide dismutase (PoMn-SOD) was cloned and successfully expressed by using the pPIC9K vector under the control of alcohol oxidase 1 promoter with a secretion signal peptide (α-factor) in Pichia pastoris. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting demonstrated that recombinant PoMn-SOD, a 21.8 kDa protein, was secreted into the culture medium. Nondenaturing PAGE experiments confirmed that recombinant PoMn-SOD was secreted in a functionally active form and the expression system did not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. The maximum enzyme activity (156.9 U/mg) was observed under the following conditions: Initial medium pH was 6.0, induction time point was at the 6th day, and methanol concentration was 0.7 % (v/v). This was the first report on secretory expression of recombinant PoMn-SOD in P. pastoris, which might provide a reference for further practical applications. PMID:25059984

  11. Endothelial KLF2 Links Local Arterial Shear Stress Levels to the Expression of Vascular Tone-Regulating Genes

    PubMed Central

    Dekker, Rob J.; van Thienen, Johannes V.; Rohlena, Jakub; de Jager, Saskia C.; Elderkamp, Yvonne W.; Seppen, Jurgen; de Vries, Carlie J.M.; Biessen, Erik A.L.; van Berkel, Theo J.C.; Pannekoek, Hans; Horrevoets, Anton J.G.

    2005-01-01

    Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than ∼5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow. PMID:16049344

  12. Identification of a new subclass of ALK-negative ALCL expressing aberrant levels of ERBB4 transcripts.

    PubMed

    Scarfò, Irene; Pellegrino, Elisa; Mereu, Elisabetta; Kwee, Ivo; Agnelli, Luca; Bergaggio, Elisa; Garaffo, Giulia; Vitale, Nicoletta; Caputo, Manuel; Machiorlatti, Rodolfo; Circosta, Paola; Abate, Francesco; Barreca, Antonella; Novero, Domenico; Mathew, Susan; Rinaldi, Andrea; Tiacci, Enrico; Serra, Sara; Deaglio, Silvia; Neri, Antonino; Falini, Brunangelo; Rabadan, Raul; Bertoni, Francesco; Inghirami, Giorgio; Piva, Roberto

    2016-01-14

    Anaplastic large-cell lymphoma (ALCL) is a clinical and biological heterogeneous disease that includes systemic anaplastic lymphoma kinase (ALK)-positive and ALK-negative entities. To discover biomarkers and/or genes involved in ALK-negative ALCL pathogenesis, we applied the cancer outlier profile analysis algorithm to a gene expression profiling data set including 249 cases of T-cell non-Hodgkin lymphoma and normal T cells. Ectopic coexpression of ERBB4 and COL29A1 genes was detected in 24% of ALK-negative ALCL patients. RNA sequencing and 5' RNA ligase-mediated rapid amplification of complementary DNA ends identified 2 novel ERBB4-truncated transcripts displaying intronic transcription start sites. By luciferase assays, we defined that the expression of ERBB4-aberrant transcripts is promoted by endogenous intronic long terminal repeats. ERBB4 expression was confirmed at the protein level by western blot analysis and immunohistochemistry. Lastly, we demonstrated that ERBB4-truncated forms show oncogenic potentials and that ERBB4 pharmacologic inhibition partially controls ALCL cell growth and disease progression in an ERBB4-positive patient-derived tumorgraft model. In conclusion, we identified a new subclass of ALK-negative ALCL characterized by aberrant expression of ERBB4-truncated transcripts carrying intronic 5' untranslated regions. PMID:26463425

  13. High-level expression and characterization of a glycosylated human cementum protein 1 with lectin activity.

    PubMed

    Romo-Arévalo, Enrique; Arzate, Higinio; Montoya-Ayala, Gonzalo; Rodríguez-Romero, Adela

    2016-01-01

    This work aims to contribute to the knowledge of human cementum protein 1 (CEMP1), its conformational characteristics and influence during the biomineralization process. The results revealed that hrCEMP1 expressed in Pichia pastoris is a 2.4% glycosylated, thermostable protein which possesses a molecular mass of 28 770 Da. The circular dichroism spectrum indicated a secondary structure content of 28.6% of alpha-helix, 9.9% of beta-sheet and 61.5% of random-coil forms. Biological activity assays demonstrated that hrCEMP1 nucleates and regulates hydroxyapatite crystal growth. Hereby, it is demonstrated for the first time that CEMP1 has a (C-type) lectin-like activity and specifically recognizes mannopyranoside. The information produced by this biochemical and structural characterization may contribute to understand more fully the biological functions of CEMP1. PMID:26763148

  14. Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA

    SciTech Connect

    Chu, G.; Chang, E. )

    1990-05-01

    Cancer treatment with the drug cisplatin is often thwarted by the emergence of drug-resistant cells. To study this phenomenon, the authors identified two independent cellular factors that recognize cisplatin-damaged DNA. One of the two factors, designated XPE binding factor, is deficient in complementation group E of xeroderma pigmentosum, an inherited disease characterized by defective repair of DNA damaged by ultraviolet radiation, cisplatin, and other agents. Human tumor cell lines selected for resistance to cisplatin showed more efficient DNA repair and increased expression of XPE binding factor. These results suggest that XPE binding factor may be responsible, at least in part, for the development of cisplatin resistance in human tumors and that the mechanism may be increased DNA repair.

  15. EVALUATION OF BIOLOGICAL ACTIVITY OF VITELLOGENIN EXPRESSION IN DIFFERENT AQUATIC MESOCOSM TROPIC LEVELS

    EPA Science Inventory

    Aquatic mesocosms were dosed with an environmentally relevant concentration of 17-a-ethinyl estradiol (EE2) to study the significance of trophic status (N, P levels) on the attenuation and bioavailability of synthetic estrogens in aquatic ecosystems. Estrogenic activity was asse...

  16. Superoxide dismutase activity and gene expression levels in Saudi women with recurrent miscarriage

    PubMed Central

    GHNEIM, HAZEM K.; AL-SHEIKH, YAZEED A.; ALSHEBLY, MASHAEL M.; ABOUL-SOUD, MOURAD A. M.

    2016-01-01

    The antioxidant activities of superoxide dismutase 1 (SOD1) and SOD2, as well as the levels of the oxidant superoxide anion (SOA) and the micronutrients zinc (Zn), copper (Cu) and manganese (Mn), were assayed in plasma, whole blood and placental tissue of non-pregnant (NP), healthy pregnant (HP) women and recurrent miscarriage (RM) patients. The results showed that SOD1 and SOD2 activities and the levels of Zn, Cu and Mn in plasma and whole blood of HP women were slightly, but significantly lower, and even more significantly decreased in RM patients compared to those observed in NP women (P<0.05 and P<0.0001, respectively). Additionally, whereas plasma SOD1 and SOD2 activities and Zn, Cu and Mn levels were significantly lower in RM patients, those of whole blood and placental tissue were significantly lower when compared to HP women (P<0.001 and P<0.0001, respectively). Concurrently, there were consistent increases of equal magnitude and statistical significance in SOA levels in all the assayed samples as identified by a comparison between the subjects. The findings thus supported oxidative metabolism and excessive reactive oxygen species generation. The resultant oxidative stress, identified in whole blood and placental tissues of RM patients, may have been a primary cause of RM. Dietary supplementation of Zn, Cu and Mn may be beneficial to these patients pre- and post-conception. PMID:26821085

  17. Changes in acetylcholinesterase expression are associated with altered presenilin-1 levels.

    PubMed

    Silveyra, María-Ximena; García-Ayllón, María-Salud; Serra-Basante, Carol; Mazzoni, Valeria; García-Gutierrez, María-Salud; Manzanares, Jorge; Culvenor, Janetta G; Sáez-Valero, Javier

    2012-03-01

    We have previously identified presenilin-1 (PS1), the active component of the γ-secretase complex, as an interacting protein of the amyloid-associated enzyme acetylcholinesterase (AChE). In this study, we have explored the consequences of AChE-PS1 interactions. Treatment of SH-SY5Y cells with the AChE-inhibitor tacrine decreased PS1 levels, in parallel with increase in the secretion of amyloid precursor protein APPα, whereas the cholinergic agonist carbachol had no effect on PS1. AChE knockdown with siRNA also decreased PS1 levels, while AChE overexpression exerted opposing effect. AChE-deficient also had decreased PS1. Mice administered with tacrine or donepezil displayed lower levels of brain PS1. However, sustained AChE inhibition failed to exert long-term effect on PS1. This limited duration of response may be due to AChE upregulation caused by chronic inhibition. Finally, we exposed SH-SY5Y cells to β-amyloid (Aβ)42 which triggered elevation of both AChE and PS1 levels. The Aβ42-induced PS1 increase was abolished by siRNA AChE pretreatment, suggesting that AChE may participate in the pathological feedback loop between PS1 and Aβ. Our results provide insight into AChE-amyloid interrelationships. PMID:21621296

  18. First Results from VIRTIS on Venus Express1. From Surface to Cloud Level

    NASA Astrophysics Data System (ADS)

    Piccioni, Giuseppe; Drossart, P.; VIRTIS/Venus Express Team

    2006-09-01

    VIRTIS is the imaging spectrometer of the ESA/Venus Express mission, in orbit around Venus since 2006, April 11th. It consists in two channels : VIRTIS-M, an imaging spectrometer with 0.25 mrad instantaneous field of view, working from 0.3 to 1 μm at 2nm spectral sampling (VIRTIS-M-vis) and from 1 to 5.2 μm at 10 nm resolution (VIRTIS-M-ir) and VIRTIS-H, a high resolution spectrometer working from 1.9 to 5.0 μm on an aperture of 0.58 x 1.74 mrad at 2000 resolving power. The main scientific objectives for the lower part of the atmosphere are the following:

    • Surface characteristics
    • Composition of the lower atmosphere, and spatial or temporal variations (night side)
    • Cloud structure (composition, scattering properties, and dynamics)
    • Thermal structure
    An important new result from VIRTIS is the first detailed study of the South polar vortex. This feature exhibits unique characteristics of essential importance for the understanding of the global dynamics of Venus. The rotation of the vortex is observed by VIRTIS at different altitudes for different wavelengths. The vortex has a complex structure, exhibiting high thermal contrasts and filamentary clouds forming an inverse "S” shape. The 1.7 μm and 2.3 μm spectral images, observed in the night side within these deep atmospheric windows, are the first observations of the deep structure of the vortex. Future observations during the Venus Express mission will allow us to continue the vortex observation to study its long term variations.

  19. The curli biogenesis genes expression level is unassociated with Enterobacter cloacae hsp60 clusters and PFGE genotypes.

    PubMed

    Akbari, Majid; Bakhshi, Bita; Najar-Peerayeh, Shahin; Behmanesh, Mehrdad

    2016-09-01

    The objective of this study was to determine the correlation between Enterobacter cloacae complex subspecies and clusters involved in UTI infections and specific pulsotypes, and to assess the contribution of major curli biogenesis genes (csgD, csgA) expression level to pathogenesis of clusters and genotypes. Based on the PFGE analysis, 37 different profiles were observed among which 8 profiles were common types. Real time PCR of csgD and csgA genes of 50 E. cloacae complex in relation to PFGE and hsp60 genotypes showed that all the genetic clusters are not equally involved in pathogenesis of urinary tract infections. It was elucidated in this study that isolates with common PFGE genotypes belonged to identical hsp60 clusters, and the foremost clusters (VI, III, and V) mainly comprised within PFGE common types. In our study, no significant correlation was detected between the specific hsp60 clusters or PFGE genotypes and the expression level of csgD and csgA genes (P-value > 0.05). This is the first study describing that unequivalent contribution of E. cloacae genotypes and clusters in pathogenesis of UTI, is not owing to varied curli biogenesis expression potential. The PFGE genotyping showed more discriminatory power than hsp60 genotyping for epidemiological studies and source tracking purpose. PMID:27354208

  20. The level of residual dispersion variation and the power of differential expression tests for RNA-Seq data.

    PubMed

    Mi, Gu; Di, Yanming

    2015-01-01

    RNA-Sequencing (RNA-Seq) has been widely adopted for quantifying gene expression changes in comparative transcriptome analysis. For detecting differentially expressed genes, a variety of statistical methods based on the negative binomial (NB) distribution have been proposed. These methods differ in the ways they handle the NB nuisance parameters (i.e., the dispersion parameters associated with each gene) to save power, such as by using a dispersion model to exploit an apparent relationship between the dispersion parameter and the NB mean. Presumably, dispersion models with fewer parameters will result in greater power if the models are correct, but will produce misleading conclusions if not. This paper investigates this power and robustness trade-off by assessing rates of identifying true differential expression using the various methods under realistic assumptions about NB dispersion parameters. Our results indicate that the relative performances of the different methods are closely related to the level of dispersion variation unexplained by the dispersion model. We propose a simple statistic to quantify the level of residual dispersion variation from a fitted dispersion model and show that the magnitude of this statistic gives hints about whether and how much we can gain statistical power by a dispersion-modeling approach. PMID:25849826

  1. Associations between joint effusion in the knee and gene expression levels in the circulation: a meta-analysis

    PubMed Central

    Peters, Marjolein J.; Ramos, Yolande F.M.; den Hollander, Wouter; Schiphof, Dieuwke; Hofman, Albert; Uitterlinden, André G.; Oei, Edwin H.G.; Slagboom, P. Eline; Kloppenburg, Margreet; Bloem, Johan L.; Bierma-Zeinstra, Sita M.A.; Meulenbelt, Ingrid; van Meurs, Joyce B.J.

    2016-01-01

    Objective: To identify molecular biomarkers for early knee osteoarthritis (OA), we examined whether joint effusion in the knee associated with different gene expression levels in the circulation. Materials and Methods: Joint effusion grades measured with magnetic resonance (MR) imaging and gene expression levels in blood were determined in women of the Rotterdam Study (N=135) and GARP (N=98). Associations were examined using linear regression analyses, adjusted for age, fasting status, RNA quality, technical batch effects, blood cell counts, and BMI. To investigate enriched pathways and protein-protein interactions, we used the DAVID and STRING webtools. Results: In a meta-analysis, we identified 257 probes mapping to 189 unique genes in blood that were nominally significantly associated with joint effusion grades in the knee. Several compelling genes were identified such as C1orf38 and NFATC1. Significantly enriched biological pathways were: response to stress, gene expression, negative regulation of intracellular signal transduction, and antigen processing and presentation of exogenous pathways. Conclusion: Meta-analyses and subsequent enriched biological pathways resulted in interesting candidate genes associated with joint effusion that require further characterization. Associations were not transcriptome-wide significant most likely due to limited power. Additional studies are required to replicate our findings in more samples, which will greatly help in understanding the pathophysiology of OA and its relation to inflammation, and may result in biomarkers urgently needed to diagnose OA at an early stage. PMID:27134727

  2. Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure

    SciTech Connect

    Tin-Tin-Win-Shwe Mitsushima, Dai; Yamamoto, Shoji; Fukushima, Atsushi; Funabashi, Toshiya; Kobayashi, Takahiro; Fujimaki, Hidekazu

    2008-01-15

    Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1{beta}, TNF-{alpha}, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 {mu}g) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 {mu}g of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1{beta} mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-{alpha} mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 {beta} mRNA expressions synergistically with LTA

  3. Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure.

    PubMed

    Tin-Tin-Win-Shwe; Mitsushima, Dai; Yamamoto, Shoji; Fukushima, Atsushi; Funabashi, Toshiya; Kobayashi, Takahiro; Fujimaki, Hidekazu

    2008-01-15

    Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1 beta, TNF-alpha, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 microg) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 mug of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1 beta mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-alpha mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 beta mRNA expressions synergistically with LTA in the

  4. Glutathione peroxidase 1 expression, malondialdehyde levels and histological alterations in the liver of Acrossocheilus fasciatus exposed to cadmium chloride.

    PubMed

    Liu, Guo-Di; Sheng, Zhang; Wang, You-Fa; Han, Ying-Li; Zhou, Yang; Zhu, Jun-Quan

    2016-03-10

    Cadmium (Cd) is known as a widespread pollutant in aquatic environment. The accumulation of reactive oxygen species (ROS) is attributed to Cd exposure, which may affect the growth, development and physiological metabolism of aquatic organisms. In response to these unfavorable damages, antioxidant systems have been developed to protect against oxidative stress. In this study, we investigated the expression pattern of glutathione peroxidase 1 genes (GPx-1a and GPx-1b) in the liver of Acrossocheilus fasciatus after Cd administration. Total RNA extraction, reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were performed in order to clone the A. fasciatus GPx-1a and GPx-1b full-length cDNA sequences and partial fragment of β-actin cDNA from the liver for the first time. Tissue-specific expression analysis proved that GPx-1 genes were widely expressed in the liver, kidney, gill, testis, muscle, spleen, heart and brain. The changes of GPx-1 mRNA and malondialdehyde (MDA) levels in the liver treated with Cd were measured. In addition, the acute toxic effects of Cd on the microstructure of the liver were studied using light microscopy. These results suggest that GPx-1, MDA and liver histology which represent molecular, biochemical and histological levels, can be used as potential biomarkers to monitor Cd pollution. The overall findings also highlight the potential use of those three bio-indicators combined together as a multi-level tool (molecular, biochemical and histological levels) when monitoring Cd contamination and other possible exogenetic pollutants in aquatic environment. PMID:26707212

  5. Dose-related gene expression changes in forebrain following acute, low-level chlorpyrifos exposure in neonatal rats

    SciTech Connect

    Ray, Anamika; Liu Jing; Ayoubi, Patricia; Pope, Carey

    2010-10-15

    /synaptic transmission and transcription/translation. Nine genes were differentially affected in all four CPF dosing groups. We conclude that the most robust, consistent changes in differential gene expression in neonatal forebrain across a range of acute CPF dosages occurred at an exposure level associated with the classical marker of OP toxicity, AChE inhibition. Disruption of multiple cellular pathways, in particular cell adhesion, may contribute to the developmental neurotoxicity potential of this pesticide.

  6. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

    SciTech Connect

    Sustarsic, Elahu G.; Junnila, Riia K.; Kopchick, John J.

    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

  7. miR-146a and miR-155 Expression Levels in Acute Graft-Versus-Host Disease Incidence

    PubMed Central

    Atarod, Sadaf; Ahmed, Mohammed Mahid; Lendrem, Clare; Pearce, Kim Frances; Cope, Wei; Norden, Jean; Wang, Xiao-Nong; Collin, Matthew; Dickinson, Anne Mary

    2016-01-01

    Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for numerous hematological malignancies. However, acute graft-versus-host disease (aGVHD) is still the major complication causing mortality. MicroRNAs (miRNAs) play a significant role in inflammation and have potential as prognostic and diagnostic biomarkers. This study investigated the role of two immune-specific miRNAs (miR-146a and miR-155) as biomarkers for aGVHD incidence in the peripheral blood of allo-HSCT patients prior to disease onset. The study showed that miR-146a and its statistical interaction with miR-155 at day +28 were predictive of aGVHD incidence. Interestingly, the expression levels of miR-146a and miR-155 negatively correlated with the transcription factor, SPI1 (PU.1gene) mRNA expression. PMID:27014257

  8. Serum level of soluble interleukin-2 receptor correlates with CD25 expression in patients with T lymphoblastic lymphoma.

    PubMed

    Toji, Tomohiro; Takata, Katsuyoshi; Sato, Yasuharu; Miyata-Takata, Tomoko; Hayashi, Eiko; Habara, Toshiyuki; Maeda, Yoshinobu; Tanimoto, Mitsune; Yoshino, Tadashi

    2015-08-01

    Acute lymphoblastic leukaemia/lymphoma (ALL/LBL) is an aggressive form of non-Hodgkin's lymphoma (NHL) affecting B-cells or T-cells, respectively. The serum level of soluble interleukin-2 receptor (sIL-2R) is known to reflect the immune activity and tumour volume in aggressive NHL; however, the release of sIL-2R in LBL has not been extensively studied. Further, the relationship between sIL-2R release and the expression level of IL-2R α subunit (CD25) remains unknown. In the present study, we examined the serum level of sIL-2R in 23 patients with T lymphoblactic lymphoma (T-LBL) and compared these with the levels in 20 patient with T acute lymphoblastic leukaemia (T-ALL), 40 patients with diffuse large B-cell lymphoma (DLBCL) and 40 patients with peripheral T-cell lymphoma (PTCL), not otherwise specified. The release of sIL-2R into the serum in patients with T-LBL was significantly lower than that for T-ALL, DLBCL and PTCL (p<0.001). Immunohistochemistry revealed that CD25 expression was correlated with the serum level of sIL-2R in T-LBL (p=0.0069), whereas no correlation was found to exist between serum sIL-2R levels and CD25 expression in patients with DLBCL (p=0.348) and PTCL (p=0.266). Furthermore, double immunohistochemical analysis revealed that CD25-positive cells were also found to be Foxp3-positive non-neoplastic T-cells. In conclusion, CD25-positive non-neoplastic T-cells in T-LBL are presumed to be the primary source of sIL-2R, and the low number of cells present results in a lower level of sIL-2R released into the serum compared with the other aggressive and highly aggressive lymphomas. PMID:25935549

  9. Chemosensory reception, behavioral expression, and ecological interactions at multiple trophic levels.

    PubMed

    Ferrer, Ryan P; Zimmer, Richard K

    2007-05-01

    Chemoreception may function throughout an entire animal lifetime, with independent, stage-specific selection pressures leading to changes in physiological properties, behavioral expression, and hence, trophic interactions. When the California newt (Taricha torosa) metamorphoses from an entirely aquatic larva to a semi-terrestrial juvenile/adult form, its chemosensory organs undergo dramatic reorganization. The relationship between newt life-history stage and chemosensory-mediated behavior was established by comparing responses of adults (as determined here) to those of conspecific larvae (as studied previously). Bioassays were performed in mountain streams, testing responses of free-ranging adults to 13 individual l-amino acids. Relative to stream water (controls), adults turned immediately upcurrent and moved to the source of arginine, glycine or alanine release. These responses were indicative of predatory search. Arginine was the strongest attractant tested, with a response threshold (median effective dose) of 8.3x10(-7) mol l(-1) (uncorrected for dilution associated with chemical release and delivery). In contrast to adult behavior, arginine suppressed cannibal-avoidance and failed to evoke search reactions in larvae. For a common set of arginine analogs, the magnitudes of adult attraction and larval suppression were not positively correlated. Suppression of cannibal-avoidance behavior in larvae was unaffected by most structural modifications of the arginine molecule. Adult behavior, on the other hand, was strongly influenced by even subtle alterations in the parent compound. Reactions to arginine in both adults and larvae were eliminated by blocking the external openings of the nasal cavity. Stimulating adult predatory search in one case and inhibiting larval cannibal avoidance in the other, arginine is a chemical signal with opposing behavioral effects and varying ecological consequences. Significant differences between responses of adults and larvae to

  10. Expression of Genes Involved in Anthocyanin Biosynthesis in Relation to Anthocyanin, Proanthocyanidin, and Flavonol Levels during Bilberry Fruit Development1

    PubMed Central

    Jaakola, Laura; Määttä, Kaisu; Pirttilä, Anna Maria; Törrönen, Riitta; Kärenlampi, Sirpa; Hohtola, Anja

    2002-01-01

    The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented. PMID:12376640

  11. TRPV1 expression level in isolectin B₄-positive neurons contributes to mouse strain difference in cutaneous thermal nociceptive sensitivity.

    PubMed

    Ono, Kentaro; Ye, Yi; Viet, Chi T; Dang, Dongmin; Schmidt, Brian L

    2015-05-01

    Differential thermal nociception across inbred mouse strains has genetic determinants. Thermal nociception is largely attributed to the heat/capsaicin receptor transient receptor potential vanilloid 1 (TRPV1); however, the contribution of this channel to the genetics of thermal nociception has not been revealed. In this study we compared TRPV1 expression levels and electrophysiological properties in primary sensory neurons and thermal nociceptive behaviors between two (C57BL/6 and BALB/c) inbred mouse strains. Using immunofluorescence and patch-clamp physiology methods, we demonstrated that TRPV1 expression was significantly higher in isolectin B4 (IB4)-positive trigeminal sensory neurons of C57BL/6 relative to BALB/c; the expression in IB4-negative neurons was similar between the strains. Furthermore, using electrophysiological cell classification (current signature method), we showed differences between the two strains in capsaicin sensitivity in IB4-positive neuronal cell types 2 and 13, which were previously reported as skin nociceptors. Otherwise electrophysiological membrane properties of the classified cell types were similar in the two mouse strains. In publicly available nocifensive behavior data and our own behavior data from the using the two mouse strains, C57BL/6 exhibited higher sensitivity to heat stimulation than BALB/c, independent of sex and anatomical location of thermal testing (the tail, hind paw, and whisker pad). The TRPV1-selective antagonist JNJ-17203212 inhibited thermal nociception in both strains; however, removing IB4-positive trigeminal sensory neurons with IB4-conjugated saporin inhibited thermal nociception on the whisker pad in C57BL/6 but not in BALB/c. These results suggest that TRPV1 expression levels in IB4-positive type 2 and 13 neurons contributed to differential thermal nociception in skin of C57BL/6 compared with BALB/c. PMID:25787958

  12. Enhanced cyclooxygenase-2 expression levels and metalloproteinase 2 and 9 activation by Hexachlorobenzene in human endometrial stromal cells.

    PubMed

    Chiappini, Florencia; Bastón, Juan Ignacio; Vaccarezza, Agustina; Singla, José Javier; Pontillo, Carolina; Miret, Noelia; Farina, Mariana; Meresman, Gabriela; Randi, Andrea

    2016-06-01

    Hexachlorobenzene (HCB) is an organochlorine pesticide that induces toxic reproductive effects in laboratory animals. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). Endometriosis is characterized by the presence of functional endometrial tissues outside the uterine cavity. Experimental studies indicate that exposure to organochlorines can interfere with both hormonal regulation and immune function to promote endometriosis. Altered expression of metalloproteinases (MMPs) in patients with endometriosis, suggests that MMPs may play a critical role. In the endometriotic lesions, prostaglandin E2 (PGE2) produced by cyclooxygenase-2 (COX-2), binds to its EP4 receptor (EP4), and via c-Src kinase induces MMPs activation, promoting endometriosis. We examined the HCB action on MMP-2 and MMP-9 activities and expression, COX-2 levels, PGE2 signaling, and the AhR involvement in HCB-induced effects. We have used different in vitro models: (1) human endometrial stromal cell line T-HESC, (2) primary cultures of Human Uterine Fibroblast (HUF), and (3) primary cultures of endometrial stromal cells from eutopic endometrium of control (CESC) and subjects with endometriosis (EESC). Our results show that HCB enhances MMP-2 and MMP-9 activities in T-HESC, HUF and ESC cells. The MMP-9 levels were elevated in all models, while the MMP-2 expression only increased in ESC cells. HCB enhanced COX-2 and EP4 expression, PGE2 secretion and the c-Src kinase activation in T-HESC. Besides, we observed that AhR is implicated in these HCB-induced effects. In conclusion, our results show that HCB exposure could contribute to endometriosis development, affecting inflammation and invasion parameters of human endometrial cells. PMID:27038655

  13. Tissue Phthalate Levels Correlate With Changes in Immune Gene Expression in a Population of Juvenile Wild Salmon.

    PubMed

    Martins, Kelly; Hagedorn, Birgit; Ali, Shareen; Kennish, John; Applegate, Ben; Leu, Matthias; Epp, Lidia; Pallister, Chris; Zwollo, Patty

    2016-07-01

    Phthalates have detrimental effects on health and have been shown to dysregulate the immune system of mammals, birds, and fish. We recently reported that di(2-ethylhexyl) phthalate exposure reduces the abundance and inhibits the proliferation of rainbow trout (Oncorhynchus mykiss) IgM(+) B lymphocytes and expression of secreted immunoglobulin heavy-chain mu transcripts in an in vitro culture system. We proposed that phthalates act as immunomodulators by modifying the normal B cell-activation pathways by accelerating B cell differentiation while suppressing plasmablast expansion, thus resulting in fewer IgM-secreting plasma cells. This hypothesis was tested here in an in vivo field study of juvenile Dolly Varden (Salvelinus malma) from a plastic-polluted lake in the Gulf of Alaska. Fish tissues were analyzed for both phthalate levels using liquid chromatography-coupled tandem mass spectrometry and for changes in immune gene expression using reverse transcriptase-real time polymerase chain reaction. Results showed that fish with higher tissue levels of di(2-ethylhexyl) phthalate, di(n-butyl) phthalate, and/or dimethyl phthalate expressed significantly fewer secreted and membrane-bound immunoglobulin heavy-chain mu and Blimp1 transcripts in their hematopoietic tissue. This suggests that in vivo uptake of phthalates in fish changes the expression of B cell-specific genes. Chronic exposure to phthalates likely dysregulates normal B-lymphoid development and antibody responses in salmonids and may increase susceptibility to infection. Given the conserved nature of B-lineage cells in vertebrate animals, other marine species may be similarly affected by chronic phthalate exposure. PMID:27177745

  14. Quiescence in Artemia franciscana embryos: reversible arrest of metabolism and gene expression at low oxygen levels.

    PubMed

    Hand, S C

    1998-04-01

    Depression of the production and consumption of cellular energy appears to be a prerequisite for the survival of prolonged bouts of anoxia. A correlation exists between the degree of metabolic depression under anoxia and the duration of anoxia tolerance. In the case of brine shrimp (Artemia franciscana) embryos, oxygen deprivation induces a reversible quiescent state that can be tolerated for several years with substantial survivorship. A global arrest of cytoplasmic translation accompanies the transition into anoxia, and rates of protein synthesis in mitochondria from these embryos appears to be markedly reduced in response to anoxia. Previous evidence suggests that the acute acidification of intracellular pH (pHi) by over 1.0 unit during the transition into anoxia contributes to the depression of biosynthesis, but message limitation does not appear to play a role in the down-regulation in either cellular compartment. The ontogenetic increase in mRNA levels for a mitochondrial-encoded subunit of cytochrome c oxidase (COX I) and for nuclear-encoded actin is blocked by anoxia and aerobic acidosis (artificial quiescence imposed by intracellular acidification under aerobic conditions). Further, the levels of COX I and actin mRNA do not decline apprecia