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Sample records for limitation increases expression

  1. Increasing Originality in Written Expression.

    ERIC Educational Resources Information Center

    Belasco, Jack Thomas

    This study partially replicated Moss's "A Study of the Effect of Selected Methods of Instruction Designed to Increase Originality in Written Expression," except for the fact that this investigator taught a 5th grade and an 11th grade class for most of a school year. Some of the conclusions of the study were: no particular teaching technique was…

  2. The Low Noise Limit in Gene Expression

    PubMed Central

    Dar, Roy D.; Razooky, Brandon S.; Weinberger, Leor S.; Cox, Chris D.; Simpson, Michael L.

    2015-01-01

    Protein noise measurements are increasingly used to elucidate biophysical parameters. Unfortunately noise analyses are often at odds with directly measured parameters. Here we show that these inconsistencies arise from two problematic analytical choices: (i) the assumption that protein translation rate is invariant for different proteins of different abundances, which has inadvertently led to (ii) the assumption that a large constitutive extrinsic noise sets the low noise limit in gene expression. While growing evidence suggests that transcriptional bursting may set the low noise limit, variability in translational bursting has been largely ignored. We show that genome-wide systematic variation in translational efficiency can–and in the case of E. coli does–control the low noise limit in gene expression. Therefore constitutive extrinsic noise is small and only plays a role in the absence of a systematic variation in translational efficiency. These results show the existence of two distinct expression noise patterns: (1) a global noise floor uniformly imposed on all genes by expression bursting; and (2) high noise distributed to only a select group of genes. PMID:26488303

  3. The low noise limit in gene expression

    SciTech Connect

    Dar, Roy D.; Weinberger, Leor S.; Cox, Chris D.; Simpson, Michael L.; Razooky, Brandon S.

    2015-10-21

    Protein noise measurements are increasingly used to elucidate biophysical parameters. Unfortunately noise analyses are often at odds with directly measured parameters. Here we show that these inconsistencies arise from two problematic analytical choices: (i) the assumption that protein translation rate is invariant for different proteins of different abundances, which has inadvertently led to (ii) the assumption that a large constitutive extrinsic noise sets the low noise limit in gene expression. While growing evidence suggests that transcriptional bursting may set the low noise limit, variability in translational bursting has been largely ignored. We show that genome-wide systematic variation in translational efficiency can-and in the case of E. coli does-control the low noise limit in gene expression. Therefore constitutive extrinsic noise is small and only plays a role in the absence of a systematic variation in translational efficiency. Lastly, these results show the existence of two distinct expression noise patterns: (1) a global noise floor uniformly imposed on all genes by expression bursting; and (2) high noise distributed to only a select group of genes.

  4. The low noise limit in gene expression

    DOE PAGESBeta

    Dar, Roy D.; Weinberger, Leor S.; Cox, Chris D.; Simpson, Michael L.; Razooky, Brandon S.

    2015-10-21

    Protein noise measurements are increasingly used to elucidate biophysical parameters. Unfortunately noise analyses are often at odds with directly measured parameters. Here we show that these inconsistencies arise from two problematic analytical choices: (i) the assumption that protein translation rate is invariant for different proteins of different abundances, which has inadvertently led to (ii) the assumption that a large constitutive extrinsic noise sets the low noise limit in gene expression. While growing evidence suggests that transcriptional bursting may set the low noise limit, variability in translational bursting has been largely ignored. We show that genome-wide systematic variation in translational efficiencymore » can-and in the case of E. coli does-control the low noise limit in gene expression. Therefore constitutive extrinsic noise is small and only plays a role in the absence of a systematic variation in translational efficiency. Lastly, these results show the existence of two distinct expression noise patterns: (1) a global noise floor uniformly imposed on all genes by expression bursting; and (2) high noise distributed to only a select group of genes.« less

  5. Aminoglycoside uptake increased by tet gene expression.

    PubMed Central

    Merlin, T L; Davis, G E; Anderson, W L; Moyzis, R K; Griffith, J K

    1989-01-01

    The expression of extrachromosomal tet genes not only confers tetracycline resistance but also increases the susceptibilities of gram-negative bacteria to commonly used aminoglycoside antibiotics. We investigated the possibility that tet expression increases aminoglycoside susceptibility by increasing bacterial uptake of aminoglycoside. Studies of [3H]gentamicin uptake in paired sets of Escherichia coli HB101 and Salmonella typhimurium LT2 expressing and not expressing tet showed that tet expression accelerates energy-dependent [3H]gentamicin uptake. Increased [3H]gentamicin uptake was accompanied by decreased bacterial protein synthesis and bacterial growth. Increased aminoglycoside uptake occurred whether tet expression was constitutive or induced, whether the tet gene was class B or C, and whether the tet gene was plasmid borne or integrated into the bacterial chromosome. tet expression produced no measurable change in membrane potential, suggesting that tet expression increases aminoglycoside uptake either by increasing the availability of specific carriers or by lowering the minimum membrane potential that is necessary for uptake. PMID:2684011

  6. Methodological Limitations in Determining Astrocytic Gene Expression

    PubMed Central

    Peng, Liang; Guo, Chuang; Wang, Tao; Li, Baoman; Gu, Li; Wang, Zhanyou

    2013-01-01

    Traditionally, astrocytic mRNA and protein expression are studied by in situ hybridization (ISH) and immunohistochemically. This led to the concept that astrocytes lack aralar, a component of the malate-aspartate-shuttle. At least similar aralar mRNA and protein expression in astrocytes and neurons isolated by fluorescence-assisted cell sorting (FACS) reversed this opinion. Demonstration of expression of other astrocytic genes may also be erroneous. Literature data based on morphological methods were therefore compared with mRNA expression in cells obtained by recently developed methods for determination of cell-specific gene expression. All Na,K-ATPase-α subunits were demonstrated by immunohistochemistry (IHC), but there are problems with the cotransporter NKCC1. Glutamate and GABA transporter gene expression was well determined immunohistochemically. The same applies to expression of many genes of glucose metabolism, whereas a single study based on findings in bacterial artificial chromosome (BAC) transgenic animals showed very low astrocytic expression of hexokinase. Gene expression of the equilibrative nucleoside transporters ENT1 and ENT2 was recognized by ISH, but ENT3 was not. The same applies to the concentrative transporters CNT2 and CNT3. All were clearly expressed in FACS-isolated cells, followed by biochemical analysis. ENT3 was enriched in astrocytes. Expression of many nucleoside transporter genes were shown by microarray analysis, whereas other important genes were not. Results in cultured astrocytes resembled those obtained by FACS. These findings call for reappraisal of cellular nucleoside transporter expression. FACS cell yield is small. Further development of cell separation methods to render methods more easily available and less animal and cost consuming and parallel studies of astrocytic mRNA and protein expression by ISH/IHC and other methods are necessary, but new methods also need to be thoroughly checked. PMID:24324456

  7. 48 CFR 217.7505 - Limitations on price increases.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Limitations on price... Acquisition of Replenishment Parts 217.7505 Limitations on price increases. This section provides implementing... award, on a sole source basis, a contract for any centrally managed replenishment part when the price...

  8. 48 CFR 217.7505 - Limitations on price increases.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 3 2014-10-01 2014-10-01 false Limitations on price... Acquisition of Replenishment Parts 217.7505 Limitations on price increases. This section provides implementing... award, on a sole source basis, a contract for any centrally managed replenishment part when the price...

  9. 48 CFR 217.7505 - Limitations on price increases.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 3 2013-10-01 2013-10-01 false Limitations on price... Acquisition of Replenishment Parts 217.7505 Limitations on price increases. This section provides implementing... award, on a sole source basis, a contract for any centrally managed replenishment part when the price...

  10. 48 CFR 217.7505 - Limitations on price increases.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 3 2012-10-01 2012-10-01 false Limitations on price... Acquisition of Replenishment Parts 217.7505 Limitations on price increases. This section provides implementing... award, on a sole source basis, a contract for any centrally managed replenishment part when the price...

  11. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish

    PubMed Central

    Horstick, Eric J.; Jordan, Diana C.; Bergeron, Sadie A.; Tabor, Kathryn M.; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A.

    2015-01-01

    Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3′ untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models. PMID:25628360

  12. Tissue Dependent Limited Pleiotropy Affects Gene Expression in Barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-synonymous coding mutations in a gene change the resulting protein no matter where it is expressed, but the effects of cis-regulatory mutations could be spatially or temporally limited, a phenomenon termed limited pleiotropy. Here we report the genome-wide occurrence of limited pleiotropy of cis...

  13. Increased evaporation following widespread tree mortality limits streamflow response

    NASA Astrophysics Data System (ADS)

    Biederman, J. A.; Harpold, A. A.; Gochis, D. J.; Ewers, B. E.; Reed, D. E.; Papuga, S. A.; Brooks, P. D.

    2014-07-01

    A North American epidemic of mountain pine beetle (MPB) has disturbed over 5 million ha of forest containing headwater catchments crucial to water resources. However, there are limited observations of MPB effects on partitioning of precipitation between vapor loss and streamflow, and to our knowledge these fluxes have not been observed simultaneously following disturbance. We combined eddy covariance vapor loss (V), catchment streamflow (Q), and stable isotope indicators of evaporation (E) to quantify hydrologic partitioning over 3 years in MPB-impacted and control sites. Annual control V was conservative, varying only from 573 to 623 mm, while MPB site V varied more widely from 570 to 700 mm. During wet periods, MPB site V was greater than control V in spite of similar above-canopy potential evapotranspiration (PET). During a wet year, annual MPB V was greater and annual Q was lower as compared to an average year, while in a dry year, essentially all water was partitioned to V. Ratios of 2H and 18O in stream and soil water showed no kinetic evaporation at the control site, while MPB isotope ratios fell below the local meteoric water line, indicating greater E and snowpack sublimation (Ss) counteracted reductions in transpiration (T) and sublimation of canopy-intercepted snow (Sc). Increased E was possibly driven by reduced canopy shading of shortwave radiation, which averaged 21 W m-2 during summer under control forest as compared to 66 W m-2 under MPB forest. These results show that abiotic vapor losses may limit widely expected streamflow increases.

  14. Increased monocyte tissue factor expression in coronary disease.

    PubMed Central

    Leatham, E. W.; Bath, P. M.; Tooze, J. A.; Camm, A. J.

    1995-01-01

    OBJECTIVE--To investigate whether monocyte expression of tissue factor is increased in patients with acute coronary syndromes and chronic stable angina. DESIGN--Cross sectional study of monocyte tissue factor expression in patients with ischaemic heart disease and control subjects. BACKGROUND--Unstable angina and myocardial infarction are associated with enhanced mononuclear cell procoagulant activity. Procoagulant activity of blood monocytes is principally mediated by tissue factor expression. Tissue factor initiates the coagulation cascade and monocyte tissue factor expression may therefore be increased in these syndromes. METHODS--Monocyte tissue factor expression was measured cytometrically in whole blood flow using a polyclonal rabbit antihuman tissue factor antibody. PATIENTS--30 patients with acute myocardial infarction, 17 with unstable angina, 13 with chronic stable angina, and 11 normal control subjects. RESULTS--Increased proportions of monocytes expressing tissue factor (> 2.5%) were found in none of 11 (0%) normal subjects, five 13 (38%) patients with stable angina, 11 of 17 (64%) patients with unstable angina, and 16 of 30 (53%) patients with myocardial infarction (2P = 0.006). Blood from all subjects showed similar monocyte tissue factor expression similar monocyte tissue factor expression (46.1 (15.1)%) after lipopolysaccharide stimulation. CONCLUSION--Hypercoagulability associated with acute myocardial infarction, unstable angina, and chronic stable angina may be induced by tissue factor expressed on circulating monocytes. PMID:7888247

  15. Oregon School Districts Respond to Increased Tax Limitations.

    ERIC Educational Resources Information Center

    O'Toole, Daniel; Stipak, Brian

    2000-01-01

    Passage of a 1990 tax limitation measure made Oregon one of few states since 1978 to mandate reductions in local property taxes. A survey of Oregon school superintendents revealed their fears about reduced local discretion and use of revenue management/forecasting tools and rational, goal-oriented cutback approaches. (Contains 36 footnotes.) (MLH)

  16. Characterization of genes with increased expression in human glioblastomas.

    PubMed

    Kavsan, V; Shostak, K; Dmitrenko, V; Zozulya, Yu; Rozumenko, V; Demotes-Mainard, J

    2005-01-01

    In the present study, we have used the gene expression data available in the SAGE database in an attempt to identify glioblastoma molecular markers. Of 129 genes with more than 5-fold difference found by comparison of nine glioblastoma with five normal brain SAGE libraries, 44 increased their expression in glioblastomas. Most corresponding proteins were involved in angiogenesis, host-tumor immune interplay, multidrug resistance, extracellular matrix (ECM) formation, IGF-signalling, or MAP-kinase pathway. Among them, 16 genes had a high expression both in glioblastomas and in glioblastoma cell lines suggesting their expression in transformed cells. Other 28 genes had an increased expression only in glioblastomas, not in glioblastoma cell lines suggesting an expression possibly originated from host cells. Many of these genes are among the top transcripts in activated macrophages, and involved in immune response and angiogenesis. This altered pattern of gene expression in both host and tumor cells, can be viewed as a molecular marker in the analysis of malignant progression of astrocytic tumors, and as possible clues for the mechanism of disease. Moreover, several genes overexpressed in glioblastomas produce extracellular proteins, thereby providing possible therapeutic targets. Further characterization of these genes will thus allow them to be exploited in molecular classification of glial tumors, diagnosis, prognosis, and anticancer therapy. PMID:16396319

  17. Whirlin increases TRPV1 channel expression and cellular stability.

    PubMed

    Ciardo, Maria Grazia; Andrés-Bordería, Amparo; Cuesta, Natalia; Valente, Pierluigi; Camprubí-Robles, María; Yang, Jun; Planells-Cases, Rosa; Ferrer-Montiel, Antonio

    2016-01-01

    The expression and function of TRPV1 are influenced by its interaction with cellular proteins. Here, we identify Whirlin, a cytoskeletal PDZ-scaffold protein implicated in hearing, vision and mechanosensory transduction, as an interacting partner of TRPV1. Whirlin associates with TRPV1 in cell lines and in primary cultures of rat nociceptors. Whirlin is expressed in 55% of mouse sensory C-fibers, including peptidergic and non-peptidergic nociceptors, and co-localizes with TRPV1 in 70% of them. Heterologous expression of Whirlin increased TRPV1 protein expression and trafficking to the plasma membrane, and promoted receptor clustering. Silencing Whirlin expression with siRNA or blocking protein translation resulted in a concomitant degradation of TRPV1 that could be prevented by inhibiting the proteasome. The degradation kinetics of TRPV1 upon arresting protein translation mirrored that of Whirlin in cells co-expressing both proteins, suggesting a parallel degradation mechanism. Noteworthy, Whirlin expression significantly reduced TRPV1 degradation induced by prolonged exposure to capsaicin. Thus, our findings indicate that Whirlin and TRPV1 are associated in a subset of nociceptors and that TRPV1 protein stability is increased through the interaction with the cytoskeletal scaffold protein. Our results suggest that the Whirlin–TRPV1 complex may represent a novel molecular target and its pharmacological disruption might be a therapeutic strategy for the treatment of peripheral TRPV1-mediated disorders. PMID:26516054

  18. Increased stathmin expression strengthens fear conditioning in epileptic rats.

    PubMed

    Zhang, Linna; Feng, Danni; Tao, Hong; DE, Xiangyan; Chang, Qing; Hu, Qikuan

    2015-01-01

    Patients with temporal lobe epilepsy have inexplicable fear attack as the aura. However, the underlying neural mechanisms of seizure-modulated fear are not clarified. Recent studies identified stathmin as one of the key controlling molecules in learning and innate fear. Stathmin binds to tubulin, inhibits microtubule assembly and promotes microtubule catastrophes. Therefore, stathmin is predicted to play a crucial role in the association of epilepsy seizures with fear conditioning. Firstly, a pilocarpine model of epilepsy in rats was established, and subsequently the fear condition training was performed. The epileptic rats with fear conditioning (epilepsy + fear) had a much longer freezing time compared to each single stimulus. The increased freezing levels revealed a significantly strengthened effect of the epileptic seizures on the learned fear of the tone-shock contextual. Subsequently, the stathmin expression was compared in the hippocampus, the amygdale, the insular cortex and the temporal lobe. The significant change of stathmin expression occurred in the insular and the hippocampus, but not in the amygdale. Stathmin expression and dendritic microtubule stability were compared between fear and epilepsy in rats. Epilepsy was found to strengthen the fear conditioning with increased expression of stathmin and a decrease in microtubule stability. Fear conditioning slightly increased the expression of stathmin, whereas epilepsy with fear conditioning increased it significantly in the hippocampus, insular cortex and hypothalamus. The phosphorylated stathmin slightly increased in the epilepsy with fear conditioning. The increased expression of stathmin was contrary to the decrease of the stathmin microtubule-associated protein (MAP2) and α-tubulin in the epileptic rats with fear conditioning in all three areas of the brain. The most significant change of the ratio of MAP2 and α-tubulin/stathmin occurred in the insular cortex and hippocampus. In conclusion

  19. Expressivity tag: a novel tool for increased expression in Escherichia coli.

    PubMed

    Hansted, Jon Gade; Pietikäinen, Laura; Hög, Friederike; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

    2011-09-20

    Protein expression in Escherichia coli is rarely trivial as low expression and insolubility are common problems. In this work we define a fusion partner, which increases expression levels similarly to the distinct function of solubility and affinity tags. This type of fusion tag we term an expressivity tag. Our work is based on earlier observations where 3' deletions of the InfB gene displays strongly increased expression levels. We have constructed progressively shortened fragments of the InfB(1-471) gene and fused gene fragments to a gfp reporter gene. A 5-fold increase in GFP expression was seen for an optimal 21 nucleotide InfB(1-21) sequence compared to gfp independently. We defined the InfB(1-21) sequence as an expressivity tag. The tag was tested for improved expression of two biotechnological important proteins streptavidin and a single chain antibody (scFv). Expression of both streptavidin and scFv(L32) was improved as evaluated by SDS-PAGE. Calculation of folding energies in the translation initiation region gave higher free energies for gfp, L32 and streptavidin when linked to InfB(1-21) than independently. InfB(1-21) did however not improve the codon usage or codon adaptation index. The expressivity tag is an important addition to the box of tools available for optimizing heterologous protein expression. PMID:21801766

  20. Cryostat Filling Limitations for Proposed Ar Dewar Pressure Increase

    SciTech Connect

    Dixon, K.; Wu, J.; /Fermilab

    1991-07-23

    In order to significantly decrease the amount of time required to fill the cryostats, it is desired to raise the setpoint of the 'operating' relief valve on the argon storage dewar to 20 psig from its existing 16 psig setting. This additional pressure increases the flow to the cryostats and will overwhelm the relief capacity if the temperature of the modules within these vessels is warm enough. Using some conservative assumptions and simple calculations within this note, the maximum average temperature that the modules within each cryostat can be at prior to filling from the storage dewar with liquid argon is at least 290 K.

  1. Increased intra- and extracellular granzyme expression in patients with tuberculosis.

    PubMed

    Garcia-Laorden, M Isabel; Blok, Dana C; Kager, Liesbeth M; Hoogendijk, Arie J; van Mierlo, Gerard J; Lede, Ivar O; Rahman, Wahid; Afroz, Rumana; Ghose, Aniruddha; Visser, Caroline E; Md Zahed, Abu Shahed; Husain, Md Anwar; Alam, Khan Mashrequl; Chandra Barua, Pravat; Hassan, Mahtabuddin; Hossain, Ahmed; Tayab, Md Abu; Day, Nick; Dondorp, Arjen M; de Vos, Alex F; van der Poll, Tom

    2015-09-01

    Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Granzymes (gzms) are proteases mainly found in cytotoxic lymphocytes, but also extracellularly. While the role of gzms in target cell death has been widely characterized, considerable evidence points towards broader roles related to infectious and inflammatory responses. To investigate the expression of the gzms in TB, intracellular gzms A, B and K were measured by flow cytometry in lymphocyte populations from peripheral blood mononuclear cells from 18 TB patients and 12 healthy donors from Bangladesh, and extracellular levels of gzmA and B were measured in serum from 58 TB patients and 31 healthy controls. TB patients showed increased expression of gzmA in CD8(+) T, CD4(+) T and CD56(+) T, but not NK, cells, and of gzmB in CD8(+) T cells, when compared to controls. GzmK expression was not altered in TB patients in any lymphocyte subset. The extracellular levels of gzmA and, to a lesser extent, of gzmB, were increased in TB patients, but did not correlate with intracellular gzm expression in lymphocyte subsets. Our results reveal enhanced intra- and extracellular expression of gzmA and B in patients with pulmonary TB, suggesting that gzms are part of the host response to tuberculosis. PMID:26156785

  2. Thyroid hormone increases bulk histones expression by enhancing translational efficiency.

    PubMed

    Zambrano, Alberto; García-Carpizo, Verónica; Villamuera, Raquel; Aranda, Ana

    2015-01-01

    The expression of canonical histones is normally coupled to DNA synthesis during the S phase of the cell cycle. Replication-dependent histone mRNAs do not contain a poly(A) tail at their 3' terminus, but instead possess a stem-loop motif, the binding site for the stem-loop binding protein (SLBP), which regulates mRNA processing, stability, and relocation to polysomes. Here we show that the thyroid hormone can increase the levels of canonical histones independent of DNA replication. Incubation of mouse embryonic fibroblasts with T3 increases the total levels of histones, and expression of the thyroid hormone receptor β induces a further increase. This is not restricted to mouse embryonic fibroblasts, because T3 also raises histone expression in other cell lines. T3 does not increase histone mRNA or SLBP levels, suggesting that T3 regulates histone expression by a posttranscriptional mechanism. Indeed, T3 enhanced translational efficiency, inducing relocation of histone mRNA to heavy polysomes. Increased translation was associated with augmented transcription of the eukaryotic translation initiation factor 4 γ2 (EIF4G2). T3 induced EIF4G2 protein and mRNA levels and the thyroid hormone receptor bound to the promoter region of the Eif4g2 gene. Induction of EIF4G2 was essential for T3-dependent histone induction, because depletion of this factor abolished histone increase. These results point out the importance of the thyroid hormones on the posttranscriptional regulation of histone biosynthesis in a cell cycle-independent manner and also suggest the potential regulation of eukaryotic translation by the modulation of the initiation factor EIF4G2, which also operates in the translation of canonical mRNAs. PMID:25422881

  3. GABA selectively increases mucin-1 expression in isolated pig jejunum.

    PubMed

    Braun, Hannah-Sophie; Sponder, Gerhard; Pieper, Robert; Aschenbach, Jörg R; Deiner, Carolin

    2015-11-01

    The inhibitory neurotransmitter GABA (γ-aminobutyric acid) is synthesized by glutamic acid decarboxylase, which is expressed in the central nervous system and in various other tissues including the intestine. Moreover, GABA can be ingested in vegetarian diets or produced by bacterial commensals in the gastrointestinal tract. As previous studies in lung have suggested a link between locally increased GABA availability and mucin 5AC production, the present study sought to test whether the presence or lack of GABA (and its precursor glutamine) has an effect on intestinal mucin expression. Porcine jejunum epithelial preparations were incubated with two different amounts of GABA or glutamine on the mucosal side for 4 h, and changes in the relative gene expression of seven different mucins, enzymes involved in mucin shedding, GABA B receptor, enzymes involved in glutamine/GABA metabolism, glutathione peroxidase 2, and interleukin 10 were examined by quantitative PCR (TaqMan(®) assays). Protein expression of mucin-1 (MUC1) was analyzed by Western blot. On the RNA level, only MUC1 was significantly up-regulated by both GABA concentrations compared with the control. Glutamine-treated groups showed the same trend. On the protein level, all treatment groups showed a significantly higher MUC1 expression than the control group. We conclude that GABA selectively increases the expression of MUC1, a cell surface mucin that prevents the adhesion of microorganisms, because of its size and negative charge, and therefore propose that the well-described positive effects of glutamine on enterocytes and intestinal integrity are partly attributable to effects of its metabolite GABA. PMID:26471792

  4. TPD52 expression increases neutral lipid storage within cultured cells.

    PubMed

    Kamili, Alvin; Roslan, Nuruliza; Frost, Sarah; Cantrill, Laurence C; Wang, Dongwei; Della-Franca, Austin; Bright, Robert K; Groblewski, Guy E; Straub, Beate K; Hoy, Andrew J; Chen, Yuyan; Byrne, Jennifer A

    2015-09-01

    Tumor protein D52 (TPD52) is amplified and/or overexpressed in cancers of diverse cellular origins. Altered cellular metabolism (including lipogenesis) is a hallmark of cancer development, and protein-protein associations between TPD52 and known regulators of lipid storage, and differential TPD52 expression in obese versus non-obese adipose tissue, suggest that TPD52 might regulate cellular lipid metabolism. We found increased lipid droplet numbers in BALB/c 3T3 cell lines stably expressing TPD52, compared with control and TPD52L1-expressing cell lines. TPD52-expressing 3T3 cells showed increased fatty acid storage in triglyceride (from both de novo synthesis and uptake) and formed greater numbers of lipid droplets upon oleic acid supplementation than control cells. TPD52 colocalised with Golgi, but not endoplasmic reticulum (ER), markers and also showed partial colocalisation with lipid droplets coated with ADRP (also known as PLIN2), with a proportion of TPD52 being detected in the lipid droplet fraction. Direct interactions between ADRP and TPD52, but not TPD52L1, were demonstrated using the yeast two-hybrid system, with ADRP-TPD52 interactions confirmed using GST pulldown assays. Our findings uncover a new isoform-specific role for TPD52 in promoting intracellular lipid storage, which might be relevant to TPD52 overexpression in cancer. PMID:26183179

  5. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

    PubMed Central

    Terrón-González, L.; Medina, C.; Limón-Mortés, M. C.; Santero, E.

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance. PMID:23346364

  6. Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels

    PubMed Central

    Dronadula, Nagadhara; Du, Liang; Flynn, Rowan; Buckler, Joshua; Kho, Jordan; Jiang, Zhilong; Tanaka, Shinji; Dichek, David A.

    2010-01-01

    The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity, and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin-10) in large vessel endothelial cells (EC) at levels as high as obtained with the CMV immediate-early promoter, while retaining EC-specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC-specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely 4 copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining—and possibly enhancing—EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large vessel EC, while minimizing systemic effects. PMID:21179172

  7. Increased expression of β-glucosidase A in Clostridium thermocellum 27405 significantly increases cellulase activity

    PubMed Central

    Maki, Miranda L.; Armstrong, Lachlan; Leung, Kam Tin; Qin, Wensheng

    2013-01-01

    β-glucosidase A (bglA) in Clostridium thermocellum 27405 was increased by expression from shuttle vector pIBglA in attempts to increase cellulase activity and ethanol titer by lowering the end product inhibition of cellulase. Through a modified electrotransformation protocol C. thermocellum transformant (+MCbglA) harbouring pIBglA was produced. The β-glucosidase activity of +MCbglA was 2.3- and 1.6-fold greater than wild-type (WT) during late log and stationary phases of growth. Similarly, total cellulase activity of +MCbglA was shown to be 1.7-, 2.3- and 1.6-fold greater than WT during, log, late log and stationary phases of growth. However, there was no significant correlation found between increased cellulase activity and increased ethanol titers for +MCbglA compared with the WT. C. thermocellum has industrial potential for consolidated bioprocessing (CBP) to make a more cost effective production of biofuels; however, the hydrolysis rate of the strain is still hindered by end product inhibition. We successfully increased total cellulase activity by increased expression of bglA and thereby increased the productivity of C. thermocellum during the hydrolysis stage in CBP. Our work also lends insights into the complex metabolism of C. thermocellum for future improvement of this strain. PMID:22922214

  8. Controversy surrounding the increased expression of TGFβ1 in asthma

    PubMed Central

    Bossé, Ynuk; Rola-Pleszczynski, Marek

    2007-01-01

    Asthma is a waxing and waning disease that leads to structural changes in the airways, such as subepithelial fibrosis, increased mass of airway smooth muscle and epithelial metaplasia. Such a remodeling of the airways futher amplifies asthma symptoms, but its etiology is unknown. Transforming growth factor β1 is a pleiotropic cytokine involved in many fibrotic, oncologic and immunologic diseases and is believed to play an essential role in airway remodeling that occurs in asthmatic patients. Since it is secreted in an inactive form, the overall activity of this cytokine is not exclusively determined by its level of expression, but also by extensive and complex post-translational mechanisms, which are all importanin modulating the magnitude of the TGFβ1 response. Even if TGFβ1 upregulation in asthma is considered as a dogma by certain investigators in the field, the overall picture of the published litterature is not that clear and the cellular origin of this cytokine in the airways of asthmatics is still a contemporaneous debate. On the other hand, it is becoming clear that TGFβ1 signaling is increased in the lungs of asthmatics, which testifies the increased activity of this cytokine in asthma pathogenesis. The current work is an impartial and exhaustive compilation of the reported papers regarding the expression of TGFβ1 in human asthmatics. For the sake of comparison, several studies performed in animal models of the disease are also included. Inconsistencies observed in human studies are discussed and conclusions as well as trends from the current state of the litterature on the matter are proposed. Finally, the different points of regulation that can affect the amplitude of the TGFβ1 response are briefly revised and the possibility that TGFβ1 is disregulated at another level in asthma, rather than simply in its expression, is highlighted. PMID:17892594

  9. Reduced Expression of MYC Increases Longevity and Enhances Healthspan

    PubMed Central

    Hofmann, Jeffrey W.; Zhao, Xiaoai; De Cecco, Marco; Peterson, Abigail L.; Pagliaroli, Luca; Manivannan, Jayameenakshi; Hubbard, Gene B.; Ikeno, Yuji; Zhang, Yongqing; Feng, Bin; Li, Xiaxi; Serre, Thomas; Qi, Wenbo; Van Remmen, Holly; Miller, Richard A.; Bath, Kevin G.; de Cabo, Rafael; Xu, Haiyan; Neretti, Nicola; Sedivy, John M.

    2015-01-01

    SUMMARY MYC is a highly pleiotropic transcription factor whose deregulation promotes cancer. In contrast, we find that Myc haploinsufficient (Myc+/−) mice exhibit increased lifespan. They show resistance to several age-associated pathologies, including osteoporosis, cardiac fibrosis and immunosenescence. They also appear to be more active, with a higher metabolic rate and healthier lipid metabolism. Transcriptomic analysis reveals a gene expression signature enriched for metabolic and immune processes. The ancestral role of MYC as a regulator of ribosome biogenesis is reflected in reduced protein translation, which is inversely correlated with longevity. We also observe changes in nutrient and energy sensing pathways, including reduced serum IGF-1, increased AMPK activity, and decreased AKT, TOR and S6K activities. In contrast to observations in other longevity models, Myc+/− mice do not show improvements in stress management pathways. Our findings indicate that MYC activity has a significant impact on longevity and multiple aspects of mammalian healthspan. PMID:25619689

  10. Reduced expression of MYC increases longevity and enhances healthspan.

    PubMed

    Hofmann, Jeffrey W; Zhao, Xiaoai; De Cecco, Marco; Peterson, Abigail L; Pagliaroli, Luca; Manivannan, Jayameenakshi; Hubbard, Gene B; Ikeno, Yuji; Zhang, Yongqing; Feng, Bin; Li, Xiaxi; Serre, Thomas; Qi, Wenbo; Van Remmen, Holly; Miller, Richard A; Bath, Kevin G; de Cabo, Rafael; Xu, Haiyan; Neretti, Nicola; Sedivy, John M

    2015-01-29

    MYC is a highly pleiotropic transcription factor whose deregulation promotes cancer. In contrast, we find that Myc haploinsufficient (Myc(+/-)) mice exhibit increased lifespan. They show resistance to several age-associated pathologies, including osteoporosis, cardiac fibrosis, and immunosenescence. They also appear to be more active, with a higher metabolic rate and healthier lipid metabolism. Transcriptomic analysis reveals a gene expression signature enriched for metabolic and immune processes. The ancestral role of MYC as a regulator of ribosome biogenesis is reflected in reduced protein translation, which is inversely correlated with longevity. We also observe changes in nutrient and energy sensing pathways, including reduced serum IGF-1, increased AMPK activity, and decreased AKT, TOR, and S6K activities. In contrast to observations in other longevity models, Myc(+/-) mice do not show improvements in stress management pathways. Our findings indicate that MYC activity has a significant impact on longevity and multiple aspects of mammalian healthspan. PMID:25619689

  11. Chronic intermittent mechanical stress increases MUC5AC protein expression.

    PubMed

    Park, Jin-Ah; Tschumperlin, Daniel J

    2009-10-01

    Increased abundance of mucin secretory cells is a characteristic feature of the epithelium in asthma and other chronic airway diseases. We showed previously that the mechanical stresses of airway constriction, both in the intact mouse lung and a cell culture model, activate the epidermal growth factor receptor (EGFR), a known modulator of mucin expression in airway epithelial cells. Here we tested whether chronic, intermittent, short-duration compressive stress (30 cm H(2)O) is sufficient to increase the abundance of MUC5AC-positive cells and intracellular mucin levels in human bronchial epithelial cells cultured at an air-liquid interface. Compressive stress applied for 1 hour per day for 14 days significantly increased the percentage of cells staining positively for MUC5AC protein (22.0 +/- 3.8%, mean +/- SD) relative to unstimulated controls (8.6 +/- 2.6%), and similarly changed intracellular MUC5AC protein levels measured by Western and slot blotting. The effect of compressive stress was gradual, with significant changes in MUC5AC-positive cell numbers evident by Day 7, but required as little as 10 minutes of compressive stress daily. Daily treatment of cells with an EGFR kinase inhibitor (AG1478, 1 muM) significantly but incompletely attenuated the response to compressive stress. Complete attenuation could be accomplished by simultaneous treatment with the combination of AG1478 and a transforming growth factor (TGF)-beta(2) (1 microg/ml)-neutralizing antibody, or with anti-TGF-beta(2) alone. Our findings demonstrate that short duration episodes of mechanical stress, representative of those occurring during bronchoconstriction, are sufficient to increase goblet cell number and MUC5AC protein expression in bronchial epithelial cells in vitro. We propose that the mechanical environment present in asthma may fundamentally bias the composition of airway epithelial lining in favor of mucin secretory cells. PMID:19168703

  12. Fast Growth Increases the Selective Advantage of a Mutation Arising Recurrently during Evolution under Metal Limitation

    PubMed Central

    Chou, Hsin-Hung; Berthet, Julia; Marx, Christopher J.

    2009-01-01

    Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation across many environments, ranging from pathogens in the human body to phytoplankton in the oceans, imposes strong selection for improved metal acquisition systems. In this study, we uncovered the genetic and physiological basis of adaptation to metal limitation using experimental populations of Methylobacterium extorquens AM1 evolved in metal-deficient growth media. We identified a transposition mutation arising recurrently in 30 of 32 independent populations that utilized methanol as a carbon source, but not in any of the 8 that utilized only succinate. These parallel insertion events increased expression of a novel transporter system that enhanced cobalt uptake. Such ability ensured the production of vitamin B12, a cobalt-containing cofactor, to sustain two vitamin B12–dependent enzymatic reactions essential to methanol, but not succinate, metabolism. Interestingly, this mutation provided higher selective advantages under genetic backgrounds or incubation temperatures that permit faster growth, indicating growth-rate–dependent epistatic and genotype-by-environment interactions. Our results link beneficial mutations emerging in a metal-limiting environment to their physiological basis in carbon metabolism, suggest that certain molecular features may promote the emergence of parallel mutations, and indicate that the selective advantages of some mutations depend generically upon changes in growth rate that can stem from either genetic or environmental influences. PMID:19763169

  13. Differential and limited expression of mutant alleles in multiple myeloma

    PubMed Central

    Rashid, Naim U.; Sperling, Adam S.; Bolli, Niccolo; Wedge, David C.; Van Loo, Peter; Tai, Yu-Tzu; Shammas, Masood A.; Fulciniti, Mariateresa; Samur, Mehmet K.; Richardson, Paul G.; Magrangeas, Florence; Minvielle, Stephane; Futreal, P. Andrew; Anderson, Kenneth C.; Avet-Loiseau, Herve; Parmigiani, Giovanni

    2014-01-01

    Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. PMID:25237203

  14. Differential and limited expression of mutant alleles in multiple myeloma.

    PubMed

    Rashid, Naim U; Sperling, Adam S; Bolli, Niccolo; Wedge, David C; Van Loo, Peter; Tai, Yu-Tzu; Shammas, Masood A; Fulciniti, Mariateresa; Samur, Mehmet K; Richardson, Paul G; Magrangeas, Florence; Minvielle, Stephane; Futreal, P Andrew; Anderson, Kenneth C; Avet-Loiseau, Herve; Campbell, Peter J; Parmigiani, Giovanni; Munshi, Nikhil C

    2014-11-13

    Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. PMID:25237203

  15. Expression of PARK7 is increased in celiac disease.

    PubMed

    Vörös, Péter; Sziksz, Erna; Himer, Leonóra; Onody, Anna; Pap, Domonkos; Frivolt, Klára; Szebeni, Beáta; Lippai, Rita; Győrffy, Hajnalka; Fekete, Andrea; Brandt, Ferenc; Molnár, Kriszta; Veres, Gábor; Arató, András; Tulassay, Tivadar; Vannay, Adám

    2013-09-01

    Recently, it has been suggested that the gene called Parkinson's disease 7 (PARK7) might be an upstream activator of hypoxia-inducible factor (HIF)-1α, which plays a major role in sustaining intestinal barrier integrity. Furthermore, PARK7 has been proposed to participate in the Toll-like receptor (TLR)-dependent regulation of the innate immune system. Our aim was to investigate the involvement of PARK7 in the pathogenesis of coeliac disease (CD). Duodenal biopsy specimens were collected from 19 children with untreated CD, five children with treated CD (maintained on gluten-free diet), and ten children with histologically normal duodenal biopsies. PARK7 mRNA expression and protein level were determined by real-time polymerase chain reaction (PCR) and Western blot, respectively. Localization of PARK7 was visualized by immunofluorescence staining. Protein level of PARK7 increased in the duodenal mucosa of children with untreated CD compared to children with treated CD or to control biopsies (p <0.03). We detected intensive PARK7 staining in the epithelial cells and lamina propria of the duodenal mucosa of children with untreated CD compared with that in control biopsies. Our finding that mucosal expression of PARK7 is increased suggests that PARK7 is involved in the pathogenesis of gastrointestinal diseases, notably CD. Our results suggest that PARK7 may alter processes mediated by HIF-1α and TLR4, which supports a role for PARK7 in the maintenance of epithelial barrier integrity, immune homeostasis, or apoptosis. PMID:23832581

  16. Investigating a Multimodal Intervention for Children With Limited Expressive Vocabularies Associated With Autism

    PubMed Central

    Storkel, Holly L.; Bushnell, Paige; Barker, R. Michael; Saunders, Kate; Daniels, Debby; Fleming, Kandace

    2015-01-01

    Purpose This study investigated a new intervention package aimed at increasing expressive word learning by school-age children with autism who have limited expressive vocabularies. This pilot investigation was intended to show proof of concept. Method Ten children between the ages of 6 and 10 years participated, with educational diagnoses of autism and limited expressive vocabularies at the outset of the study. A multimodal intervention composed of speech sound practice and augmentative and alternative communication was used to teach individualized vocabulary words that were selected on the basis of initial speech sound repertoires and principles of phonotactic probability and neighborhood density. A multiple-probe design was used to evaluate learning outcomes. Results Five children showed gains in spoken-word learning across successive word sets (high responders). Five children did not meet learning criteria (low responders). Comparisons of behaviors measured prior to intervention indicated that high responders had relatively higher skills in receptive language, prelinguistic communication, vocal/verbal imitation, adaptive behavior, and consonant productions. Conclusions The intervention package holds promise for improving spoken word productions for some children with autism who have limited expressive vocabularies. Further research is needed to better describe who may most benefit from this approach as well as investigate generalized benefits to untaught contexts and targets. PMID:25910710

  17. MiR-224 expression increases radiation sensitivity of glioblastoma cells

    SciTech Connect

    Upraity, Shailendra; Kazi, Sadaf; Padul, Vijay; Shirsat, Neelam Vishwanath

    2014-05-30

    Highlights: • MiR-224 expression in established glioblastoma cell lines and sporadic tumor tissues is low. • Exogenous miR-224 expression was found to increase radiation sensitivity of glioblastoma cells. • MiR-224 expression brought about 55–60% reduction in API5 expression levels. • Transfection with API5 siRNA increased radiation sensitivity of glioblastoma cells. • Low miR-224 and high API5 expression correlated with worse survival of GBM patients. - Abstract: Glioblastoma (GBM) is the most common and highly aggressive primary malignant brain tumor. The intrinsic resistance of this brain tumor limits the efficacy of administered treatment like radiation therapy. In the present study, effect of miR-224 expression on growth characteristics of established GBM cell lines was analyzed. MiR-224 expression in the cell lines as well as in primary GBM tumor tissues was found to be low. Exogenous transient expression of miR-224 using either synthetic mimics or stable inducible expression using doxycycline inducible lentiviral vector carrying miR-224 gene, was found to bring about 30–55% reduction in clonogenic potential of U87 MG cells. MiR-224 expression reduced clonogenic potential of U87 MG cells by 85–90% on irradiation at a dose of 6 Gy, a dose that brought about 50% reduction in clonogenic potential in the absence of miR-224 expression. MiR-224 expression in glioblastoma cells resulted in 55–65% reduction in the expression levels of API5 gene, a known target of miR-224. Further, siRNA mediated down-regulation of API5 was also found to have radiation sensitizing effect on glioblastoma cell lines. Analysis of the Cancer Genome Atlas data showed lower miR-224 expression levels in male GBM patients to correlate with poorer survival. Higher expression levels of miR-224 target API5 also showed significant correlation with poorer survival of GBM patients. Up-regulation of miR-224 or down-regulation of its target API5 in combination with radiation therapy

  18. Increased caspase-3 expression and activity contribute to reduced CD3zeta expression in systemic lupus erythematosus T cells.

    PubMed

    Krishnan, Sandeep; Kiang, Juliann G; Fisher, Carolyn U; Nambiar, Madhusoodana P; Nguyen, Hang T; Kyttaris, Vasileios C; Chowdhury, Bhabadeb; Rus, Violeta; Tsokos, George C

    2005-09-01

    T cells isolated from patients with systemic lupus erythematosus (SLE) express low levels of CD3zeta-chain, a critical molecule involved in TCR-mediated signaling, but the involved mechanisms are not fully understood. In this study we examined caspase-3 as a candidate for cleaving CD3zeta in SLE T cells. We demonstrate that SLE T cells display increased expression and activity of caspase-3. Treatment of SLE T cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-FMK reduced proteolysis of CD3zeta and enhanced its expression. In addition, Z-Asp-Glu-Val-Asp-FMK treatment increased the association of CD3zeta with lipid rafts and simultaneously reversed the abnormal lipid raft preclustering, heightened TCR-induced calcium responses, and reduced the expression of FcRgamma-chain exclusively in SLE T cells. We conclude that caspase-3 inhibitors can normalize SLE T cell function by limiting the excessive digestion of CD3zeta-chain and suggest that such molecules can be considered in the treatment of this disease. PMID:16116236

  19. Increased expression of the nicotinic acetylcholine receptor in stimulated muscle.

    PubMed

    O'Reilly, Clare; Pette, Dirk; Ohlendieck, Kay

    2003-01-10

    Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts. PMID:12504123

  20. 26 CFR 1.402(g)-2 - Increased limit for catch-up contributions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 5 2010-04-01 2010-04-01 false Increased limit for catch-up contributions. 1... Increased limit for catch-up contributions. (a) General rule. Under section 402(g)(1)(C), in determining the amount of elective deferrals that are includible in gross income under section 402(g) for a...

  1. 26 CFR 1.402(g)-2 - Increased limit for catch-up contributions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 5 2011-04-01 2011-04-01 false Increased limit for catch-up contributions. 1.... § 1.402(g)-2 Increased limit for catch-up contributions. (a) General rule. Under section 402(g)(1)(C...(g) for a catch-up eligible participant (within the meaning of § 1.414(v)-1(g)), the...

  2. 26 CFR 1.402(g)-2 - Increased limit for catch-up contributions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 5 2013-04-01 2013-04-01 false Increased limit for catch-up contributions. 1.... § 1.402(g)-2 Increased limit for catch-up contributions. (a) General rule. Under section 402(g)(1)(C...(g) for a catch-up eligible participant (within the meaning of § 1.414(v)-1(g)), the...

  3. 26 CFR 1.402(g)-2 - Increased limit for catch-up contributions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 5 2014-04-01 2014-04-01 false Increased limit for catch-up contributions. 1.... § 1.402(g)-2 Increased limit for catch-up contributions. (a) General rule. Under section 402(g)(1)(C...(g) for a catch-up eligible participant (within the meaning of § 1.414(v)-1(g)), the...

  4. Living to the range limit: consumer isotopic variation increases with environmental stress

    PubMed Central

    O’Connor, Nessa E.

    2016-01-01

    Background: Theoretically, each species’ ecological niche is phylogenetically-determined and expressed spatially as the species’ range. However, environmental stress gradients may directly or indirectly decrease individual performance, such that the precise process delimiting a species range may not be revealed simply by studying abundance patterns. In the intertidal habitat the vertical ranges of marine species may be constrained by their abilities to tolerate thermal and desiccation stress, which may act directly or indirectly, the latter by limiting the availability of preferred trophic resources. Therefore, we expected individuals at greater shore heights to show greater variation in diet alongside lower indices of physiological condition. Methods: We sampled the grazing gastropod Echinolittorina peruviana from the desert coastline of northern Chile at three shore heights, across eighteen regionally-representative shores. Stable isotope values (δ13C and δ15N) were extracted from E. peruviana and its putative food resources to estimate Bayesian ellipse area, carbon and nitrogen ranges and diet. Individual physiological condition was tracked by muscle % C and % N. Results: There was an increase in isotopic variation at high shore levels, where E. peruviana’s preferred resource, tide-deposited particulate organic matter (POM), appeared to decrease in dietary contribution, and was expected to be less abundant. Both muscle % C and % N of individuals decreased with height on the shore. Discussion: Individuals at higher stress levels appear to be less discriminating in diet, likely because of abiotic forcing, which decreases both consumer mobility and the availability of a preferred resource. Abiotic stress might be expected to increase trophic variation in other selective dietary generalist species. Where this coincides with a lower physiological condition may be a direct factor in setting their range limit. PMID:27280067

  5. Carnitine Palmitoyltransferase 1 Increases Lipolysis, UCP1 Protein Expression and Mitochondrial Activity in Brown Adipocytes

    PubMed Central

    Calderon-Dominguez, María; Sebastián, David; Fucho, Raquel; Weber, Minéia; Mir, Joan F.; García-Casarrubios, Ester; Obregón, María Jesús; Zorzano, Antonio; Valverde, Ángela M.; Serra, Dolors

    2016-01-01

    The discovery of active brown adipose tissue (BAT) in adult humans and the fact that it is reduced in obese and diabetic patients have put a spotlight on this tissue as a key player in obesity-induced metabolic disorders. BAT regulates energy expenditure through thermogenesis; therefore, harnessing its thermogenic fat-burning power is an attractive therapeutic approach. We aimed to enhance BAT thermogenesis by increasing its fatty acid oxidation (FAO) rate. Thus, we expressed carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown adipocyte (rBA) cell line through adenoviral infection. We found that CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Additionally, enhanced FAO reduced the palmitate-induced increase in triglyceride content and the expression of obese and inflammatory markers. Thus, CPT1AM-expressing rBA had enhanced fat-burning capacity and improved lipid-induced derangements. This indicates that CPT1AM-mediated increase in brown adipocytes FAO may be a new approach to the treatment of obesity-induced disorders. PMID:27438137

  6. p53 increases caspase-6 expression and activation in muscle tissue expressing mutant huntingtin.

    PubMed

    Ehrnhoefer, Dagmar E; Skotte, Niels H; Ladha, Safia; Nguyen, Yen T N; Qiu, Xiaofan; Deng, Yu; Huynh, Khuong T; Engemann, Sabine; Nielsen, Signe M; Becanovic, Kristina; Leavitt, Blair R; Hasholt, Lis; Hayden, Michael R

    2014-02-01

    Activation of caspase-6 in the striatum of both presymptomatic and affected persons with Huntington's disease (HD) is an early event in the disease pathogenesis. However, little is known about the role of caspase-6 outside the central nervous system (CNS) and whether caspase activation might play a role in the peripheral phenotypes, such as muscle wasting observed in HD. We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A. Using mouse embryonic fibroblasts (MEFs) from YAC128 mice, we show that this increase in caspase-6 activity can be mitigated by pifithrin-α (pifα), an inhibitor of p53 transcriptional activity, but not through the inhibition of p53's mitochondrial pro-apoptotic function. Remarkably, the p53-mediated increase in caspase-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase-6. Furthermore, these results suggest that this pathway is activated both within and outside the CNS in HD and may contribute to both loss of CNS neurons and muscle atrophy. PMID:24070868

  7. Limitations of allotopic expression of mitochondrial genes in mammalian cells.

    PubMed Central

    Oca-Cossio, Jose; Kenyon, Lesley; Hao, Huiling; Moraes, Carlos T

    2003-01-01

    The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria. PMID:14573482

  8. Increased expression of Talin1 in the eutopic and ectopic endometria of women with adenomyosis.

    PubMed

    Jiang, Jianfa; Sun, Aijun; Wang, Yanfang; Deng, Yan

    2016-06-01

    Adenomyosis is a prevalent gynecologic benign disease in women. Despite its significance, there is only a limited understanding of its pathological mechanisms. Talin1, a cytoskeletal protein, plays an important role in cell survival, proliferation, invasion and migration. The objective of this study was to investigate the mRNA and protein expression of talin1 in both the eutopic and ectopic endometria of women with adenomyosis. Higher talin1 mRNA levels were observed in both ectopic and eutopic endometria from the adenomyosis subjects compared with the eutopic endometria from women without adenomyosis. Immunohistochemistry revealed increased epithelial expression of talin1 in the ectopic and eutopic endometria from patients with adenomyosis compared with those without adenomyosis. When tests were performed on matched samples of eutopic and ectopic endometria of adenomyosis subjects, the mRNA and protein expression of talin1 was much higher in the ectopic endometria than in the eutopic endometria. The results reveal that the expression pattern of talin1 in the eutopic and ectopic endometria is enhanced in women with adenomyosis. Increased talin1 expression may play a role in the pathogenesis and development of adenomyosis. PMID:26759065

  9. Evaluating the safety impact of increased speed limits on rural highways in British Columbia.

    PubMed

    Sayed, Tarek; Sacchi, Emanuele

    2016-10-01

    Maximum speed limits are usually set to inform drivers of the highest speed that it is safe and appropriate for ideal traffic, road and weather conditions. Many previous studies were conducted to investigate the relationship between changed speed limits and safety. The results of these studies generally show that relaxing speed limits can negatively affect safety, especially with regard to fatal and injury crashes. Despite these results, several road jurisdictions in North America continue to raise the maximum speed limits. In 2013, the British Columbia Ministry of Transportation and Infrastructure initiated a speed limits review. The review found that the 85th percentile speed on many highway segments was 10km/h higher than corresponding posted speed limits and 1300km of rural provincial highway segments were recommended for higher speed limits. Most of the highway segments had 10km/h speed limit increase with a small section having 20km/h speed limit increase. As speed limit changes can have a substantial impact on safety, the main objective of this study is to estimate the effect of the increased speed limits on crash occurrence. A before-after evaluation was undertaken with the full Bayesian technique. Overall, the evaluation showed that changed speed limits led to a statistically significant increase in fatal-plus-injury (severe) crashes of 11.1%. A crash modification function that includes changes in the treatment effect over time showed that the initial increase of the first post-implementation period may slightly decrease over time. PMID:27447060

  10. Increased Expression of PHGDH and Prognostic Significance in Colorectal Cancer.

    PubMed

    Jia, Xiao-Qin; Zhang, Shu; Zhu, Hui-Jun; Wang, Wei; Zhu, Jin-Hong; Wang, Xu-Dong; Qiang, Jian-Feng

    2016-06-01

    Phosphoglycerate dehydrogenase (PHGDH) plays an essential role in cancer-specific metabolic reprogramming. It has been reported as a putative metabolic oncogene in several types of human malignant tumors, such as breast cancer and melanoma. To date, PHGDH expression in colorectal cancer (CRC) as well as its association with clinicopathological characteristics and prognostic implication remain undetermined. In this study, we determined the PHGDH protein expression using tissue microarray immunohistochemistry (TMA-IHC) on 193 pairs of formalin-fixed, paraffin-embedded specimens of CRC and adjacent tissues, 25 chronic colitis, 41 low-, and 19 high-grade intraepithelial neoplasia specimens, and we also determined PHGDH mRNA level using quantitative reverse transcription PCR (qRT-PCR) on additional 23 pairs of fresh CRC tissues and adjacent tissues. We found that both PHGDH mRNA and protein was highly expressed in tumor tissues in comparison with matched adjacent non-tumor tissues, and high PHGDH protein expression was correlated with advanced TNM stage (P = .038) and larger tumor (P = .001). Multivariate Cox regression analysis showed that PHGDH protein expression (HR = 2.285, 95% CI = 1.18 to 4.41, P = .014), tumor differentiation (HR = .307, 95% CI = .154 to 0.609, P = .001), and TNM stage (HR = 1.791, 95% CI = 1.125 to 2.85, P = .014) were independent prognostic factors in CRC. Kaplan-Meier survival curves and log rank test showed that high PHGDH protein expression contributed to poor outcome in CRC patients (P < .001). In conclusion, these results suggest that assessment of PHGDH expression could be useful in identifying a high-risk subgroup of CRC. PMID:27267836

  11. Increased H+ efflux is sufficient to induce dysplasia and necessary for viability with oncogene expression

    PubMed Central

    Grillo-Hill, Bree K; Choi, Changhoon; Jimenez-Vidal, Maite; Barber, Diane L

    2015-01-01

    Intracellular pH (pHi) dynamics is increasingly recognized as an important regulator of a range of normal and pathological cell behaviors. Notably, increased pHi is now acknowledged as a conserved characteristic of cancers and in cell models is confirmed to increase proliferation and migration as well as limit apoptosis. However, the significance of increased pHi for cancer in vivo remains unresolved. Using Drosophila melanogaster, we show that increased pHi is sufficient to induce dysplasia in the absence of other transforming cues and potentiates growth and invasion with oncogenic Ras. Using a genetically encoded biosensor we also confirm increased pHi in situ. Moreover, in Drosophila models and clonal human mammary cells we show that limiting H+ efflux with oncogenic Raf or Ras induces acidosis and synthetic lethality. Further, we show lethality in invasive primary tumor cell lines with inhibiting H+ efflux. Synthetic lethality with reduced H+ efflux and activated oncogene expression could be exploited therapeutically to restrain cancer progression while limiting off-target effects. DOI: http://dx.doi.org/10.7554/eLife.03270.001 PMID:25793441

  12. Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation

    PubMed Central

    Chen, Huiyong; Choesang, Tenzin; Li, Huihui; Sun, Shuming; Pham, Petra; Bao, Weili; Feola, Maria; Westerman, Mark; Li, Guiyuan; Follenzi, Antonia; Blanc, Lionel; Rivella, Stefano; Fleming, Robert E.; Ginzburg, Yelena Z.

    2016-01-01

    Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbbth1/th1 (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes. PMID:26635037

  13. Regret Expression and Social Learning Increases Delay to Sexual Gratification

    PubMed Central

    Quisenberry, Amanda J.; Eddy, Celia R.; Patterson, David L.; Franck, Christopher T.; Bickel, Warren K.

    2015-01-01

    Objective Modification and prevention of risky sexual behavior is important to individuals’ health and public health policy. This study employed a novel sexual discounting task to elucidate the effects of social learning and regret expression on delay to sexual gratification in a behavioral task. Methods Amazon Mechanical Turk Workers were assigned to hear one of three scenarios about a friend who engages in similar sexual behavior. The scenarios included a positive health consequence, a negative health consequence or a negative health consequence with the expression of regret. After reading one scenario, participants were asked to select from 60 images, those with whom they would have casual sex. Of the selected images, participants chose one image each for the person they most and least want to have sex with and person most and least likely to have a sexually transmitted infection. They then answered questions about engaging in unprotected sex now or waiting some delay for condom-protected sex in each partner condition. Results Results indicate that the negative health outcome scenario with regret expression resulted in delayed sexual gratification in the most attractive and least STI partner conditions, whereas in the least attractive and most STI partner conditions the negative health outcome with and without regret resulted in delayed sexual gratification. Conclusions Results suggest that the sexual discounting task is a relevant laboratory measure and the framing of information to include regret expression may be relevant for prevention of risky sexual behavior. PMID:26280349

  14. 26 CFR 1.402(g)-2 - Increased limit for catch-up contributions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 5 2012-04-01 2011-04-01 true Increased limit for catch-up contributions. 1.402.... § 1.402(g)-2 Increased limit for catch-up contributions. (a) General rule. Under section 402(g)(1)(C...(g) for a catch-up eligible participant (within the meaning of § 1.414(v)-1(g)), the...

  15. Increased fibroblast telomerase expression precedes myofibroblast α-smooth muscle actin expression in idiopathic pulmonary fibrosis

    PubMed Central

    Waisberg, Daniel Reis; Parra, Edwin Roger; Barbas-Filho, João Valente; Fernezlian, Sandra; Capelozzi, Vera Luiza

    2012-01-01

    OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast α-smooth muscle actin expression and the tissue expression of interleukin-4, transforming growth factor-β, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-β expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast α-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast α-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast α-smooth muscle actin expression predominates in areas of late remodeling. These events seem to be

  16. Parkin degrades estrogen-related receptors to limit the expression of monoamine oxidases.

    PubMed

    Ren, Yong; Jiang, Houbo; Ma, Dingyuan; Nakaso, Kazuhiro; Feng, Jian

    2011-03-15

    Parkin, whose mutations cause Parkinson disease (PD), controls oxidative stress by limiting the expression of monoamine oxidases (MAO)--mitochondrial enzymes responsible for the oxidative de-amination of dopamine. Here, we show that parkin performed this function by increasing the ubiquitination and degradation of estrogen-related receptors (ERR), orphan nuclear receptors that play critical roles in the transcription regulation of many nuclear-encoded mitochondrial proteins. All three ERRs (α, β and γ) increased the transcription of MAOs A and B; the effects were abolished by parkin, but not by its PD-linked mutants. Parkin bound to ERRs and increased their ubiquitination and degradation. In fibroblasts from PD patients with parkin mutations or brain slices from parkin knockout mice, degradation of ERRs was significantly attenuated. The results reveal the molecular mechanism by which parkin suppresses the transcription of MAOs to control oxidative stress induced by dopamine oxidation. PMID:21177257

  17. Parkin degrades estrogen-related receptors to limit the expression of monoamine oxidases

    PubMed Central

    Ren, Yong; Jiang, Houbo; Ma, Dingyuan; Nakaso, Kazuhiro; Feng, Jian

    2011-01-01

    Parkin, whose mutations cause Parkinson disease (PD), controls oxidative stress by limiting the expression of monoamine oxidases (MAO)—mitochondrial enzymes responsible for the oxidative de-amination of dopamine. Here, we show that parkin performed this function by increasing the ubiquitination and degradation of estrogen-related receptors (ERR), orphan nuclear receptors that play critical roles in the transcription regulation of many nuclear-encoded mitochondrial proteins. All three ERRs (α, β and γ) increased the transcription of MAOs A and B; the effects were abolished by parkin, but not by its PD-linked mutants. Parkin bound to ERRs and increased their ubiquitination and degradation. In fibroblasts from PD patients with parkin mutations or brain slices from parkin knockout mice, degradation of ERRs was significantly attenuated. The results reveal the molecular mechanism by which parkin suppresses the transcription of MAOs to control oxidative stress induced by dopamine oxidation. PMID:21177257

  18. Interleukin-6-driven progranulin expression increases cholangiocarcinoma growth by an Akt-dependent mechanism

    PubMed Central

    Frampton, Gabriel; Invernizzi, Pietro; Bernuzzi, Francesca; Pae, Hae Yong; Quinn, Matthew; Horvat, Darijana; Galindo, Cheryl; Huang, Li; McMillin, Matthew; Cooper, Brandon; Rimassa, Lorenza; DeMorrow, Sharon

    2015-01-01

    Background and objectives Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. The growth factor, progranulin, is overexpressed in a number of tumours. The study aims were to assess the expression of progranulin in cholangiocarcinoma and to determine its effects on tumour growth. Methods The expression and secretion of progranulin were evaluated in multiple cholangiocarcinoma cell lines and in clinical samples from patients with cholangiocarcinoma. The role of interleukin 6 (IL-6)-mediated signalling in the expression of progranulin was assessed using a combination of specific inhibitors and shRNA knockdown techniques. The effect of progranulin on proliferation and Akt activation and subsequent effects of FOXO1 phosphorylation were assessed in vitro. Progranulin knockdown cell lines were established, and the effects on cholangiocarcinoma growth were determined. Results Progranulin expression and secretion were upregulated in cholangiocarcinoma cell lines and tissue, which were in part via IL-6-mediated activation of the ERK1/2/RSK1/C/EBPβ pathway. Blocking any of these signalling molecules, by either pharmacological inhibitors or shRNA, prevented the IL-6-dependent activation of progranulin expression. Treatment of cholangiocarcinoma cells with recombinant progranulin increased cell proliferation in vitro by a mechanism involving Akt phosphorylation leading to phosphorylation and nuclear extrusion of FOXO1. Knockdown of progranulin expression in cholangiocarcinoma cells decreased the expression of proliferating cellular nuclear antigen, a marker of proliferative capacity, and slowed tumour growth in vivo. Conclusions Evidence is presented for a role for progranulin as a novel growth factor regulating cholangiocarcinoma growth. Specific targeting of progranulin may represent an alternative for the development of therapeutic strategies. PMID:22068162

  19. Increased RUNX1 expression in patients with immune thrombocytopenia.

    PubMed

    Zhong, Xiaomin; Wu, Yulu; Liu, Yun; Zhu, Feng; Li, Xiaoqian; Li, Depeng; Li, Zhenyu; Zeng, Lingyu; Qiao, Jianlin; Chen, Xiaofei; Xu, Kailin

    2016-08-01

    Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disease, characterized by dysregulation of cellular immunity. Th17 and associated IL-17 were involved in the pathogenesis of ITP. Runt-related transcription factor 1 (RUNX1), a member of the runt domain-containing family of transcription factors, is required for Th17 differentiation. Whether RUNX1 was involved in the pathogenesis of ITP remains poorly understood. In this study, 30 active ITP patients, 20 ITP in remission and 20 age and gender matched healthy controls were included. Peripheral blood mononuclear cells (PBMCs) were isolated to measure mRNA level of RUNX1 and retinoic acid receptor-related orphan receptor-γt (RORγt) by quantitative real-time PCR and Th17 cells by flow cytometry. Meanwhile, plasma was extracted for measurement of IL-17 level by ELISA. Our results showed a significantly higher expression of RUNX1, RORγt, Th17 cells and plasma level of IL-17 in active ITP patients than that in healthy controls. No differences of expression of RUNX1, RORγt and Th17 cells were observed between remission patients and controls. Furthermore, a significantly positive correlation of RUNX1 with RORγt was found in active ITP patients. In conclusion, RUNX1 was associated with the pathogenesis of ITP possibly through regulation of Th17 cell differentiation and therapeutically targeting it might be a novel approach in ITP treatment. PMID:27288310

  20. 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

  1. Increased hydrophobicity in Malassezia species correlates with increased proinflammatory cytokine expression in human keratinocytes.

    PubMed

    Akaza, Narifumi; Akamatsu, Hirohiko; Takeoka, Shiori; Mizutani, Hiroshi; Nakata, Satoru; Matsunaga, Kayoko

    2012-11-01

    Malassezia cells stimulate cytokine production by keratinocytes, although this ability differs among Malassezia species for unknown reasons. The aim of this study was to clarify the factors determining the ability to induce cytokine production by human keratinocytes in response to Malassezia species. M. furfur NBRC 0656, M. sympodialis CBS 7222, M. dermatis JCM 11348, M. globosa CBS 7966, M. restricta CBS 7877, and three strains each of M. globosa, M. restricta, M. dermatis, M. sympodialis, and M. furfur maintained under various culture conditions were used. Normal human epidermal keratinocytes (NHEKs) (1 × 10(5) cells) and the Malassezia species (1 × 10(6) cells) were co-cultured, and IL-1α, IL-6, and IL-8 mRNA levels were determined. Moreover, the hydrophobicity and β-1,3-glucan expression at the surface of Malassezia cells were analyzed. The ability of Malassezia cells to trigger the mRNA expression of proinflammatory cytokines in NHEKs differed with the species and conditions and was dependent upon the hydrophobicity of Malassezia cells not β-1,3-glucan expression. PMID:22548238

  2. 12 CFR 226.55 - Limitations on increasing annual percentage rates, fees, and charges.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Applicable to Credit Card Accounts and Open-End Credit Offered to College Students § 226.55 Limitations on... required to be disclosed under § 226.6(b)(2)(ii), (b)(2)(iii), or (b)(2)(xii) on a credit card account under an open-end (not home-secured) consumer credit plan. (b) Exceptions. A card issuer may increase...

  3. 12 CFR 226.55 - Limitations on increasing annual percentage rates, fees, and charges.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Applicable to Credit Card Accounts and Open-End Credit Offered to College Students § 226.55 Limitations on... required to be disclosed under § 226.6(b)(2)(ii), (b)(2)(iii), or (b)(2)(xii) on a credit card account under an open-end (not home-secured) consumer credit plan. (b) Exceptions. A card issuer may increase...

  4. 24 CFR 236.2 - Increased distributions to certain limited distribution mortgagors.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... of 24 CFR, parts 220 to 499, or permitted by the Commissioner under this section is preempted to the... 24 Housing and Urban Development 2 2011-04-01 2011-04-01 false Increased distributions to certain limited distribution mortgagors. 236.2 Section 236.2 Housing and Urban Development Regulations Relating...

  5. 24 CFR 236.2 - Increased distributions to certain limited distribution mortgagors.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... of 24 CFR, parts 220 to 499, or permitted by the Commissioner under this section is preempted to the... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Increased distributions to certain limited distribution mortgagors. 236.2 Section 236.2 Housing and Urban Development Regulations Relating...

  6. 20 CFR 641.870 - Under what circumstances may the administrative cost limitation be increased?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Under what circumstances may the administrative cost limitation be increased? 641.870 Section 641.870 Employees' Benefits EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR PROVISIONS GOVERNING THE SENIOR COMMUNITY SERVICE EMPLOYMENT...

  7. NITROGEN LIMITATION INCREASES BREVETOXINS IN KARENIA BREVIS (DINOPHYCEAE): IMPLICATIONS FOR BLOOM TOXICITY(1).

    PubMed

    Ransom Hardison, D; Sunda, William G; Wayne Litaker, R; Shea, Damian; Tester, Patricia A

    2012-08-01

    Laboratory and field measurements of the toxin content in Karenia brevis cells vary by >4-fold. These differences have been largely attributed to genotypic variations in toxin production among strains. We hypothesized that nutrient limitation of growth rate is equally or more important in controlling the toxicity of K. brevis, as has been documented for other toxic algae. To test this hypothesis, we measured cellular growth rate, chlorophyll a, cellular carbon and nitrogen, cell volume, and brevetoxins in four strains of K. brevis grown in nutrient-replete and nitrogen (N)-limited semi-continuous cultures. N-limitation resulted in reductions of chlorophyll a, growth rate, volume per cell and nirtogen:carbon (N:C) ratios as well as a two-fold increase (1%-4% to 5%-9%) in the percentage of cellular carbon present as brevetoxins. The increase in cellular brevetoxin concentrations was consistent among genetically distinct strains. Normalizing brevetoxins to cellular volume instead of per cell eliminated much of the commonly reported toxin variability among strains. These results suggest that genetically linked differences in cellular volume may affect the toxin content of K. brevis cells as much or more than innate genotypic differences in cellular toxin content per unit of biomass. Our data suggest at least some of the >4-fold difference in toxicity per cell reported from field studies can be explained by limitation by nitrogen or other nutrients and by differences in cell size. The observed increase in brevetoxins in nitrogen limited cells is consistent with the carbon:nutrient balance hypothesis for increases in toxins and other plant defenses under nutrient limitation. PMID:27008996

  8. Increased Toxicity of Karenia brevis during Phosphate Limited Growth: Ecological and Evolutionary Implications

    PubMed Central

    Hardison, Donnie Ransom; Sunda, William G.; Shea, Damian; Litaker, Richard Wayne

    2013-01-01

    Karenia brevis is the dominant toxic red tide algal species in the Gulf of Mexico. It produces potent neurotoxins (brevetoxins [PbTxs]), which negatively impact human and animal health, local economies, and ecosystem function. Field measurements have shown that cellular brevetoxin contents vary from 1–68 pg/cell but the source of this variability is uncertain. Increases in cellular toxicity caused by nutrient-limitation and inter-strain differences have been observed in many algal species. This study examined the effect of P-limitation of growth rate on cellular toxin concentrations in five Karenia brevis strains from different geographic locations. Phosphorous was selected because of evidence for regional P-limitation of algal growth in the Gulf of Mexico. Depending on the isolate, P-limited cells had 2.3- to 7.3-fold higher PbTx per cell than P-replete cells. The percent of cellular carbon associated with brevetoxins (%C-PbTx) was ∼ 0.7 to 2.1% in P-replete cells, but increased to 1.6–5% under P-limitation. Because PbTxs are potent anti-grazing compounds, this increased investment in PbTxs should enhance cellular survival during periods of nutrient-limited growth. The %C-PbTx was inversely related to the specific growth rate in both the nutrient-replete and P-limited cultures of all strains. This inverse relationship is consistent with an evolutionary tradeoff between carbon investment in PbTxs and other grazing defenses, and C investment in growth and reproduction. In aquatic environments where nutrient supply and grazing pressure often vary on different temporal and spatial scales, this tradeoff would be selectively advantageous as it would result in increased net population growth rates. The variation in PbTx/cell values observed in this study can account for the range of values observed in the field, including the highest values, which are not observed under N-limitation. These results suggest P-limitation is an important factor regulating cellular

  9. Direct intracerebral delivery of a miR-33 antisense oligonucleotide into mouse brain increases brain ABCA1 expression. [Corrected].

    PubMed

    Jan, Asad; Karasinska, Joanna M; Kang, Martin H; de Haan, Willeke; Ruddle, Piers; Kaur, Achint; Connolly, Colum; Leavitt, Blair R; Sorensen, Poul H; Hayden, Michael R

    2015-06-26

    The ATP-binding cassette transporter A1 (ABCA1) is a membrane bound protein that serves to efflux cholesterol and phospholipids onto lipid poor apolipoproteins during HDL biogenesis. Increasing the expression and activity of ABCA1 have beneficial effects in experimental models of various neurologic and cardiovascular diseases including Alzheimer's disease. Despite the beneficial effects of liver X receptor (LXR) agonists--compounds that increase ABCA1 expression--in preclinical studies, their therapeutic utility is limited by systemic adverse effects on lipid metabolism. Interestingly, microRNA-33 (miR-33) inhibition increases ABCA1 expression and activity in rodents and non-human primates without severe metabolic adverse effects. Herein, we demonstrate that treatment of cultured mouse neurons, astrocytes and microglia with an antisense oligonucleotide (ASO) targeting miR-33 increased ABCA1 expression, which was accompanied by increased cholesterol efflux and apoE secretion in astrocytic cultures. We also show that intracerebral delivery of an ASO targeting miR-33 leads to increased ABCA1 expression in cerebral cortex or subcortical structures such as hippocampus. These findings highlight an effective strategy for increasing brain ABCA1 expression/activity for relevant mechanistic studies. [Corrected] PMID:25957561

  10. Hyperoxia increases hepatic arginase expression and ornithine production in mice

    SciTech Connect

    Malleske, Daniel T.; Rogers, Lynette K.; Velluci, Sean M.; Young, Tamara L.; Park, Min S.; Long, Donald W.; Welty, Stephen E.; Smith, Charles V.; Nelin, Leif D. . E-mail: NelinL@pediatrics.ohio-state.edu

    2006-08-15

    Hyperoxic exposure affects the levels and activities of some hepatic proteins. We tested the hypothesis that hyperoxic exposure would result in greater hepatic .NO concentrations. C3H/HeN mice were exposed to >95% O{sub 2} for 72 or 96 h and compared to room air-breathing controls. In contrast to our working hypothesis, exposure to >95% O{sub 2} for 96 h decreased hepatic nitrite/nitrate NO {sub X} concentrations (10.9 {+-} 2.2 nmol/g liver versus 19.3 {+-} 2.4 nmol/g liver in room air, P < 0.05). The hepatic levels of endothelial NO synthase (eNOS) and inducible NOS (iNOS) proteins were not different among the groups. The arginases, which convert L-arginine to urea and L-ornithine, may affect hepatic NOS activities by decreasing L-arginine bioavailability. Hepatic ornithine concentrations were greater in hyperoxic animals than in controls (318 {+-} 18 nmol/g liver in room air, and 539 {+-} 64, and 475 {+-} 40 at 72 and 96 h of hyperoxia, respectively, P < 0.01). Hepatic arginase I protein levels were greater in hyperoxic animals than in controls. Hepatic carbamoyl phosphate synthetase (CPS) protein levels and activities were not different among groups. These results indicate that increases in hepatic levels of arginase I in mice exposed to hyperoxia may diminish .NO production, as reflected by lower liver levels of NO {sub X}. The resultant greater hepatic ornithine concentrations may represent a mechanism to facilitate tissue repair, by favoring the production of polyamines and/or proline.

  11. Increased expression of the multidrug efflux genes acrAB occurs during slow growth of Escherichia coli.

    PubMed

    Rand, Jonathan D; Danby, Simon G; Greenway, David L A; England, Reg R

    2002-01-22

    Intrinsic antimicrobial resistance of Escherichia coli is elicited by the gene products of the multidrug efflux acrAB-tolC operon. In this paper, we have shown that acrAB is regulated as a function of the growth rate of E. coli during growth in batch and chemostat culture. In chemostat culture, expression of acrAB is inversely related to growth rate irrespective of the limiting nutrient. The level of expression of acrAB is greater under glucose limitation compared with either iron or nitrogen limitation. Increase in expression of acrAB confers a greater resistance to ciprofloxacin, and the implications for a clinical situation are discussed. Slow growth rate regulation of acrAB transcription does not require the presence of the stationary-phase sigma factor. A putative gearbox consensus sequence was identified at the -10 region of the acrAB promoter. PMID:11886757

  12. CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression

    PubMed Central

    Liu, S-C; Chuang, S-M; Hsu, C-J; Tsai, C-H; Wang, S-W; Tang, C-H

    2014-01-01

    Connective tissue growth factor (CTGF, a.k.a. CCN2) is inflammatory mediator and abundantly expressed in osteoarthritis (OA). Angiogenesis is essential for OA progression. Here, we investigated the role of CTGF in vascular endothelial growth factor (VEGF) production and angiogenesis in OA synovial fibroblasts (OASFs). We showed that expression of CTGF and VEGF in synovial fluid were higher in OA patients than in controls. Directly applying CTGF to OASFs increased VEGF production then promoted endothelial progenitor cells tube formation and migration. CTGF induced VEGF by raising miR-210 expression via PI3K, AKT, ERK, and nuclear factor-κB (NF-κB)/ELK1 pathways. CTGF-mediating miR-210 upregulation repressed glycerol-3-phosphate dehydrogenase 1-like (GPD1L) expression and PHD activity and subsequently promoted hypoxia-inducible factor (HIF)-1α-dependent VEGF expression. Knockdown of CTGF decreased VEGF expression and abolished OASF-conditional medium-mediated angiogenesis in vitro as well as angiogenesis in chick chorioallantoic membrane and Matrigel-plug nude mice model in vivo. Taken together, our results suggest CTGF activates PI3K, AKT, ERK, and NF-κB/ELK1 pathway, leading to the upregulation of miR-210, contributing to inhibit GPD1L expression and prolyl hydroxylases 2 activity, promoting HIF-1α-dependent VEGF expression and angiogenesis in human synovial fibroblasts. PMID:25341039

  13. Radial basis function interpolation in the limit of increasingly flat basis functions

    NASA Astrophysics Data System (ADS)

    Kindelan, Manuel; Moscoso, Miguel; González-Rodríguez, Pedro

    2016-02-01

    We propose a new approach to study Radial Basis Function (RBF) interpolation in the limit of increasingly flat functions. The new approach is based on the semi-analytical computation of the Laurent series of the inverse of the RBF interpolation matrix described in a previous paper [3]. Once the Laurent series is obtained, it can be used to compute the limiting polynomial interpolant, the optimal shape parameter of the RBFs used for interpolation, and the weights of RBF finite difference formulas, among other things.

  14. Limited up-regulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation.

    PubMed Central

    Lee, P J; Washer, L L; Law, D J; Boland, C R; Horon, I L; Feinberg, A P

    1996-01-01

    Epigenetic alterations in the genome of tumor cells have attracted considerable attention since the discovery of widespread alterations in DNA methylation of colorectal cancers over 10 years ago. However, the mechanism of these changes has remained obscure. el-Deiry and coworkers [el-Deiry, W. S., Nelkin, B. D., Celano, P., Yen, R. C., Falco, J. P., Hamilton, S. R. & Baylin, S. B. (1991) Proc. Natl. Acad. Sci. USA 88, 3470-3474], using a quantitative reverse transcription-PCR assay, reported 15-fold increased expression of DNA methyltransferase (MTase) in colon cancer, compared with matched normal colon mucosa, and a 200-fold increase in MTase mRNA levels compared with mucosa of unaffected patients. These authors suggested that increases in MTase mRNA levels play a direct pathogenetic role in colon carcinogenesis. To test this hypothesis, we developed a sensitive quantitative RNase protection assay of MTase, linear over three orders of magnitude. Using this assay on 12 colorectal carcinomas and matched normal mucosal specimens, we observed a 1.8- to 2.5-fold increase in MTase mRNA levels in colon carcinoma compared with levels in normal mucosa from the same patients. There was no significant difference between the normal mucosa of affected and unaffected patients. Furthermore, when the assay was normalized to histone H4 expression, a measure of S-phase-specific expression, the moderate increase in tumor MTase mRNA levels was no longer observed. These data are in contrast to the previously reported results, and they indicate that changes in MTase mRNA levels in colon cancer are nonspecific and compatible with other markers of cell proliferation. Images Fig. 2 PMID:8816806

  15. Cadmium exposure and phosphorus limitation increases metal content in the freshwater alga Chlamydomonas reinhardtii.

    PubMed

    Webster, Rachel E; Dean, Andrew P; Pittman, Jon K

    2011-09-01

    The characteristics of metal accumulation in freshwater microalgae are important to elucidate for a full understanding of metal cycling and toxicity in a freshwater system. This study has utilized an elemental profiling approach to investigate the impacts of Cd exposure and phosphorus (P) availability on metal accumulation after 7 days in batch culture-grown Chlamydomonas reinhardtii. Multivariate statistical analysis of the elemental data demonstrated distinct responses between both stresses. Sublethal concentrations of Cd (up to 15 μM) caused increased accumulation of Co. Cu, Fe, and Zn content also increased in response to enhanced Cd concentrations but only when P availability was low. While Cd exposure effected the accumulation of a few specific metals, P limitation increased the accumulation of all essential trace metals and macronutrients analyzed including Co, Fe, K, Na, and Zn but not Mn. The accumulation of Cd also markedly increased in response to P limitation. The impact of P availability on essential metal accumulation was the same when either inorganic P or an organic P source (glycerophosphate) was used. These results highlight the potential risks of metal toxicity for freshwater microalgae and aquatic food chains when P availability is limiting and which can be exacerbated by Cd pollution. PMID:21809879

  16. Mild Airflow Limitation during N2 Sleep Increases K-complex Frequency and Slows Electroencephalographic Activity

    PubMed Central

    Nguyen, Chinh D.; Wellman, Andrew; Jordan, Amy S.; Eckert, Danny J.

    2016-01-01

    Study Objectives: To determine the effects of mild airflow limitation on K-complex frequency and morphology and electroencephalogram (EEG) spectral power. Methods: Transient reductions in continuous positive airway pressure (CPAP) during stable N2 sleep were performed to induce mild airflow limitation in 20 patients with obstructive sleep apnea (OSA) and 10 healthy controls aged 44 ± 13 y. EEG at C3 and airflow were measured in 1-min windows to quantify K-complex properties and EEG spectral power immediately before and during transient reductions in CPAP. The frequency and morphology (amplitude and latency of P200, N550 and N900 components) of K-complexes and EEG spectral power were compared between conditions. Results: During mild airflow limitation (18% reduction in peak inspiratory airflow from baseline, 0.38 ± 0.11 versus 0.31 ± 0.1 L/sec) insufficient to cause American Academy of Sleep Medicine-defined cortical arousal, K-complex frequency (9.5 ± 4.5 versus 13.7 ± 6.4 per min, P < 0.01), N550 amplitude (25 ± 3 versus 27 ± 3 μV, P < 0.01) and EEG spectral power (delta: 147 ± 48 versus 230 ± 99 μV2, P < 0.01 and theta bands: 31 ± 14 versus 34 ± 13 μV2, P < 0.01) significantly increased whereas beta band power decreased (14 ± 5 versus 11 ± 4 μV2, P < 0.01) compared to the preceding non flow-limited period on CPAP. K-complex frequency, morphology, and timing did not differ between patients and controls. Conclusion: Mild airflow limitation increases K-complex frequency, N550 amplitude, and spectral power of delta and theta bands. In addition to providing mechanistic insight into the role of mild airflow limitation on K-complex characteristics and EEG activity, these findings may have important implications for respiratory conditions in which airflow limitation during sleep is common (e.g., snoring and OSA). Citation: Nguyen CD, Wellman A, Jordan AS, Eckert DJ. Mild airflow limitation during N2 sleep increases k-complex frequency and slows

  17. Aggravated phosphorus limitation on biomass production under increasing nitrogen loading: a meta-analysis.

    PubMed

    Li, Yong; Niu, Shuli; Yu, Guirui

    2016-02-01

    Nitrogen (N) and phosphorus (P), either individually or in combination, have been demonstrated to limit biomass production in terrestrial ecosystems. Field studies have been extensively synthesized to assess global patterns of N impacts on terrestrial ecosystem processes. However, to our knowledge, no synthesis has been done so far to reveal global patterns of P impacts on terrestrial ecosystems, especially under different nitrogen (N) levels. Here, we conducted a meta-analysis of impacts of P addition, either alone or with N addition, on aboveground (AGB) and belowground biomass production (BGB), plant and soil P concentrations, and N : P ratio in terrestrial ecosystems. Overall, our meta-analysis quantitatively confirmed existing notions: (i) colimitation of N and P on biomass production and (ii) more P limitation in tropical forest than other ecosystems. More importantly, our analysis revealed new findings: (i) P limitation on biomass production was aggravated by N enrichment and (ii) plant P concentration was a better indicator of P limitation than soil P availability. Specifically, P addition increased AGB and BGB by 34% and 13%, respectively. The effect size of P addition on biomass production was larger in tropical forest than grassland, wetland, and tundra and varied with P fertilizer forms, P addition rates, or experimental durations. The P-induced increase in biomass production and plant P concentration was larger under elevated than ambient N. Our findings suggest that the global limitation of P on biomass production will become severer under increasing N fertilizer and deposition in the future. PMID:26463578

  18. Pushing the Photon Limit: Nanoantennas Increase Maximal Photon Stream and Total Photon Number

    PubMed Central

    2016-01-01

    Nanoantennas are well-known for their effective role in fluorescence enhancement, both in excitation and emission. Enhancements of 3–4 orders of magnitude have been reported. Yet in practice, the photon emission is limited by saturation due to the time that a molecule spends in singlet and especially triplet excited states. The maximal photon stream restricts the attainable enhancement. Furthermore, the total number of photons emitted is limited by photobleaching. The limited brightness and observation time are a drawback for applications, especially in biology. Here we challenge this photon limit, showing that nanoantennas can actually increase both saturation intensity and photostability. So far, this limit-shifting role of nanoantennas has hardly been explored. Specifically, we demonstrate that single light-harvesting complexes, under saturating excitation conditions, show over a 50-fold antenna-enhanced photon emission stream, with 10-fold more total photons, up to 108 detected photons, before photobleaching. This work shows yet another facet of the great potential of nanoantennas in the world of single-molecule biology. PMID:27082249

  19. Expression of Nemo-like kinase was increased and negatively correlated with the expression of TCF4 in lung cancers

    PubMed Central

    Zhang, Xiu-Wei; Chen, Song-Yan; Xue, Dong-Wei; Xu, Hui-Hui; Yang, Lian-He; Xu, Hong-Tao; Wang, En-Hua

    2015-01-01

    Nemo-like kinase (NLK), as a mitogen activated protein kinase (MAPK)-like kinase, is involved in the development of several human cancers. In this study, we explored the expression of NLK in lung squamous cell carcinoma (SCC) and adenocarcinoma tissues, and investigated the associations among NLK, β-catenin, T-cell factor 4 (TCF4), and the clinicopathological factors of lung cancers. The expressions of NLK, β-catenin, TCF4 were examined in 109 cases of lung cancers using immunohistochemistry method. The expression of NLK was observed in the nuclei of lung cancer tissues, and was significantly higher in lung cancer tissues than that in corresponding normal lung tissues (t = 21.636, n = 109, P < 0.001). The high expression of NLK was found in 45 cases of lung SCCs (45/49, 91.84%), which was much more than that in adenocarcinomas (38/60, 63.33%) (P = 0.001). Furthermore, the high expression of NLK was negatively correlated with TCF4 expression and positively correlated with the membranous expression of β-catenin. In conclusion, the present study demonstrated that the expression of NLK was localized in nucleus and significantly increased in lung cancers. The expression of NLK was negatively correlated with TCF4 expression and positively correlated with β-catenin membranous expression in lung cancers. PMID:26823848

  20. Elevated carbon dioxide increases soil nitrogen and phosphorus availability in a phosphorus-limited Eucalyptus woodland.

    PubMed

    Hasegawa, Shun; Macdonald, Catriona A; Power, Sally A

    2016-04-01

    Free-air CO2 enrichment (FACE) experiments have demonstrated increased plant productivity in response to elevated (e)CO2, with the magnitude of responses related to soil nutrient status. Whilst understanding nutrient constraints on productivity responses to eCO2 is crucial for predicting carbon uptake and storage, very little is known about how eCO2 affects nutrient cycling in phosphorus (P)-limited ecosystems. Our study investigates eCO2 effects on soil N and P dynamics at the EucFACE experiment in Western Sydney over an 18-month period. Three ambient and three eCO2 (+150 ppm) FACE rings were installed in a P-limited, mature Cumberland Plain Eucalyptus woodland. Levels of plant accessible nutrients, evaluated using ion exchange resins, were increased under eCO2, compared to ambient, for nitrate (+93%), ammonium (+12%) and phosphate (+54%). There was a strong seasonality to responses, particularly for phosphate, resulting in a relatively greater stimulation in available P, compared to N, under eCO2 in spring and summer. eCO2 was also associated with faster nutrient turnover rates in the first six months of the experiment, with higher N (+175%) and P (+211%) mineralization rates compared to ambient rings, although this difference did not persist. Seasonally dependant effects of eCO2 were seen for concentrations of dissolved organic carbon in soil solution (+31%), and there was also a reduction in bulk soil pH (-0.18 units) observed under eCO2. These results demonstrate that CO2 fertilization increases nutrient availability - particularly for phosphate - in P-limited soils, likely via increased plant belowground investment in labile carbon and associated enhancement of microbial turnover of organic matter and mobilization of chemically bound P. Early evidence suggests that there is the potential for the observed increases in P availability to support increased ecosystem C-accumulation under future predicted CO2 concentrations. PMID:26546164

  1. Expression of β-glucosidase increases trichome density and artemisinin content in transgenic Artemisia annua plants.

    PubMed

    Singh, Nameirakpam Dolendro; Kumar, Shashi; Daniell, Henry

    2016-03-01

    Artemisinin is highly effective against multidrug-resistant strains of Plasmodium falciparum, the aetiological agent of the most severe form of malaria. However, a low level of accumulation of artemisinin in Artemisia annua is a major limitation for its production and delivery to malaria endemic areas of the world. While several strategies to enhance artemisinin have been extensively explored, enhancing storage capacity in trichome has not yet been considered. Therefore, trichome density was increased with the expression of β-glucosidase (bgl1) gene in A. annua through Agrobacterium-mediated transformation. Transgene (bgl1) integration and transcript were confirmed by molecular analysis. Trichome density increased up to 20% in leaves and 66% in flowers of BGL1 transgenic plants than Artemisia control plants. High-performance liquid chromatography, time of flight mass spectrometer data showed that artemisinin content increased up to 1.4% in leaf and 2.56% in flowers (per g DW), similar to the highest yields achieved so far through metabolic engineering. Artemisinin was enhanced up to five-fold in BGL1 transgenic flowers. This study opens the possibility of increasing artemisinin content by manipulating trichomes' density, which is a major reservoir of artemisinin. Combining biosynthetic pathway engineering with enhancing trichome density may further increase artemisinin yield in A. annua. Because oral feeding of Artemisia plant cells reduced parasitemia more efficiently than the purified drug, reduced drug resistance and cost of prohibitively expensive purification process, enhanced expression should play a key role in making this valuable drug affordable to treat malaria in a large global population that disproportionally impacts low-socioeconomic areas and underprivileged children. PMID:26360801

  2. Increases in the evolutionary potential of upper thermal limits under warmer temperatures in two rainforest Drosophila species.

    PubMed

    van Heerwaarden, Belinda; Malmberg, Michelle; Sgrò, Carla M

    2016-02-01

    Tropical and subtropical species represent the majority of biodiversity. These species are predicted to lack the capacity to evolve higher thermal limits in response to selection imposed by climatic change. However, these assessments have relied on indirect estimates of adaptive capacity, using conditions that do not reflect environmental changes projected under climate change. Using a paternal half-sib full-sib breeding design, we estimated the additive genetic variance and narrow-sense heritability for adult upper thermal limits in two rainforest-restricted species of Drosophila reared under two thermal regimes, reflecting increases in seasonal temperature projected for the Wet Tropics of Australia and under standard laboratory conditions (constant 25°C). Estimates of additive genetic variation and narrow-sense heritability for adult heat tolerance were significantly different from zero in both species under projected summer, but not winter or constant, thermal regimes. In contrast, significant broad-sense genetic variation was apparent in all thermal regimes for egg-to-adult viability. Environment-dependent changes in the expression of genetic variation for adult upper thermal limits suggest that predicting adaptive responses to climate change will be difficult. Estimating adaptive capacity under conditions that do not reflect future environmental conditions may provide limited insight into evolutionary responses to climate change. PMID:26703976

  3. Effect of Drought on Herbivore-Induced Plant Gene Expression: Population Comparison for Range Limit Inferences

    PubMed Central

    Gill, Gunbharpur Singh; Haugen, Riston; Matzner, Steven L.; Barakat, Abdelali; Siemens, David H.

    2016-01-01

    Low elevation “trailing edge” range margin populations typically face increases in both abiotic and biotic stressors that may contribute to range limit development. We hypothesize that selection may act on ABA and JA signaling pathways for more stable expression needed for range expansion, but that antagonistic crosstalk prevents their simultaneous co-option. To test this hypothesis, we compared high and low elevation populations of Boechera stricta that have diverged with respect to constitutive levels of glucosinolate defenses and root:shoot ratios; neither population has high levels of both traits. If constraints imposed by antagonistic signaling underlie this divergence, one would predict that high constitutive levels of traits would coincide with lower plasticity. To test this prediction, we compared the genetically diverged populations in a double challenge drought-herbivory growth chamber experiment. Although a glucosinolate defense response to the generalist insect herbivore Spodoptera exigua was attenuated under drought conditions, the plastic defense response did not differ significantly between populations. Similarly, although several potential drought tolerance traits were measured, only stomatal aperture behavior, as measured by carbon isotope ratios, was less plastic as predicted in the high elevation population. However, RNAseq results on a small subset of plants indicated differential expression of relevant genes between populations as predicted. We suggest that the ambiguity in our results stems from a weaker link between the pathways and the functional traits compared to transcripts. PMID:27135233

  4. Effect of Drought on Herbivore-Induced Plant Gene Expression: Population Comparison for Range Limit Inferences.

    PubMed

    Gill, Gunbharpur Singh; Haugen, Riston; Matzner, Steven L; Barakat, Abdelali; Siemens, David H

    2016-01-01

    Low elevation "trailing edge" range margin populations typically face increases in both abiotic and biotic stressors that may contribute to range limit development. We hypothesize that selection may act on ABA and JA signaling pathways for more stable expression needed for range expansion, but that antagonistic crosstalk prevents their simultaneous co-option. To test this hypothesis, we compared high and low elevation populations of Boechera stricta that have diverged with respect to constitutive levels of glucosinolate defenses and root:shoot ratios; neither population has high levels of both traits. If constraints imposed by antagonistic signaling underlie this divergence, one would predict that high constitutive levels of traits would coincide with lower plasticity. To test this prediction, we compared the genetically diverged populations in a double challenge drought-herbivory growth chamber experiment. Although a glucosinolate defense response to the generalist insect herbivore Spodoptera exigua was attenuated under drought conditions, the plastic defense response did not differ significantly between populations. Similarly, although several potential drought tolerance traits were measured, only stomatal aperture behavior, as measured by carbon isotope ratios, was less plastic as predicted in the high elevation population. However, RNAseq results on a small subset of plants indicated differential expression of relevant genes between populations as predicted. We suggest that the ambiguity in our results stems from a weaker link between the pathways and the functional traits compared to transcripts. PMID:27135233

  5. Tipping the mutation-selection balance: Limited migration increases the frequency of deleterious mutants.

    PubMed

    Cooper, Jacob D; Neuhauser, Claudia; Dean, Antony M; Kerr, Benjamin

    2015-09-01

    Typical mutation-selection models assume well-mixed populations, but dispersal and migration within many natural populations is spatially limited. Such limitations can lead to enhanced variation among locations as different types become clustered in different places. Such clustering weakens competition between unlike types relative to competition between like types; thus, the rate by which a fitter type displaces an inferior competitor can be affected by the spatial scale of movement. In this paper, we use a birth-death model to show that limited migration can affect asexual populations by creating competitive refugia. We use a moment closure approach to show that as population structure is introduced by limiting migration, the equilibrial frequency of deleterious mutants increases. We support and extend the model through stochastic simulation, and we use a spatially explicit cellular automaton approach to corroborate the results. We discuss the implications of these results for standing variation in structured populations and adaptive valley crossing in Wright's "shifting balance" process. PMID:25983046

  6. Increased risk of phosphorus limitation at higher temperatures for Daphnia magna.

    PubMed

    Persson, Jonas; Wojewodzic, Marcin Włodzimierz; Hessen, Dag Olav; Andersen, Tom

    2011-01-01

    Invertebrate herbivores frequently face growth rate constraints due to their high demands for phosphorus (P) and nitrogen (N). Temperature is a key modulator of growth rate, yet the interaction between temperature and P limitation on somatic growth rate is scarcely known. To investigate this interaction, we conducted a study on the somatic growth rate (SGR) of the cladoceran Daphnia magna, known to be susceptible to P-limitation. We determined the SGR across a broad range of dietary P content of algae (carbon (C):P ratios (125-790), and at different temperatures (10-25°C). There was a strong impact of both temperature and C:P ratio on the SGR of D. magna, and also a significant interaction between both factors was revealed. The negative effect of dietary C:P on growth rate was reduced with decreased temperature. We found no evidence of P limitation at lowest temperature, suggesting that enzyme kinetics or other measures of food quality overrides the demands for P to RNA and protein synthesis at low temperatures. These findings also indicate an increased risk of P limitation and thus reduced growth efficiency at high temperatures. PMID:20803219

  7. Effects of increased hypothalamic leptin gene expression on ovariectomy-induced bone loss in rats

    PubMed Central

    Jackson, M.A.; Iwaniec, U.T.; Turner, R.T.; Wronski, T.J.; Kalra, S.P.

    2011-01-01

    Estrogen deficiency results in accelerated bone turnover with a net increase in bone resorption. Subcutaneous administration of leptin attenuates bone loss in ovariectomized (ovx) rats by reducing bone resorption. However, in addition to its direct beneficial effects, leptin has been reported to have indirect (central nervous system-mediated) antiosteogenic effects on bone, which may limit the efficacy of elevated serum leptin to prevent estrogen deficiency-associated bone loss. The present study evaluated the long-term effects of increased hypothalamic leptin transgene expression, using recombinant adeno-associated virus-leptin (rAAV-Lep) gene therapy, on bone mass, architecture, and cellular endpoints in sexually mature ovx Sprague-Dawley rats. Ovx rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained for 10 weeks. Additional controls consisted of ovary-intact rats and ovx rats pair-fed to rAAV-Lep rats. Lumbar vertebrae were analyzed by micro-computed tomography and tibiae by histomorphometry. Cancellous bone volume was lower and osteoclast perimeter, osteoblast perimeter, and bone marrow adipocyte density were greater in ovx rats compared to ovary-intact controls. In contrast, differences among ovx groups were not detected for any endpoint evaluated. In conclusion, whereas estrogen deficiency resulted in marked cancellous osteopenia, increased bone turnover and marrow adiposity, increasing hypothalamic leptin transgene expression in ovx rats had neither detrimental nor beneficial effects on bone mass, architecture, or cellular endpoints. These findings demonstrate that the antiresorptive effects of subcutaneous leptin administration in ovx rats are mediated through leptin targets in the periphery. PMID:21640774

  8. A cautionary note on cosmetics containing ingredients that increase aquaporin-3 expression.

    PubMed

    Verkman, A S

    2008-10-01

    Aquaporin-3 (AQP3) is a membrane transport protein that facilitates water and glycerol transport across cell plasma membranes in the basal layer of keratinocytes in normal skin. Motivated by a relation between AQP3 expression and skin water content, several companies have marketed cosmetics containing ingredients that increase AQP3 expression. However, caution seems warranted in targeting AQP3 to increase skin moisturization based on a recently discovered association in mice between epidermal AQP3 expression and skin tumor formation. PMID:18312385

  9. Increasing P limitation and viral infection impact lipid remodeling of the picophytoplankter Micromonas pusilla

    NASA Astrophysics Data System (ADS)

    Maat, Douwe S.; Bale, Nicole J.; Hopmans, Ellen C.; Sinninghe Damsté, Jaap S.; Schouten, Stefan; Brussaard, Corina P. D.

    2016-03-01

    The intact polar lipid (IPL) composition of phytoplankton is plastic and dependent on environmental factors. Previous studies have shown that phytoplankton under low phosphorus (P) availability substitutes phosphatidylglycerols (PGs) with sulfoquinovosyldiacylglycerols (SQDGs) and digalactosyldiacylglycerols (DGDGs). However, these studies focused merely on P depletion, while phytoplankton in the natural environment often experience P limitation whereby the strength depends on the supply rate of the limiting nutrient. Here we report on the IPL composition of axenic cultures of the picophotoeukaryote Micromonas pusilla under different degrees of P limitation, i.e., P-controlled chemostats at 97 and 32 % of the maximum growth rate, and P starvation (obtained by stopping P supply to these chemostats). P-controlled cultures were also grown at elevated partial carbon dioxide pressure (pCO2) to mimic a future scenario of strengthened vertical stratification in combination with ocean acidification. Additionally, we tested the influence of viral infection for this readily infected phytoplankton host species. Results show that both SQDG : PG and DGDG : PG ratios increased with enhanced P limitation. Lipid composition was, however, not affected by enhanced (750 vs. 370 µatm) pCO2. In the P-starved virally infected cells the increase in SQDG : PG and DGDG : PG ratios was lower, whereby the extent depended on the growth rate of the host cultures before infection. The lipid membrane of the virus MpV-08T itself lacked some IPLs (e.g., monogalactosyldiacylglycerols; MGDGs) in comparison with its host. This study demonstrates that, besides P concentration, also the P supply rate, viral infection and even the history of the P supply rate can affect phytoplankton lipid composition (i.e., the non-phospholipid : phospholipid ratio), with possible consequences for the nutritional quality of phytoplankton.

  10. Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

    SciTech Connect

    Takahashi, Nobuhiko; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. Black-Right-Pointing-Pointer Adipose lipin-1 expression is reduced in obesity. Black-Right-Pointing-Pointer Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. Black-Right-Pointing-Pointer Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-{kappa}B activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

  11. Late-Postnatal Cannabinoid Exposure Persistently Increases FoxP2 Expression within Zebra Finch Striatum

    PubMed Central

    Soderstrom, Ken; Luo, Bin

    2010-01-01

    Prior work has shown that cannabinoid exposure of zebra finches during sensorimotor stages of vocal development alters song patterns produced in adulthood. We are currently working to identify physiological substrates for this altered song learning. FoxP2 is a transcription factor associated with altered vocal development in both zebra finches and humans. This protein shows a distinct pattern of expression within Area X of striatum that coincides with peak expression of CB1 cannabinoid receptors during sensorimotor learning. Coincident expression in a brain region essential for song learning led us to test for a potential signaling interaction. We have found that cannabinoid agonists acutely increase expression of FoxP2 throughout striatum. When administered during sensorimotor song learning, cannabinoids increase basal levels of striatal FoxP2 expression in adulthood. Thus, song-altering cannabinoid treatments are associated with persistent increases in basal expression of FoxP2 in zebra finch striatum. PMID:20017118

  12. Ethanol increases osteoclastogenesis associated with the increased expression of RANK, PU.1 and MITF in vitro and in vivo.

    PubMed

    Iitsuka, Natsumi; Hie, Mamiko; Nakanishi, Atsuko; Tsukamoto, Ikuyo

    2012-07-01

    Ethanol has been known to induce osteopenia. However, the cellular and molecular mechanisms responsible for its effect have not been well characterized. This study investigated the effects of ethanol on bone metabolism and osteoclastogenesis using rats fed an ethanol-containing liquid diet (35% of calories from ethanol) for 3 weeks. Ethanol increased the activities of bone tartrate-resistant acid phosphatase (TRAP) and cathepsin K, without affecting the levels of serum osteocalcin or bone alkaline phosphatase activity. Histological analysis showed an increased number of osteoclasts in the proximal tibia, but no significant change in the number of osteoblasts. The mRNA levels of receptor for activation of NF-κB (RANK), c-fos, c-jun, TRAP and cathepsin K were significantly increased, although those of macrophage colony-stimulating factor and c-fms were unaltered. The mRNA and protein levels of PU.1 and microphthalmia-associated trascription factor (MITF) also increased. Further, the osteoclastic differentiation of bone marrow-derived macrophage/monocyte precursor cells (BMMs) in vitro was stimulated by ethanol. The increased osteoclastogenesis of BMMs was associated with increased levels of RANK, PU.1 and MITF expression, activated extracellular signal-regulated kinase (ERK), and reactive oxygen species (ROS). Higher lipid peroxide levels and lower glutathione levels were also observed in the serum of the ethanol-fed rats. These results suggested that ethanol promoted osteoclastogenesis by increasing RANK expression through increases in the production of ROS, activation of ERK and expression of PU.1 and MITF. PMID:22576626

  13. Traumatic Brain Injury Results in Disparate Regions of Chondroitin Sulfate Proteoglycan Expression That Are Temporally Limited

    PubMed Central

    Harris, N.G.; Carmichael, S.T.; Hovda, D.A.; Sutton, R.L.

    2010-01-01

    Axonal injury is a major hallmark of traumatic brain injury (TBI), and it seems likely that therapies directed toward enhancing axon repair could potentially improve functional outcomes. One potential target is chondroitin sulfate proteoglycans (CSPGs), which are major axon growth inhibitory molecules that are generally, but not always, up-regulated after central nervous system injury. The current study was designed to determine temporal changes in cerebral cortical mRNA or protein expression levels of CSPGs and to determine their regional localization and cellular association by using immunohistochemistry in a controlled cortical impact model of TBI. The results showed significant increases in versican mRNA at 4 and 14 days after TBI but no change in neurocan, aggrecan, or phosphacan. Semiquantitative Western blot (WB) analysis of cortical CSPG protein expression revealed a significant ipsilateral decrease of all CSPGs at 1 day after TBI. Lower CSPG protein levels were sustained until at least 14 days, after which the levels began to normalize. Immunohistochemistry data confirm previous reports of regional increases in CSPG proteins after CNS injury, seen primarily within the developing glial scar after TBI, but also corroborate the WB data by revealing wide areas of pericontusional tissue that are deficient in both extracellular and perineuronal net-associated CSPGs. Given the evidence that CSPGs are largely inhibitory to axonal growth, we interpret these data to indicate a potential for regional spontaneous plasticity after TBI. If this were the case, the gradual normalization of CSPG proteins over time postinjury would suggest that this may be temporally as well as regionally limited. PMID:19437549

  14. Using probabilistic estimation of expression residuals (PEER) to obtain increased power and interpretability of gene expression analyses.

    PubMed

    Stegle, Oliver; Parts, Leopold; Piipari, Matias; Winn, John; Durbin, Richard

    2012-03-01

    We present PEER (probabilistic estimation of expression residuals), a software package implementing statistical models that improve the sensitivity and interpretability of genetic associations in population-scale expression data. This approach builds on factor analysis methods that infer broad variance components in the measurements. PEER takes as input transcript profiles and covariates from a set of individuals, and then outputs hidden factors that explain much of the expression variability. Optionally, these factors can be interpreted as pathway or transcription factor activations by providing prior information about which genes are involved in the pathway or targeted by the factor. The inferred factors are used in genetic association analyses. First, they are treated as additional covariates, and are included in the model to increase detection power for mapping expression traits. Second, they are analyzed as phenotypes themselves to understand the causes of global expression variability. PEER extends previous related surrogate variable models and can be implemented within hours on a desktop computer. PMID:22343431

  15. Candidate genes for limiting cholestatic intestinal injury identified by gene expression profiling

    PubMed Central

    Alaish, Samuel M; Timmons, Jennifer; Smith, Alexis; Buzza, Marguerite S; Murphy, Ebony; Zhao, Aiping; Sun, Yezhou; Turner, Douglas J; Shea-Donahue, Terez; Antalis, Toni M; Cross, Alan; Dorsey, Susan G

    2013-01-01

    The lack of bile flow from the liver into the intestine can have devastating complications including hepatic failure, sepsis, and even death. This pathologic condition known as cholestasis can result from etiologies as diverse as total parenteral nutrition (TPN), hepatitis, and pancreatic cancer. The intestinal injury associated with cholestasis has been shown to result in decreased intestinal resistance, increased bacterial translocation, and increased endotoxemia. Anecdotal clinical evidence suggests a genetic predisposition to exaggerated injury. Recent animal research on two different strains of inbred mice demonstrating different rates of bacterial translocation with different mortality rates supports this premise. In this study, a microarray analysis of intestinal tissue following common bile duct ligation (CBDL) performed under general anesthesia on these same two strains of inbred mice was done with the goal of identifying the potential molecular mechanistic pathways responsible. Over 500 genes were increased more than 2.0-fold following CBDL. The most promising candidate genes included major urinary proteins (MUPs), serine protease-1-inhibitor (Serpina1a), and lipocalin-2 (LCN-2). Quantitative polymerase chain reaction (qPCR) validated the microarray results for these candidate genes. In an in vitro experiment using differentiated intestinal epithelial cells, inhibition of MUP-1 by siRNA resulted in increased intestinal epithelial cell permeability. Diverse novel mechanisms involving the growth hormone pathway, the acute phase response, and the innate immune response are thus potential avenues for limiting cholestatic intestinal injury. Changes in gene expression were at times found to be not only due to the CBDL but also due to the murine strain. Should further studies in cholestatic patients demonstrate interindividual variability similar to what we have shown in mice, then a “personalized medicine” approach to cholestatic patients may become

  16. Limiter

    DOEpatents

    Cohen, S.A.; Hosea, J.C.; Timberlake, J.R.

    1984-10-19

    A limiter with a specially contoured front face is provided. The front face of the limiter (the plasma-side face) is flat with a central indentation. In addition, the limiter shape is cylindrically symmetric so that the limiter can be rotated for greater heat distribution. This limiter shape accommodates the various power scrape-off distances lambda p, which depend on the parallel velocity, V/sub parallel/, of the impacting particles.

  17. [Four-week simulated weightlessness increases the expression of atrial natriuretic peptide in the myocardium].

    PubMed

    Zhang, Wen-Cheng; Lu, Yuan-Ming; Yang, Huai-Zhang; Xu, Peng-Tao; Chang, Hui; Yu, Zhi-Bin

    2013-04-25

    One of the major circulatory changes that occur in human during space flight and simulated weightlessness is a cerebral redistribution of body fluids, which is accompanied by an increase of blood volume in the upper body. Therefore, atrial myocardium should increase the secretion of atrial natriuretic peptide (ANP), but the researches lack common conclusion until now. The present study was to investigate the expression level of ANP in simulated weightlessness rats, and to confirm the changes of ANP by observing the associated proteins of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The tail-suspended rat model was used to simulate weightlessness. Western blots were carried out to examine the expression levels of ANP and SNARE proteins in atrial and left ventricular myocardium. The results showed that ANP expression in atrial myocardium showed an increase in 4-week tail-suspended rats (SUS) compared with that in the synchronous control rats (CON). We only detected a trace amount of ANP in the left ventricular myocardium of the CON, but found an enhanced expression of ANP in left ventricular myocardium of the SUS. Expression of VAMP-1/2 (vesicle associated SNARE) increased significantly in both atrial and left ventricular myocardium in the SUS compared with that in the CON. There was no difference of the expression of syntaxin-4 (target compartment associated SNARE) between the CON and SUS, but the expression of SNAP-23 showed an increase in atrial myocardium of the SUS compared with that in the CON. Synip and Munc-18c as regulators of SNAREs did not show significant difference between the CON and SUS. These results suggest that the expression of ANP shows an increase in atrial and left ventricular myocardium of 4-week tail-suspended rats. Enhanced expression of VAMP-1/2 associated with ANP vesicles confirms the increased expression of ANP in atrial and left ventricular myocardium. PMID:23598869

  18. Increased GADD gene expression in human colon epithelial cells exposed to deoxycholate.

    PubMed

    Scott, David W; Mutamba, Sophia; Hopkins, Robin G; Loo, George

    2005-01-01

    The colonic epithelium is often exposed to high concentrations of secondary bile acids, which stresses the epithelial cells, leading potentially to activation of stress-response genes. To examine this possibility in vitro, the purpose of this study was to determine if expression of certain growth arrest and DNA damage-inducible genes (GADD) is upregulated in human colonic epithelial cells exposed to deoxycholate (DOC). DNA macroarray screening of a small cluster of stress/apoptosis-related genes in DOC-treated HCT-116 colonocytes revealed clearly higher expression of only GADD45, which was confirmed by gene-specific relative RT-PCR analysis. Subsequently, it was found that DOC also increased GADD34 mRNA expression. However, mRNA expression of GADD153 was increased most markedly in DOC-treated HCT-116 colonocytes, which express wild-type p53. However, the upregulation of GADD34, GADD45, and GADD153 mRNA expression apparently did not require p53, based on the finding that DOC increased expression of all three GADD genes in HCT-15 colonocytes, which express mutant p53. In further studying GADD153 in particular, the effect of DOC on GADD153 mRNA was prevented by actinomycin-D (Act-D), but not by antioxidants or MAPK inhibitors. DOC also caused GADD153 protein to be expressed in close parallel with increased GADD153 mRNA expression. Induction of GADD153 protein by DOC was prevented by either anisomycin or cycloheximide. These findings suggest that DOC-induced upregulation of GADD153 mRNA expression occurred at the level of transcription without involving reactive oxygen species and MAPK signaling, and that the expression of GADD153 protein was due also to translation of pre-existing, and not just newly synthesized, mRNA. PMID:15316935

  19. Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli.

    PubMed

    Kim, Jinyeong; Kim, Seul I; Hong, Eunsoo; Ryu, Yeonwoo

    2016-11-01

    Heterologous proteins expressed in bacteria are used for numerous biotechnological applications. Escherichia coli is the most commonly used host for heterologous protein expression because of its many advantages. Researchers have been studying proteins from extremophiles heterologously expressed in E. coli because the proteins of extremophiles are strongly resistant to extreme conditions. In a previous study, a thermostable esterase Est-AF was isolated from Archaeoglobus fulgidus and expressed in E. coli. However, further studies of Est-AF were difficult owing to its low expression levels in E. coli. In this study, we used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 31.50 ± 0.35 mg/L to 61.75 ± 0.28 mg/L. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimized conditions were induction with 0.4 mM isopropyl-b-d-1-thiogalactoside (IPTG) at 37 °C for 5 h. Under these conditions, the expression level of Est-AF was increased from 31.5 ± 0.35 mg/L to 119.52 ± 0.34 mg/L. PMID:27449918

  20. Azidothymidine and cisplatin increase p14ARF expression in OVCAR-3 ovarian cancer cell line

    SciTech Connect

    Vaskivuo, Liisa; Rysae, Jaana; Koivuperae, Johanna; Myllynen, Paeivi; Vaskivuo, Tommi; Chvalova, Katerina; Serpi, Raisa; Savolainen, Eeva-Riitta; Puistola, Ulla; Vaehaekangas, Kirsi . E-mail: kirsi.vahakangas@uku.fi

    2006-10-01

    p14{sup ARF} tumor suppressor protein regulates p53 by interfering with mdm2-p53 interaction. p14{sup ARF} is activated in response to oncogenic stimuli but little is known of the responses of endogenous p14{sup ARF} to different types of cellular stress or DNA damage. Azidothymidine (AZT) is being tested in several clinical trials as an enhancer of anticancer chemotherapy. However, the knowledge of the relationship between AZT and cellular pathways, e.g. p53 pathway, is very limited. In this study, we show that AZT, cisplatin (CDDP) and docetaxel (DTX) all induce unique molecular responses in OVCAR-3 ovarian carcinoma cells carrying a mutated p53, while in A2780, ovarian carcinoma and MCF-7 breast carcinoma cells with wild type p53, all of these drugs cause similar p53 responses. We found that endogenous p14{sup ARF} protein in OVCAR-3 cells is down-regulated by DTX but induced by AZT and a short CDDP pulse treatment. In HT-29 colon carcinoma cells with a mutated p53, all treatments down-regulated p14{sup ARF} protein. Both CDDP and AZT increased the expression of p14ARF mRNA in OVCAR-3 cells. Differences in cell death induced by these drugs did not explain the differences in protein and mRNA expressions. No increase in the level of either c-Myc or H-ras oncoproteins was seen in OVCAR-3 cells after AZT or CDDP-treatment. These results suggest that p14{sup ARF} can respond to DNA damage without oncogene activation in cell lines without functional p53.

  1. Increased Expression of Regulatory T Cells and Down-Regulatory Molecules in Lepromatous Leprosy

    PubMed Central

    Palermo, Maria L.; Pagliari, Carla; Trindade, Maria Angela B.; Yamashitafuji, Tania M.; Duarte, Alberto José S.; Cacere, Camila R.; Benard, Gil

    2012-01-01

    T regulatory cells (Tregs) play an important role in the mechanism of host's failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-γ (IFN-γ) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-β (TGF-β), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-β. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth. PMID:22556091

  2. Increased expression of regulatory T cells and down-regulatory molecules in lepromatous leprosy.

    PubMed

    Palermo, Maria L; Pagliari, Carla; Trindade, Maria Angela B; Yamashitafuji, Tania M; Duarte, Alberto José S; Cacere, Camila R; Benard, Gil

    2012-05-01

    T regulatory cells (Tregs) play an important role in the mechanism of host's failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-γ (IFN-γ) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-β (TGF-β), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-β. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth. PMID:22556091

  3. PU.1 Expression in T Follicular Helper Cells Limits CD40L-Dependent Germinal Center B Cell Development.

    PubMed

    Awe, Olufolakemi; Hufford, Matthew M; Wu, Hao; Pham, Duy; Chang, Hua-Chen; Jabeen, Rukhsana; Dent, Alexander L; Kaplan, Mark H

    2015-10-15

    PU.1 is an ETS family transcription factor that is important for the development of multiple hematopoietic cell lineages. Previous work demonstrated a critical role for PU.1 in promoting Th9 development and in limiting Th2 cytokine production. Whether PU.1 has functions in other Th lineages is not clear. In this study, we examined the effects of ectopic expression of PU.1 in CD4(+) T cells and observed decreased expression of genes involved with the function of T follicular helper (Tfh) cells, including Il21 and Tnfsf5 (encoding CD40L). T cells from conditional mutant mice that lack expression of PU.1 in T cells (Sfpi1(lck-/-)) demonstrated increased production of CD40L and IL-21 in vitro. Following adjuvant-dependent or adjuvant-independent immunization, we observed that Sfpi1(lck-/-) mice had increased numbers of Tfh cells, increased germinal center B cells (GCB cells), and increased Ab production in vivo. This correlated with increased expression of IL-21 and CD40L in Tfh cells from Sfpi1(lck-/-) mice compared with control mice. Finally, although blockade of IL-21 did not affect GCB cells in Sfpi1(lck-/-) mice, anti-CD40L treatment of immunized Sfpi1(lck-/-) mice decreased GCB cell numbers and Ag-specific Ig concentrations. Together, these data indicate an inhibitory role for PU.1 in the function of Tfh cells, germinal centers, and Tfh-dependent humoral immunity. PMID:26363052

  4. Calpain activity and expression are increased in splenic inflammatory cells associated with experimental allergic encephalomyelitis.

    PubMed

    Shields, D C; Schaecher, K E; Goust, J M; Banik, N L

    1999-09-01

    Since calcium-activated neutral proteinase (calpain) activity and expression are significantly increased in activated glial/inflammatory cells in the central nervous system of animals with autoimmune demyelinating diseases, this enzyme may also play a role in peripheral organ systems in these diseases. In this study, the activity and expression of calpain and the endogenous inhibitor, calpastatin, were evaluated at transcriptional and translational levels in spleens of Lewis rats with acute experimental allergic encephalomyelitis (EAE) prior to the onset of clinical symptoms. Calpain activity and translational expression were increased by 475.5% and 44.3% respectively, on day 4 post-induction in adjuvant controls and animals with EAE. These levels remained elevated compared to normal controls on days 8 and 12. Calpastatin translational expression was similarly increased at these time points although transcriptional expression was not significantly altered at any time following induction of EAE. Likewise, transcriptional expression of mu-calpain was unchanged following induction, while small increases in m-calpain transcriptional expression were observed on days 2 and 8. Most calpain expression was observed in activated splenic macrophages at day 8 post-induction even though activated T cells were also calpain positive. In spinal cords of animals with EAE, calpain expression was significantly increased in rats with severe disease compared to those exhibiting only mild symptoms at day 12 post-induction. Thus, prior to symptomatic EAE, increased calpain activity and expression in peripheral lymphoid organs may play an important role in T cell migration and subsequent disease progression. PMID:10496171

  5. Warming rate drives microbial limitation and enzyme expression during peat decomposition

    NASA Astrophysics Data System (ADS)

    Inglett, P.; Sihi, D.; Inglett, K. S.

    2015-12-01

    Recent developments of enzyme-based decomposition models highlight the importance of enzyme kinetics with warming, but most modeling exercises are based on studies with a step-wise warming. This approach may mask the effect of temperature in controlling in-situ activities as in most ecosystems soil temperature change more gradually than air temperature. We conducted an experiment to test the effects of contrasting warming rates on the kinetics of C, N, and P degradation enzymes in subtropical peat soils. We also wanted to evaluate if the stoichiometry of enzyme kinetics shifts under contrasting warming rates and if so, how does it relate to the stoichiometry in microbial biomass. Contrasting warming rates altered microbial biomass stoichiometry leading to differing patterns of enzyme expression and microbial nutrient limitation. Activity (higher Vmax) and efficiency (lower Km) of C acquisition enzymes were greater in the step treatment; however, expressions of nutrient (N and P) acquiring enzymes were enhanced in the ramp treatment at the end of the experiment. In the step treatment, there was a typical pattern of an initial peak in the Vmax and drop in the Km for all enzyme groups followed by later adjustments. On the other hand, a consistent increase in Vmax and decline in Km of all enzyme groups were observed in the slow warming treatment. These changes were sufficient to alter microbial identity (as indicated by enzyme Km and biomass stoichiometry) with two apparently stable endpoints under contrasting warming rates. This observation resembles the concept of alternate stable states and highlights a need for improved representation of warming in models.

  6. Increased gastrin gene expression provides a physiological advantage to mice under hypoxic conditions.

    PubMed

    Laval, Marie; Baldwin, Graham S; Shulkes, Arthur; Marshall, Kathryn M

    2015-01-15

    Hypoxia, or a low concentration of O2, is encountered in humans undertaking activities such as mountain climbing and scuba diving and is important pathophysiologically as a limiting factor in tumor growth. Although data on the interplay between hypoxia and gastrins are limited, gastrin expression is upregulated by hypoxia in gastrointestinal cancer cell lines, and gastrins counterbalance hypoxia by stimulating angiogenesis in vitro and in vivo. The aim of this study was to determine if higher concentrations of the gastrin precursor progastrin are protective against hypoxia in vivo. hGAS mice, which overexpress progastrin in the liver, and mice of the corresponding wild-type FVB/N strain were exposed to normoxia or hypoxia. Iron status was assessed by measurement of serum iron parameters, real-time PCR for mRNAs encoding critical iron regulatory proteins, and Perls' stain and atomic absorption spectrometry for tissue iron concentrations. FVB/N mice lost weight at a faster rate and had higher sickness scores than hGAS mice exposed to hypoxia. Serum iron levels were lower in hGAS than FVB/N mice and decreased further when the animals were exposed to hypoxia. The concentration of iron in the liver was strikingly lower in hGAS than FVB/N mice. We conclude that increased circulating concentrations of progastrin provide a physiological advantage against systemic hypoxia in mice, possibly by increasing the availability of iron stores. This is the first report of an association between progastrin overexpression, hypoxia, and iron homeostasis. PMID:25394662

  7. The numerical response: rate of increase and food limitation in herbivores and predators.

    PubMed Central

    Bayliss, Peter; Choquenot, David

    2002-01-01

    Two types of numerical response function have evolved since Solomon first introduced the term to generalize features of Lotka-Volterra predator-prey models: (i) the demographic numerical response, which links change in consumer demographic rates to food availability; and (ii) the isocline numerical response, which links consumer abundance per se to food availability. These numerical responses are interchangeable because both recognize negative feedback loops between consumer and food abundance resulting in population regulation. We review how demographic and isocline numerical responses have been used to enhance our understanding of population regulation of kangaroos and possums, and argue that their utility may be increased by explicitly accounting for non-equilibrium dynamics (due to environmental variability and/or biological interactions) and the existence of multiple limiting factors. Interferential numerical response functions may help bridge three major historical dichotomies in population ecology (equilibrium versus non-equilibrium dynamics, extrinsic versus intrinsic regulation and demographic versus isocline numerical responses). PMID:12396515

  8. Neurotensin Decreases the Proinflammatory Status of Human Skin Fibroblasts and Increases Epidermal Growth Factor Expression

    PubMed Central

    Miguel Neves, Bruno; Cruz, Maria Teresa; Carvalho, Eugénia

    2014-01-01

    Fibroblasts colonization into injured areas during wound healing (WH) is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH. PMID:25180119

  9. Limiter

    DOEpatents

    Cohen, Samuel A.; Hosea, Joel C.; Timberlake, John R.

    1986-01-01

    A limiter with a specially contoured front face accommodates the various power scrape-off distances .lambda..sub.p, which depend on the parallel velocity, V.sub..parallel., of the impacting particles. The front face of the limiter (the plasma-side face) is flat with a central indentation. In addition, the limiter shape is cylindrically symmetric so that the limiter can be rotated for greater heat distribution.

  10. TNF-α increases endothelial progenitor cell adhesion to the endothelium by increasing bond expression and affinity

    PubMed Central

    Prisco, Anthony R.; Prisco, Michael R.; Carlson, Brian E.

    2014-01-01

    Endothelial progenitor cells (EPCs) are a rare population of cells that participate in angiogenesis. To effectively use EPCs for regenerative therapy, the mechanisms by which they participate in tissue repair must be elucidated. This study focused on the process by which activated EPCs bind to a target tissue. It has been demonstrated that EPCs can bind to endothelial cells (ECs) through the tumore necrosis factor-α (TNF-α)-regulated vascular cell adhesion molecule 1/very-late antigen 4 (VLA4) interaction. VLA4 can bind in a high or low affinity state, a process that is difficult to experimentally isolate from bond expression upregulation. To separate these processes, a new parallel plate flow chamber was built, a detachment assay was developed, and a mathematical model was created that was designed to analyze the detachment assay results. The mathematical model was developed to predict the relative expression of EPC/EC bonds made for a given bond affinity distribution. EPCs treated with TNF-α/vehicle were allowed to bind to TNF-α/vehicle-treated ECs in vitro. Bound cells were subjected to laminar flow, and the cellular adherence was quantified as a function of shear stress. Experimental data were fit to the mathematical model using changes in bond expression or affinity as the only free parameter. It was found that TNF-α treatment of ECs increased adhesion through bond upregulation, whereas TNF-α treatment of EPCs increased adhesion by increasing bond affinity. These data suggest that injured tissue could potentially increase recruitment of EPCs for tissue regeneration via the secretion of TNF-α. PMID:25539711

  11. Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells.

    PubMed

    Walter, B A; Purmessur, D; Moon, A; Occhiogrosso, J; Laudier, D M; Hecht, A C; Iatridis, J C

    2016-01-01

    The mechanical behaviour and cellular metabolism of intervertebral discs (IVDs) and articular cartilage are strongly influenced by their proteoglycan content and associated osmotic properties. This osmotic environment is a biophysical signal that changes with disease and may contribute to the elevated matrix breakdown and altered biologic response to loading observed in IVD degeneration and osteoarthritis. This study tested the hypothesis that changes in osmo-sensation by the transient receptor potential vallinoid-4 (TRPV4) ion channel occur with disease and contribute to the inflammatory environment found during degeneration. Immunohistochemistry on bovine IVDs from an inflammatory organ culture model were used to investigate if TRPV4 is expressed in the IVD and how expression changes with degeneration. Western blot, live-cell calcium imaging, and qRT-PCR were used to investigate whether osmolarity changes or tumour necrosis factor α (TNFα) regulate TRPV4 expression, and how altered TRPV4 expression influences calcium signalling and pro-inflammatory cytokine expression. TRPV4 expression correlated with TNFα expression, and was increased when cultured in reduced medium osmolarity and unaltered with TNFα-stimulation. Increased TRPV4 expression increased the calcium flux following TRPV4 activation and increased interleukin-1β (IL-1β) and IL-6 gene expression in IVD cells. TRPV4 expression was qualitatively elevated in regions of aggrecan depletion in degenerated human IVDs. Collectively, results suggest that reduced tissue osmolarity, likely following proteoglycan degradation, can increase TRPV4 signalling and enhance pro-inflammatory cytokine production, suggesting changes in TRPV4 mediated osmo-sensation may contribute to the progressive matrix breakdown in disease. PMID:27434269

  12. Cultivar Mixture Cropping Increased Water Use Efficiency in Winter Wheat under Limited Irrigation Conditions

    PubMed Central

    Wang, Yunqi; Zhang, Yinghua; Ji, Wei; Yu, Peng; Wang, Bin; Li, Jinpeng; Han, Meikun; Xu, Xuexin; Wang, Zhimin

    2016-01-01

    The effects of cultivar mixture cropping on yield, biomass, and water use efficiency (WUE) in winter wheat (Triticum aestivum L.) were investigated under non-irrigation (W0, no irrigation during growth stage), one time irrigation (W1, irrigation applied at stem elongation) and two times irrigation (W2, irrigation applied at stem elongation and anthesis) conditions. Nearly 90% of cultivar mixture cropping treatments experienced an increase in grain yield as compared with the mean of the pure stands under W0, those for W1 and W2 were 80% and 85%, respectively. Over 75% of cultivar mixture cropping treatments got greater biomass than the mean of the pure stands under the three irrigation conditions. Cultivar mixture cropping cost more water than pure stands under W0 and W1, whereas the water consumption under W2 decreased by 5.9%–6.8% as compared with pure stands. Approximately 90% of cultivar mixtures showed an increase of 5.4%–34.5% in WUE as compared with the mean of the pure stands, and about 75% of cultivar mixtures had 0.8%–28.5% higher WUE than the better pure stands under W0. Similarly, there were a majority of mixture cropping treatments with higher WUE than the mean and the better one of the pure stands under W1 and W2. On the whole, proper cultivar mixture cropping could increase yield and WUE, and a higher increase in WUE occurred under limited irrigation condition. PMID:27362563

  13. Epidermal growth factor increases LRF/Pokemon expression in human prostate cancer cells.

    PubMed

    Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K

    2011-10-01

    Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis. PMID:21640721

  14. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages.

    PubMed

    Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok; Jeong, Tae Cheon; Kim, Sang-Hyun; Park, Pil-Hoon

    2013-11-15

    Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages. PMID:23978445

  15. Limited impact on decadal-scale climate change from increased use of natural gas.

    PubMed

    McJeon, Haewon; Edmonds, Jae; Bauer, Nico; Clarke, Leon; Fisher, Brian; Flannery, Brian P; Hilaire, Jérôme; Krey, Volker; Marangoni, Giacomo; Mi, Raymond; Riahi, Keywan; Rogner, Holger; Tavoni, Massimo

    2014-10-23

    The most important energy development of the past decade has been the wide deployment of hydraulic fracturing technologies that enable the production of previously uneconomic shale gas resources in North America. If these advanced gas production technologies were to be deployed globally, the energy market could see a large influx of economically competitive unconventional gas resources. The climate implications of such abundant natural gas have been hotly debated. Some researchers have observed that abundant natural gas substituting for coal could reduce carbon dioxide (CO2) emissions. Others have reported that the non-CO2 greenhouse gas emissions associated with shale gas production make its lifecycle emissions higher than those of coal. Assessment of the full impact of abundant gas on climate change requires an integrated approach to the global energy-economy-climate systems, but the literature has been limited in either its geographic scope or its coverage of greenhouse gases. Here we show that market-driven increases in global supplies of unconventional natural gas do not discernibly reduce the trajectory of greenhouse gas emissions or climate forcing. Our results, based on simulations from five state-of-the-art integrated assessment models of energy-economy-climate systems independently forced by an abundant gas scenario, project large additional natural gas consumption of up to +170 per cent by 2050. The impact on CO2 emissions, however, is found to be much smaller (from -2 per cent to +11 per cent), and a majority of the models reported a small increase in climate forcing (from -0.3 per cent to +7 per cent) associated with the increased use of abundant gas. Our results show that although market penetration of globally abundant gas may substantially change the future energy system, it is not necessarily an effective substitute for climate change mitigation policy. PMID:25317557

  16. Gene expression analysis of tuberous sclerosis complex cortical tubers reveals increased expression of adhesion and inflammatory factors

    PubMed Central

    Boer, Karin; Crino, Peter B.; Gorter, Jan A.; Nellist, Mark; Jansen, Floor E.; Spliet, Wim G.M.; van Rijen, Peter C.; Wittink, Floyd R.A.; Breit, Timo M.; Troost, Dirk; Wadman, Wytse J.; Aronica, Eleonora

    2009-01-01

    Cortical tubers in patients with tuberous sclerosis complex are associated with disabling neurological manifestations, including intractable epilepsy. While these malformations are believed to result from the effects of TSC1 or TSC2 gene mutations, the molecular mechanisms leading to tuber formation, as well as the onset of seizures remain largely unknown. We used the Affymetrix Gene Chip platform to provide the first genome wide investigation of gene expression in surgically resected tubers, compared with histological normal perituberal tissue from the same patients or autopsy control tissue. We identified 2501 differentially expressed genes in cortical tubers compared with autopsy controls. Expression of genes associated with cell adhesion e.g., VCAM1, integrins and CD44, or with the inflammatory response, including complement factors, serpinA3, CCL2 and several cytokines, was increased in cortical tubers, whereas genes related to synaptic transmission e.g., the glial glutamate transporter GLT-1, and voltage-gated channel activity, exhibited lower expression. Gene expression in perituberal cortex was distinct from autopsy control cortex suggesting that even in the absence of tissue pathology the transcriptome is altered in TSC. Changes in gene expression yield insights into new candidate genes that may contribute to tuber formation or seizure onset, representing new targets for potential therapeutic development. PMID:19912235

  17. Prenatal stress decreases Bdnf expression and increases methylation of Bdnf exon IV in rats

    PubMed Central

    Boersma, Gretha J; Lee, Richard S; Cordner, Zachary A; Ewald, Erin R; Purcell, Ryan H; Moghadam, Alexander A; Tamashiro, Kellie L

    2014-01-01

    There is ample evidence that exposure to stress during gestation increases the risk of the offspring to develop mood disorders. Brain-derived neurotrophic factor (Bdnf) plays a critical role during neuronal development and is therefore a prime candidate to modulate neuronal signaling in adult offspring of rat dams that were stressed during gestation. In the current study, we tested the hypothesis that alterations in Bdnf expression in prenatally stressed (PNS) offspring are mediated by changes in DNA methylation in exons IV and VI of the Bdnf gene. We observed decreased Bdnf expression in the amygdala and hippocampus of prenatally stressed rats both at weaning and in adulthood. This decrease in Bdnf expression was accompanied by increased DNA methylation in Bdnf exon IV in the amygdala and hippocampus, suggesting that PNS-induced reduction in Bdnf expression may, at least in part, be mediated by increased DNA methylation of Bdnf exon IV. Expression of DNA methyltransferases (Dnmt) 1 and 3a was increased in PNS rats in the amygdala and hippocampus. Our data suggest that PNS induces decreases in Bdnf expression that may at least in part be mediated by increased DNA methylation of Bdnf exon IV. PMID:24365909

  18. Increased expression of dermatopontin and its implications for testicular dysfunction in mice

    PubMed Central

    CAI, JUN; LIU, WEIJIA; HAO, JIE; CHEN, MAOXIN; LI, GANG

    2016-01-01

    An array of specific and non-specific molecules, which are expressed in the testis, have been demonstrated to be responsible for testicular function. Our previous study revealed that dermatopontin (DPT) is expressed in Sertoli cells of the testis, however, its roles in testicular function remains somewhat elusive. In the present study, CdCl2- and busulfan-induced testicular dysfunction models were used to investigate the implications of DPT expression for testicular function. The mRNA and protein expression levels of DPT were detected using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. A negative correlation was observed between testicular damage and the expression of DPT, which suggested that an increase in DPT expression may be a marker for testicular dysfunction. This result was corroborated by the finding that transgenic mice exhibiting Sertoli cell-specific overexpression of DPT exhibited damage to their testicular morphology. Additionally, DPT overexpression in the testis affected the expression levels of claudin-11 and zonula occludens-1, which indicated that DPT may affect testicular function by affecting the integrity of the blood-testis barrier (BTB). In conclusion, the present study provided evidence to suggest that DPT may be indicative of mouse testicular dysfunction, since increased expression may be associated with damage to the BTB. PMID:26861869

  19. Increased NY-ESO-1 expression and reduced infiltrating CD3+ T cells in cutaneous melanoma.

    PubMed

    Giavina-Bianchi, Mara; Giavina-Bianchi, Pedro; Sotto, Mirian Nacagami; Muzikansky, Alona; Kalil, Jorge; Festa-Neto, Cyro; Duncan, Lyn M

    2015-01-01

    NY-ESO-1 is a cancer-testis antigen aberrantly expressed in melanomas, which may serve as a robust and specific target in immunotherapy. NY-ESO-1 antigen expression, tumor features, and the immune profile of tumor infiltrating lymphocytes were assessed in primary cutaneous melanoma. NY-ESO-1 protein was detected in 20% of invasive melanomas (16/79), rarely in in situ melanoma (1/10) and not in benign nevi (0/20). Marked intratumoral heterogeneity of NY-ESO-1 protein expression was observed. NY-ESO-1 expression was associated with increased primary tumor thickness (P = 0.007) and inversely correlated with superficial spreading melanoma (P < 0.02). NY-ESO-1 expression was also associated with reduced numbers and density of CD3+ tumor infiltrating lymphocytes (P = 0.017). When NY-ESO-1 protein was expressed, CD3+ T cells were less diffusely infiltrating the tumor and were more often arranged in small clusters (P = 0.010) or as isolated cells (P = 0.002) than in large clusters of more than five lymphocytes. No correlation of NY-ESO-1 expression with gender, age, tumor site, ulceration, lymph node sentinel status, or survival was observed. NY-ESO-1 expression in melanoma was associated with tumor progression, including increased tumor thickness, and with reduced tumor infiltrating lymphocytes. PMID:25954764

  20. PD-L1 Expression Is Increased in a Subset of Basal Type Breast Cancer Cells

    PubMed Central

    Soliman, Hatem; Khalil, Farah; Antonia, Scott

    2014-01-01

    Background Tumor cells express programmed death ligand 1 (PD-L1) and is a key immune evasion mechanism. PD-L1 expression in multiple breast cancer cell lines was evaluated to identify intrinsic differences that affect their potential for immune evasion. Methods PD-L1 expression was analyzed in six breast cancer cell lines: AU565&MCF7 (luminal), BT20&HCC1143 (basal A), MDA231&HCC38 (basal B). Surface and intracellular PD-L1 expression +/− interferon γ for 48 hours was measured by flow cytometry. PD-L1 gene expression data for all breast cancer cell lines in the Comprehensive Cell Line Encyclopedia (CCLE) was analyzed. Correlation between PD-L1 levels and clinicopathologic parameters was analyzed within Oncomine datasets. A tissue microarray containing 61 invasive breast cancer primary tumor cores was stained for PD-L1 expression and analyzed. Results Basal breast cancer cells constitutively express the highest levels of PD-L1. All cell lines increased PD-L1 expression with interferon γ, but basal B cells (MDA-231 and HCC38) demonstrated the largest increases. There were no differences in protein localization between cell lines. In the CCLE data, basal cell lines demonstrated higher mean PD-L1 expression compared to luminal cell lines. High PD-L1 expressing basal cell lines over-express genes involved in invasion, proliferation, and chemoresistance compared to low PD-L1 basal cell lines. High PD-L1 basal cell lines had lower expression of IRF2BP2 and higher STAT1 levels compared to low PD-L1 expressing cell lines. Within Oncomine datasets PDL1 mRNA levels were higher in basal type tumors. The TMA analysis demonstrated that lymph node positive cases had higher levels of PD-L1 protein expression compared to lymph node negative cases. Conclusions Basal type breast cancer (especially basal B) express greater levels of PD-L1 constitutively and with IFN γ. High PD-L1 basal cells over-express genes involved in invasion, motility, and chemoresistance. Targeting PD-L1

  1. Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

    PubMed

    Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

    2015-12-01

    Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese. PMID:26341925

  2. Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate.

    PubMed

    Tang, Xiaoyun; Zhao, Yuan Y; Dewald, Jay; Curtis, Jonathan M; Brindley, David N

    2016-04-01

    Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 μg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs. PMID:26884614

  3. Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion.

    PubMed

    Collins, Stephan C; Do, Hyun Woong; Hastoy, Benoit; Hugill, Alison; Adam, Julie; Chibalina, Margarita V; Galvanovskis, Juris; Godazgar, Mahdieh; Lee, Sheena; Goldsworthy, Michelle; Salehi, Albert; Tarasov, Andrei I; Rosengren, Anders H; Cox, Roger; Rorsman, Patrik

    2016-07-01

    The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1 In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca(2+) signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose-induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-βH2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6 These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy. PMID:26993066

  4. Increased expression of the diabetes gene SOX4 reduces insulin secretion by impaired fusion pore expansion

    PubMed Central

    Collins, Stephan C.; Do, Hyun Woong; Hastoy, Benoit; Hugill, Alison; Adam, Julie; Chibalina, Margarita V.; Galvanovskis, Juris; Godazgar, Mahdieh; Lee, Sheena; Goldsworthy, Michelle; Salehi, Albert; Tarasov, Andrei I.; Rosengren, Anders H.; Cox, Roger; Rorsman, Patrik

    2016-01-01

    The transcription factor Sox4 has been proposed to underlie the increased type-2 diabetes risk linked to an intronic SNP in CDKAL1. In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca2+ signalling and depolarization-evoked exocytosis. This paradox is explained by a 4-fold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements), in which the fusion pore connecting the granule lumen to the exterior only expands to a diameter of 2 nm that does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n=63), STXBP6 expression and GIIS correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-βH2 interfered with granule emptying and inhibited hormone release, the latter effect was reversed by silencing of STXBP6. These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy. PMID:26993066

  5. Increased FasL expression correlates with apoptotic changes in granulocytes cultured with oxidized clozapine

    SciTech Connect

    Husain, Zaheed; Almeciga, Ingrid; Delgado, Julio C.; Clavijo, Olga P.; Castro, Januario E.; Belalcazar, Viviana; Pinto, Clara; Zuniga, Joaquin; Romero, Viviana; Yunis, Edmond J. . E-mail: edmond_yunis@dfci.harvard.edu

    2006-08-01

    Clozapine has been associated with a 1% incidence of agranulocytosis. The formation of an oxidized intermediate clozapine metabolite has been implicated in direct polymorphonuclear (PMN) toxicity. We utilized two separate systems to analyze the role of oxidized clozapine in inducing apoptosis in treated cells. Human PMN cells incubated with clozapine (0-10 {mu}M) in the presence of 0.1 mM H{sub 2}O{sub 2} demonstrated a progressive decrease of surface CD16 expression along with increased apoptosis. RT-PCR analysis showed decreased CD16 but increased FasL gene expression in clozapine-treated PMN cells. No change in constitutive Fas expression was observed in treated cells. In HL-60 cells induced to differentiate with retinoic acid (RA), a similar increase in FasL expression, but no associated changes in CD16 gene expression, was observed following clozapine treatments. Our results demonstrate increased FasL gene expression in oxidized clozapine-induced apoptotic neutrophils suggesting that apoptosis in granulocytes treated with clozapine involves Fas/FasL interaction that initiates a cascade of events leading to clozapine-induced agranulocytosis.

  6. Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Frye, S. R.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

  7. ELMO1 increases expression of extracellular matrix proteins and inhibits cell adhesion to ECMs.

    PubMed

    Shimazaki, A; Tanaka, Y; Shinosaki, T; Ikeda, M; Watada, H; Hirose, T; Kawamori, R; Maeda, S

    2006-11-01

    We have previously identified the engulfment and cell motility 1 (ELMO1) as a susceptibility gene for diabetic nephropathy. To elucidate the role of ELMO1 in the pathogenesis of chronic renal injury, we examined the expression of Elmo1 in the kidney of a rat model for chronic glomerulonephritis (uninephrectomy plus anti-Thy1.1 antibody [E30] injection). We found that the expression of the Elmo1 was significantly increased in the renal cortex and glomeruli of uninephrectomized rats injected with E30 compared to controls. By in situ hybridization, the expression of Elmo1 was shown to be elevated in the diseased kidney, especially in glomerular epithelial cells. In COS cells, the overexpression of ELMO1 resulted in a substantial increase in fibronectin expression, whereas the depletion of the ELMO1 by small interfering RNA (siRNA) targeting ELMO1 significantly suppressed the fibronectin expression in ELMO1 overexpressing and control cells. We also found that the expression of integrin-linked kinase (ILK) was significantly increased in ELMO1 overexpressing cells, and the ELMO1-induced increase in fibronectin was partially, but significantly, inhibited by siRNA targeting ILK. Furthermore, we identified that the cell adhesion to ECMs was considerably inhibited in cells overexpressing ELMO1. These results suggest that the ELMO1 contributes to the development and progression of chronic glomerular injury through the dysregulation of ECM metabolism and the reduction in cell adhesive properties to ECMs. PMID:17021600

  8. Optimization of cold rolling process parameters in order to increasing rolling speed limited by chatter vibrations

    PubMed Central

    Heidari, Ali; Forouzan, Mohammad R.

    2012-01-01

    Chatter has been recognized as major restriction for the increase in productivity of cold rolling processes, limiting the rolling speed for thin steel strips. It is shown that chatter has close relation with rolling conditions. So the main aim of this paper is to attain the optimum set points of rolling to achieve maximum rolling speed, preventing chatter to occur. Two combination methods were used for optimization. First method is done in four steps: providing a simulation program for chatter analysis, preparing data from simulation program based on central composite design of experiment, developing a statistical model to relate system tendency to chatter and rolling parameters by response surface methodology, and finally optimizing the process by genetic algorithm. Second method has analogous stages. But central composite design of experiment is replaced by Taguchi method and response surface methodology is replaced by neural network method. Also a study on the influence of the rolling parameters on system stability has been carried out. By using these combination methods, new set points were determined and significant improvement achieved in rolling speed. PMID:25685398

  9. Optimization of cold rolling process parameters in order to increasing rolling speed limited by chatter vibrations.

    PubMed

    Heidari, Ali; Forouzan, Mohammad R

    2013-01-01

    Chatter has been recognized as major restriction for the increase in productivity of cold rolling processes, limiting the rolling speed for thin steel strips. It is shown that chatter has close relation with rolling conditions. So the main aim of this paper is to attain the optimum set points of rolling to achieve maximum rolling speed, preventing chatter to occur. Two combination methods were used for optimization. First method is done in four steps: providing a simulation program for chatter analysis, preparing data from simulation program based on central composite design of experiment, developing a statistical model to relate system tendency to chatter and rolling parameters by response surface methodology, and finally optimizing the process by genetic algorithm. Second method has analogous stages. But central composite design of experiment is replaced by Taguchi method and response surface methodology is replaced by neural network method. Also a study on the influence of the rolling parameters on system stability has been carried out. By using these combination methods, new set points were determined and significant improvement achieved in rolling speed. PMID:25685398

  10. Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

    SciTech Connect

    Kim, Mi Jin; Nepal, Saroj; Lee, Eung-Seok; Jeong, Tae Cheon; Kim, Sang-Hyun; Park, Pil-Hoon

    2013-11-15

    Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages. - Highlights: • Ethanol increases ROS production through up-regulation of Nox2 in macrophages. • Enhanced oxidative stress contributes to ethanol

  11. A Brucella spp. Protease Inhibitor Limits Antigen Lysosomal Proteolysis, Increases Cross-Presentation, and Enhances CD8+ T Cell Responses.

    PubMed

    Coria, Lorena M; Ibañez, Andrés E; Tkach, Mercedes; Sabbione, Florencia; Bruno, Laura; Carabajal, Marianela V; Berguer, Paula M; Barrionuevo, Paula; Schillaci, Roxana; Trevani, Analía S; Giambartolomei, Guillermo H; Pasquevich, Karina A; Cassataro, Juliana

    2016-05-15

    In this study, we demonstrate that the unlipidated (U) outer membrane protein (Omp) 19 from Brucella spp. is a competitive inhibitor of human cathepsin L. U-Omp19 inhibits lysosome cathepsins and APC-derived microsome activity in vitro and partially inhibits lysosomal cathepsin L activity within live APCs. Codelivery of U-Omp19 with the Ag can reduce intracellular Ag digestion and increases Ag half-life in dendritic cells (DCs). U-Omp19 retains the Ag in Lamp-2(+) compartments after its internalization and promotes a sustained expression of MHC class I/peptide complexes in the cell surface of DCs. Consequently, U-Omp19 enhances Ag cross-presentation by DCs to CD8(+) T cells. U-Omp19 s.c. delivery induces the recruitment of CD11c(+)CD8α(+) DCs and monocytes to lymph nodes whereas it partially limits in vivo Ag proteolysis inside DCs. Accordingly, this protein is able to induce CD8(+) T cell responses in vivo against codelivered Ag. Antitumor responses were elicited after U-Omp19 coadministration, increasing survival of mice in a murine melanoma challenge model. Collectively, these results indicate that a cysteine protease inhibitor from bacterial origin could be a suitable component of vaccine formulations against tumors. PMID:27084100

  12. Increased Expression of miR-23a Mediates a Loss of Expression in the RAF Kinase Inhibitor Protein RKIP

    PubMed Central

    Hatzl, Stefan; Geiger, Olivia; Kuepper, Maja Kim; Caraffini, Veronica; Seime, Till; Furlan, Tobias; Nussbaumer, Erika; Wieser, Rotraud; Pichler, Martin; Scheideler, Marcel; Nowek, Katarzyna; Jongen-Lavrencic, Mojca; Quehenberger, Franz; Wölfler, Albert; Troppmair, Jakob; Sill, Heinz; Zebisch, Armin

    2016-01-01

    RAF kinase inhibitor protein (RKIP) is a seminal regulator of intracellular signaling and exhibits both antimetastatic and antitumorigenic properties. Decreased expression of RKIP has been described in several human malignancies, including acute myelogenous leukemia (AML). As the mechanisms leading to RKIP loss in AML are still unclear, we aimed to analyze the potential involvement of miRNAs within this study. miRNA microarray and qPCR data of more than 400 AML patient specimens revealed correlation between decreased expression of RKIP and increased expression of miR-23a, a member of the miR-23a/27a/24-2 cluster. In functional experiments, overexpression of miR-23a decreased RKIP mRNA and protein expression, whereas miR-23a inhibition caused the opposite effect. By using an RKIP 3′-untranslated region luciferase reporter construct with and without mutation or deletion of the putative miR-23a–binding site, we could show that RKIP modulation by miR-23a is mediated via direct binding to this region. Importantly, miR-23a overexpression induced a significant increase of proliferation in hematopoietic cells. Simultaneous transfection of an RKIP expression construct lacking the miR-23a–binding sites reversed this phenotype, indicating that this effect is truly mediated via downregulation of RKIP. Finally, by analyzing more than 4,300 primary patient specimens via database retrieval from The Cancer Genome Atlas, we could highlight the importance of the miR-23a/RKIP axis in a broad range of human cancer entities. In conclusion, we have identified miR-23a as a negative regulator of RKIP expression in AML and have provided data that suggest the importance of our observation beyond this tumor entity. PMID:27197200

  13. Increased Expression of miR-23a Mediates a Loss of Expression in the RAF Kinase Inhibitor Protein RKIP.

    PubMed

    Hatzl, Stefan; Geiger, Olivia; Kuepper, Maja Kim; Caraffini, Veronica; Seime, Till; Furlan, Tobias; Nussbaumer, Erika; Wieser, Rotraud; Pichler, Martin; Scheideler, Marcel; Nowek, Katarzyna; Jongen-Lavrencic, Mojca; Quehenberger, Franz; Wölfler, Albert; Troppmair, Jakob; Sill, Heinz; Zebisch, Armin

    2016-06-15

    RAF kinase inhibitor protein (RKIP) is a seminal regulator of intracellular signaling and exhibits both antimetastatic and antitumorigenic properties. Decreased expression of RKIP has been described in several human malignancies, including acute myelogenous leukemia (AML). As the mechanisms leading to RKIP loss in AML are still unclear, we aimed to analyze the potential involvement of miRNAs within this study. miRNA microarray and qPCR data of more than 400 AML patient specimens revealed correlation between decreased expression of RKIP and increased expression of miR-23a, a member of the miR-23a/27a/24-2 cluster. In functional experiments, overexpression of miR-23a decreased RKIP mRNA and protein expression, whereas miR-23a inhibition caused the opposite effect. By using an RKIP 3'-untranslated region luciferase reporter construct with and without mutation or deletion of the putative miR-23a-binding site, we could show that RKIP modulation by miR-23a is mediated via direct binding to this region. Importantly, miR-23a overexpression induced a significant increase of proliferation in hematopoietic cells. Simultaneous transfection of an RKIP expression construct lacking the miR-23a-binding sites reversed this phenotype, indicating that this effect is truly mediated via downregulation of RKIP. Finally, by analyzing more than 4,300 primary patient specimens via database retrieval from The Cancer Genome Atlas, we could highlight the importance of the miR-23a/RKIP axis in a broad range of human cancer entities. In conclusion, we have identified miR-23a as a negative regulator of RKIP expression in AML and have provided data that suggest the importance of our observation beyond this tumor entity. Cancer Res; 76(12); 3644-54. ©2016 AACR. PMID:27197200

  14. Overtone Mobility Spectrometry: Part 5. Simulations and Analytical Expressions Describing Overtone Limits

    PubMed Central

    Ewing, Michael A.; Zucker, Steven M.; Valentine, Stephen J.; Clemmer, David E.

    2015-01-01

    Mathematical expressions for the analytical duty cycle associated with different overtones in overtone mobility spectrometry are derived from the widths of the transmitted packets of ions under different instrumental operating conditions. Support for these derivations is provided through ion trajectory simulations. The outcome of the theory and simulations indicates that under all operating conditions there exists a limit or maximum observable overtone that will result in ion transmission. Implications of these findings on experimental design are discussed. PMID:23468094

  15. The Expression of stlA in Photorhabdus luminescens Is Controlled by Nutrient Limitation

    PubMed Central

    Lango-Scholey, Lea; Brachmann, Alexander O.; Bode, Helge B.; Clarke, David J.

    2013-01-01

    Photorhabdus is a genus of Gram-negative entomopathogenic bacteria that also maintain a mutualistic association with nematodes from the family Heterorhabditis. Photorhabdus has an extensive secondary metabolism that is required for the interaction between the bacteria and the nematode. A major component of this secondary metabolism is a stilbene molecule, called ST. The first step in ST biosynthesis is the non-oxidative deamination of phenylalanine resulting in the production of cinnamic acid. This reaction is catalyzed by phenylalanine-ammonium lyase, an enzyme encoded by the stlA gene. In this study we show, using a stlA-gfp transcriptional fusion, that the expression of stlA is regulated by nutrient limitation through a regulatory network that involves at least 3 regulators. We show that TyrR, a LysR-type transcriptional regulator that regulates gene expression in response to aromatic amino acids in E. coli, is absolutely required for stlA expression. We also show that stlA expression is modulated by σS and Lrp, regulators that are implicated in the regulation of the response to nutrient limitation in other bacteria. This work is the first that describes pathway-specific regulation of secondary metabolism in Photorhabdus and, therefore, our study provides an initial insight into the complex regulatory network that controls secondary metabolism, and therefore mutualism, in this model organism. PMID:24278476

  16. Changes in gene expression of Prymnesium parvum induced by nitrogen and phosphorus limitation

    PubMed Central

    Liu, Zhenfeng; Koid, Amy E.; Terrado, Ramon; Campbell, Victoria; Caron, David A.; Heidelberg, Karla B.

    2015-01-01

    Prymnesium parvum is a globally distributed prymnesiophyte alga commonly found in brackish water marine ecosystems and lakes. It possesses a suite of toxins with ichthyotoxic, cytotoxic and hemolytic effects which, along with its mixotrophic nutritional capabilities, allows it to form massive Ecosystem Disruptive Algal Blooms (EDABs). While blooms of high abundance coincide with high levels of nitrogen (N) and phosphorus (P), reports of field and laboratory studies have noted that P. parvum toxicity appears to be augmented at high N:P ratios or P-limiting conditions. Here we present the results of a comparative analysis of P. parvum RNA-Seq transcriptomes under nutrient replete conditions, and N or P deficiency to understand how this organism responds at the transcriptional level to varying nutrient conditions. In nutrient limited conditions we found diverse transcriptional responses for genes involved in nutrient uptake, protein synthesis and degradation, photosynthesis, and toxin production. As anticipated, when either N or P was limiting, transcription levels of genes encoding transporters for the respective nutrient were higher than those under replete condition. Ribosomal and lysosomal protein genes were expressed at higher levels under either nutrient-limited condition compared to the replete condition. Photosynthesis genes and polyketide synthase genes were more highly expressed under P-limitation but not under N-limitation. These results highlight the ability of P. parvum to mount a coordinated and varied cellular and physiological response to nutrient limitation. Results also provide potential marker genes for further evaluating the physiological response and toxin production of P. parvum populations during bloom formation or to changing environmental conditions. PMID:26157435

  17. Changes in gene expression of Prymnesium parvum induced by nitrogen and phosphorus limitation.

    PubMed

    Liu, Zhenfeng; Koid, Amy E; Terrado, Ramon; Campbell, Victoria; Caron, David A; Heidelberg, Karla B

    2015-01-01

    Prymnesium parvum is a globally distributed prymnesiophyte alga commonly found in brackish water marine ecosystems and lakes. It possesses a suite of toxins with ichthyotoxic, cytotoxic and hemolytic effects which, along with its mixotrophic nutritional capabilities, allows it to form massive Ecosystem Disruptive Algal Blooms (EDABs). While blooms of high abundance coincide with high levels of nitrogen (N) and phosphorus (P), reports of field and laboratory studies have noted that P. parvum toxicity appears to be augmented at high N:P ratios or P-limiting conditions. Here we present the results of a comparative analysis of P. parvum RNA-Seq transcriptomes under nutrient replete conditions, and N or P deficiency to understand how this organism responds at the transcriptional level to varying nutrient conditions. In nutrient limited conditions we found diverse transcriptional responses for genes involved in nutrient uptake, protein synthesis and degradation, photosynthesis, and toxin production. As anticipated, when either N or P was limiting, transcription levels of genes encoding transporters for the respective nutrient were higher than those under replete condition. Ribosomal and lysosomal protein genes were expressed at higher levels under either nutrient-limited condition compared to the replete condition. Photosynthesis genes and polyketide synthase genes were more highly expressed under P-limitation but not under N-limitation. These results highlight the ability of P. parvum to mount a coordinated and varied cellular and physiological response to nutrient limitation. Results also provide potential marker genes for further evaluating the physiological response and toxin production of P. parvum populations during bloom formation or to changing environmental conditions. PMID:26157435

  18. Increased aquaporin 1 and 5 membrane expression in the lens epithelium of cataract patients.

    PubMed

    Barandika, Olatz; Ezquerra-Inchausti, Maitane; Anasagasti, Ander; Vallejo-Illarramendi, Ainara; Llarena, Irantzu; Bascaran, Lucia; Alberdi, Txomin; De Benedetti, Giacomo; Mendicute, Javier; Ruiz-Ederra, Javier

    2016-10-01

    In this work we have analyzed the expression levels of the main aquaporins (AQPs) expressed in human lens epithelial cells (HLECs) using 112 samples from patients treated with cataract surgery and 36 samples from individuals treated with refractive surgery, with transparent lenses as controls. Aquaporin-1 (AQP1) is the main AQP, representing 64.1% of total AQPs in HLECs, with aquaporin-5 (AQP5) representing 35.9% in controls. A similar proportion of each AQP in cataract was found. Although no differences were found at the mRNA level compared to controls, a significant 1.65-fold increase (p=0.001) in AQP1protein expression was observed in HLECs from cataract patients, with the highest differences being found for nuclear cataracts (2.1-fold increase; p<0.001). A similar trend was found for AQP5 (1.47-fold increase), although the difference was not significant (p=0.161). Moreover we have shown increased membrane AQP5 protein expression in HLECs of patients with cataracts. No association of AQP1 or AQP5 expression levels with age or sex was observed in either group. Our results suggest regulation of AQP1 and AQP5 at the post-translational level and support previous observations on the implication of AQP1 and 5 in maintenance of lens transparency in animal models. Our results likely reflect a compensatory response of the crystalline lens to delay cataract formation by increasing the water removal rate. PMID:27497833

  19. OCT4 increases BIRC5 and CCND1 expression and promotes cancer progression in hepatocellular carcinoma

    PubMed Central

    2013-01-01

    Background OCT4 and BIRC5 are preferentially expressed in human cancer cells and mediate cancer cell survival and tumor maintenance. However, the molecular mechanism that regulates OCT4 and BIRC5 expression is not well characterized. Methods By manipulating OCT4 and BIRC5 expression in hepatocellular carcinoma (HCC) cell lines, the regulatory mechanism of OCT4 on BIRC5 and CCND1 were investigated. Results Increasing or decreasing OCT4 expression could enhance or suppress BIRC5 expression, respectively, by regulating the activity of BIRC5 promoter. Because there is no binding site for OCT4 within BIRC5 promoter, the effect of OCT4 on BIRC5 promoter is indirect. An octamer motif for OCT4 in the CCND1 promoter has directly and partly participated in the regulation of CCND1 promoter activity, suggesting that OCT4 also could upregulated the expression of CCND1. Co-suppression of OCT4 and BIRC5 induced cancer cell apoptosis and cell cycle arrest, thereby efficiently inhibiting the proliferative activity of cancer cells and suppressing the growth of HCC xenogrfts in nude mice. Conclusion OCT4 can upregulate BIRC5 and CCND1 expression by increasing their promoter activity. These factors collusively promotes HCC cell proliferation, and co-suppression of OCT4 and BIRC5 is potentially beneficial for HCC treatment. PMID:23433354

  20. Increased Expression of Several Collagen Genes is Associated with Drug Resistance in Ovarian Cancer Cell Lines

    PubMed Central

    Januchowski, Radosław; Świerczewska, Monika; Sterzyńska, Karolina; Wojtowicz, Karolina; Nowicki, Michał; Zabel, Maciej

    2016-01-01

    Ovarian cancer is the most lethal gynaecological cancer. The main reason for the high mortality among ovarian cancer patients is the development of drug resistance. The expression of collagen genes by cancer cells can increase drug resistance by inhibiting the penetration of the drug into the cancer tissue as well as increase apoptosis resistance. In this study, we present data that shows differential expression levels of collagen genes and proteins in cisplatin- (CIS), paclitaxel- (PAC), doxorubicin- (DOX), topotecan- (TOP), vincristine- (VIN) and methotrexate- (MTX) resistant ovarian cancer cell lines. Quantitative real-time polymerase chain reactions were performed to determine the mRNA levels. Protein expression was detected using Western blot and immunocytochemistry assays. In the drug resistant cell lines, we observed the upregulation of eight collagen genes at the mRNA level and based on these expression levels, we divided the collagen genes into the following three groups: 1. Genes with less than a 50-fold increase in expression: COL1A1, COL5A2, COL12A1 and COL17A1. 2. Genes with greater than a 50-fold increase in expression: COL1A2, COL15A1 and COL21A1. 3. Gene with a very high level of expression: COL3A1. Expression of collagen (COL) proteins from groups 2 and 3 were also confirmed using immunocytochemistry. Western blot analysis showed very high expression levels of COL3A1 protein, and immunocytochemistry analysis showed the presence of extracellular COL3A1 in the W1TR cell line. The cells mainly responsible for the extracellular COL3A1 production are aldehyde dehydrogenase-1A1 (ALDH1A1) positive cells. All correlations between the types of cytostatic drugs and the expression levels of different COL genes were studied, and our results suggest that the expression of fibrillar collagens may be involved in the TOP and PAC resistance of the ovarian cancer cells. The expression pattern of COL genes provide a preliminary view into the role of these proteins in

  1. Increased Expression of Several Collagen Genes is Associated with Drug Resistance in Ovarian Cancer Cell Lines.

    PubMed

    Januchowski, Radosław; Świerczewska, Monika; Sterzyńska, Karolina; Wojtowicz, Karolina; Nowicki, Michał; Zabel, Maciej

    2016-01-01

    Ovarian cancer is the most lethal gynaecological cancer. The main reason for the high mortality among ovarian cancer patients is the development of drug resistance. The expression of collagen genes by cancer cells can increase drug resistance by inhibiting the penetration of the drug into the cancer tissue as well as increase apoptosis resistance. In this study, we present data that shows differential expression levels of collagen genes and proteins in cisplatin- (CIS), paclitaxel- (PAC), doxorubicin- (DOX), topotecan- (TOP), vincristine- (VIN) and methotrexate- (MTX) resistant ovarian cancer cell lines. Quantitative real-time polymerase chain reactions were performed to determine the mRNA levels. Protein expression was detected using Western blot and immunocytochemistry assays. In the drug resistant cell lines, we observed the upregulation of eight collagen genes at the mRNA level and based on these expression levels, we divided the collagen genes into the following three groups: 1. Genes with less than a 50-fold increase in expression: COL1A1, COL5A2, COL12A1 and COL17A1. 2. Genes with greater than a 50-fold increase in expression: COL1A2, COL15A1 and COL21A1. 3. Gene with a very high level of expression: COL3A1. Expression of collagen (COL) proteins from groups 2 and 3 were also confirmed using immunocytochemistry. Western blot analysis showed very high expression levels of COL3A1 protein, and immunocytochemistry analysis showed the presence of extracellular COL3A1 in the W1TR cell line. The cells mainly responsible for the extracellular COL3A1 production are aldehyde dehydrogenase-1A1 (ALDH1A1) positive cells. All correlations between the types of cytostatic drugs and the expression levels of different COL genes were studied, and our results suggest that the expression of fibrillar collagens may be involved in the TOP and PAC resistance of the ovarian cancer cells. The expression pattern of COL genes provide a preliminary view into the role of these proteins in

  2. Increased cardiogenesis in P19-GFP teratocarcinoma cells expressing the propeptide IGF-1Ea

    SciTech Connect

    Poudel, Bhawana; Bilbao, Daniel; Sarathchandra, Padmini; Germack, Renee; Rosenthal, Nadia; Santini, Maria Paola

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer In this study, we explored the function of IGF-1Ea propeptide in inducing cardiogenesis of stem cells. Black-Right-Pointing-Pointer IGF-1Ea promoted cardiac mesodermal induction in uncommitted cells. Black-Right-Pointing-Pointer Under differentiation condition, IGF-1Ea increased expression of cardiac differentiation markers. Black-Right-Pointing-Pointer Furthermore, it promoted formation of finely organized sarcomeric structure. Black-Right-Pointing-Pointer IGF-1Ea propeptide may be a good candidate to improve production of cardiomyocytes from pluripotent cells. -- Abstract: The mechanism implicated in differentiation of endogenous cardiac stem cells into cardiomyocytes to regenerate the heart tissue upon an insult remains elusive, limiting the therapeutical goals to exogenous cell injection and/or gene therapy. We have shown previously that cardiac specific overexpression of the insulin-like growth factor 1 propeptide IGF-1Ea induces beneficial myocardial repair after infarct. Although the mechanism is still under investigation, the possibility that this propeptide may be involved in promoting stem cell differentiation into the cardiac lineage has yet to be explored. To investigate whether IGF-1Ea promote cardiogenesis, we initially modified P19 embryonal carcinoma cells to express IGF-1Ea. Taking advantage of their cardiomyogenic nature, we analyzed whether overexpression of this propeptide affected cardiac differentiation program. The data herein presented showed for the first time that constitutively overexpressed IGF-1Ea increased cardiogenic differentiation program in both undifferentiated and DMSO-differentiated cells. In details, IGF-1Ea overexpression promoted localization of alpha-actinin in finely organized sarcomeric structure compared to control cells and upregulated the cardiac mesodermal marker NKX-2.5 and the ventricular structural protein MLC2v. Furthermore, activated IGF-1 signaling promoted cardiac

  3. High glucose and palmitate increases bone morphogenic protein 4 expression in human endothelial cells

    PubMed Central

    Hong, Oak-Kee; Yoo, Soon-Jib; Son, Jang-Won; Kim, Mee-Kyoung; Baek, Ki-Hyun; Song, Ki-Ho; Cha, Bong-Yun; Jo, Hanjoong

    2016-01-01

    Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) aff ect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this eff ect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of infl ammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL. PMID:26937213

  4. Geranylgeranylacetone attenuates hepatic fibrosis by increasing the expression of heat shock protein 70.

    PubMed

    He, Wei; Zhuang, Yun; Wang, Liangzhi; Qi, Lei; Chen, Binfang; Wang, Mei; Shao, Dong; Chen, Jianping

    2015-10-01

    Increasing evidence has demonstrated that the heat shock protein 70 (HSP70) gene may be closely associated with tissue fibrosis; however, the association between HSP70 and liver fibrosis remains to be fully elucidated. The present study hypothesized that geranylgeranylacetone (GGA) exerts beneficial effects on liver fibrosis though upregulation of the expression of HSP70. Liver fibrosis was induced in rats using carbon tetrachloride (CCl4). The rats were subsequently divided into three groups: Control group, CCl4 model group and CCl4 model + GGA group. Liver fibrosis in the rats was evaluated using hematoxylin and eosin staining, Masson's trichrome staining and Sirius red staining. The levels of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin were determined using an automated biochemistry analyzer. The levels of total hepatic hydroxyproline were also determined. The expression levels of α‑smooth muscle actin (α‑SMA) and transforming growth factor‑β1 (TGF‑β1) were determined using immunofluorescence staining and western blotting, and the protein expression levels of HSP70 were determined using western blotting. The CCl4‑induced rats exhibited liver fibrosis, increased hydroxyproline content, impaired liver function, upregulated expression levels of the α‑SMA and TGF‑β1 pro‑fibrogenic proteins, and increased expression of HSP70, compared with the control group. These changes were attenuated by treatment with GGA. These results demonstrated that GGA exerted beneficial effects in CCl4‑induced liver fibrosis via upregulating the expression of HSP70. PMID:26165998

  5. Oxytocin Increases Neurite Length and Expression of Cytoskeletal Proteins Associated with Neuronal Growth.

    PubMed

    Lestanova, Z; Bacova, Z; Kiss, A; Havranek, T; Strbak, V; Bakos, J

    2016-06-01

    Neuropeptide oxytocin acts as a growth and differentiation factor; however, its effects on neurite growth are poorly understood. The aims of the present study were (1) to evaluate time effects of oxytocin on expression of nestin and MAP2; (2) to measure the effect of oxytocin on gene expression of β-actin, vimentin, cofilin, and drebrin; and (3) to measure changes in neurite length and number in response to oxytocin/oxytocin receptor antagonist L-371,257. Exposure of SH-SY5Y cells to 1 μM oxytocin resulted in a significant increase in gene expression and protein levels of nestin after 12, 24, and 48 h. Oxytocin treatment induced no changes in gene expression of MAP2; however, a decrease of protein levels was observed in all time intervals. Gene expression of β-actin, vimentin, and drebrin increased in response to oxytocin. Oxytocin induced significant elongation of neurites after 12, 24, and 48 h. No change in neurite length was observed in the presence of the combination of retinoic acid and oxytocin receptor antagonist L-371,257. Oxytocin treatment for 12 h increased the number of neurites. Overall, the present data suggest that oxytocin contributes to the regulation of expression of cytoskeletal proteins associated with growth of neuronal cones and induces neurite elongation mediated by oxytocin receptors at least in certain types of neuronal cells. PMID:26474566

  6. UDP-glucuronosyltransferase expression in mouse liver is increased in obesity- and fasting-induced steatosis.

    PubMed

    Xu, Jialin; Kulkarni, Supriya R; Li, Liya; Slitt, Angela L

    2012-02-01

    UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lep(ob/ob) (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance. PMID:22031624

  7. 39 CFR 3010.11 - Limit on size of rate increases.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... in any 12-month period are limited. (b) Rates of general applicability are subject to an inflation-based limitation computed using CPI-U values as detailed in § 3010.12. (c) An exception to the inflation... authority is measured separately for each class of mail. (d) In any 12-month period the...

  8. 39 CFR 3010.11 - Limit on size of rate increases.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... in any 12-month period are limited. (b) Rates of general applicability are subject to an inflation-based limitation computed using CPI-U values as detailed in § 3010.12. (c) An exception to the inflation... authority is measured separately for each class of mail. (d) In any 12-month period the...

  9. 39 CFR 3010.11 - Limit on size of rate increases.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... in any 12-month period are limited. (b) Rates of general applicability are subject to an inflation-based limitation computed using CPI-U values as detailed in § 3010.12. (c) An exception to the inflation... authority is measured separately for each class of mail. (d) In any 12-month period the...

  10. 39 CFR 3010.11 - Limit on size of rate increases.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... in any 12-month period are limited. (b) Rates of general applicability are subject to an inflation-based limitation computed using CPI-U values as detailed in § 3010.12. (c) An exception to the inflation... authority is measured separately for each class of mail. (d) In any 12-month period the...

  11. NFX1-123 and Human Papillomavirus 16E6 Increase Notch Expression in Keratinocytes

    PubMed Central

    Vliet-Gregg, Portia A.; Hamilton, Jennifer R.

    2013-01-01

    The high-risk human papillomavirus (HR HPV) E6 oncoprotein binds host cell proteins to dysregulate multiple regulatory pathways, including apoptosis and senescence. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and together they posttranscriptionally increase hTERT expression, the catalytic subunit of telomerase. NFX1-123 interacts with hTERT mRNA and stabilizes it, leading to greater telomerase activity and the avoidance of cellular senescence. Little is known regarding what other transcripts are dependent on or augmented by the association of NFX1-123 with 16E6. Microarray analysis revealed enhanced expression of Notch1 mRNA in 16E6-expressing keratinocytes when NFX1-123 was overexpressed. A moderate increase in Notch1 mRNA was seen with overexpression of NFX1-123 alone, but with 16E6 coexpression the increase in Notch1 was enhanced. The PAM2 motif and R3H protein domains in NFX1-123, which were important for increased hTERT expression, were also important in the augmentation of Notch1 expression by 16E6. These findings identify a second gene coregulated by 16E6 and NFX1-123 and the protein motifs in NFX1-123 that are important for this effect. PMID:24109236

  12. Increased vitamin D receptor expression in dorsal root ganglia neurons of diabetic rats.

    PubMed

    Filipović, Natalija; Ferhatović, Lejla; Marelja, Ivana; Puljak, Livia; Grković, Ivica

    2013-08-01

    The effects of vitamin D on the nervous system have been studied extensively. In spite of accumulating data about the substantial changes in the vitamin D receptor (VDR) signaling system, during different types of neuroinflammatory diseases, its role in diabetic neuropathy has not been investigated in detail. To assess the role of VDR signaling in diabetic neuropathy, we examined expression of VDRs in dorsal root ganglia (DRG) neurons in a rat model of streptozotocin-induced diabetes mellitus type 1. Diabetes mellitus (DM) type 1 was induced with streptozotocin in male Sprague-Dawley rats. After two months, expression of VDRs was analyzed immunohistochemically in the cytoplasm of L4 and L5 DRG neurons of diabetic rats. Semi-quantitative analysis for the determination of staining in nuclei and plasma-membranes of DRG neurons was performed. A significant increase in VDR expression was observed in DRG neurons of diabetic rats. Expression of VDRs was increased in the cytoplasm, nuclei and in cell membranes of neurons. An increase in VDR expression occurred in all neurons, but the greatest increase of fluorescence intensity in cytoplasm was observed in neurons of small diameter. Results of the present study indicate that the VDR signaling system could be a potential therapeutic target for diabetic neuropathy. PMID:23684983

  13. NOD1 expression is increased in the adipose tissue of women with gestational diabetes.

    PubMed

    Lappas, Martha

    2014-07-01

    Maternal peripheral insulin resistance and increased inflammation are two features of pregnancies, complicated by gestational diabetes mellitus (GDM). The nucleotide-binding oligomerisation domain (NOD) intracellular molecules recognise a wide range of microbial products, as well as other intracellular danger signals, thereby initiating inflammation through activation of nuclear factor κB (NFκB). The aim of this study was to determine whether levels of NOD1 and NOD2 are increased in adipose tissue of women with GDM. The effect of NOD1 and NOD2 activation on inflammation and the insulin signalling pathway was also assessed. NOD1, but not NOD2, expression was higher in omental and subcutaneous adipose tissues obtained from women with GDM when compared with those from women with normal glucose tolerance (NGT). In both omental and subcutaneous adipose tissues from NGT and GDM women, the NOD1 ligand g-d-glutamyl-meso-diaminopimelic acid (iE-DAP) significantly induced the expression and secretion of the pro-inflammatory cytokine interleukin 6 (IL6) and chemokine IL8; COX2 (PTGS2) gene expression and subsequent prostaglandin production; the expression and secretion of the extracellular matrix remodelling enzyme matrix metalloproteinase 9 (MMP9) and the gene expression and secretion of the adhesion molecules ICAM1 and VCAM1. There was no effect of the NOD2 ligand muramyl dipeptide on any of the endpoints tested. The effects of the NOD1 ligand iE-DAP were mediated via NFκB, as the NFκB inhibitor BAY 11-7082 significantly attenuated iE-DAP-induced expression and secretion of pro-inflammatory cytokines, COX2 gene expression and subsequent prostaglandin production, MMP9 expression and secretion and ICAM1 and VCAM1 gene expression and secretion. In conclusion, the present findings describe an important role for NOD1 in the development of insulin resistance and inflammation in pregnancies complicated by GDM. PMID:24829218

  14. Effect of ploidy increase on transgene expression: example from Citrus diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene.

    PubMed

    Xu, Shi-Xiao; Cai, Xiao-Dong; Tan, Bin; Li, Ding-Li; Guo, Wen-Wu

    2011-07-01

    Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from 'Murcott' tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic 'Valencia' orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy

  15. TMPRSS2- Driven ERG Expression In Vivo Increases Self-Renewal and Maintains Expression in a Castration Resistant Subpopulation

    PubMed Central

    Abou-Kheir, Wassim G.; Martin, Philip L.; Tillman, Heather S.; Petrovics, Gyorgy; Awwad, Hibah O.; Ward, Yvona; Lake, Ross; Zhang, Luhua; Kelly, Kathleen

    2012-01-01

    Genomic rearrangements commonly occur in many types of cancers and often initiate or alter the progression of disease. Here we describe an in vivo mouse model that recapitulates the most frequent rearrangement in prostate cancer, the fusion of the promoter region of TMPRSS2 with the coding region of the transcription factor, ERG. A recombinant bacterial artificial chromosome including an extended TMPRSS2 promoter driving genomic ERG was constructed and used for transgenesis in mice. TMPRSS2-ERG expression was evaluated in tissue sections and FACS-fractionated prostate cell populations. In addition to the anticipated expression in luminal cells, TMPRSS2-ERG was similarly expressed in the Sca-1hi/EpCAM+ basal/progenitor fraction, where expanded numbers of clonogenic self-renewing progenitors were found, as assayed by in vitro sphere formation. These clonogenic cells increased intrinsic self renewal in subsequent generations. In addition, ERG dependent self-renewal and invasion in vitro was demonstrated in prostate cell lines derived from the model. Clinical studies have suggested that the TMPRSS2-ERG translocation occurs early in prostate cancer development. In the model described here, the presence of the TMPRSS2-ERG fusion alone was not transforming but synergized with heterozygous Pten deletion to promote PIN. Taken together, these data suggest that one function of TMPRSS2-ERG is the expansion of self-renewing cells, which may serve as targets for subsequent mutations. Primary prostate epithelial cells demonstrated increased post transcriptional turnover of ERG compared to the TMPRSS2-ERG positive VCaP cell line, originally isolated from a prostate cancer metastasis. Finally, we determined that TMPRSS2-ERG expression occurred in both castration-sensitive and resistant prostate epithelial subpopulations, suggesting the existence of androgen-independent mechanisms of TMPRSS2 expression in prostate epithelium. PMID:22860005

  16. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    PubMed Central

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041

  17. SPARCL1 Expression Increases With Preoperative Radiation Therapy and Predicts Better Survival in Rectal Cancer Patients

    SciTech Connect

    Kotti, Angeliki Holmqvist, Annica; Albertsson, Maria; Sun, Xiao-Feng

    2014-04-01

    Purpose: The secreted protein acidic and rich in cysteine-like 1 (SPARCL1) is expressed in various normal tissues and many types of cancers. The function of SPARCL1 and its relationship to a patient's prognosis have been studied, whereas its relationship to radiation therapy (RT) is not known. Our aim was to investigate the expression of SPARCL1 in rectal cancer patients who participated in a clinical trial of preoperative RT. Methods and Materials: The study included 136 rectal cancer patients who were randomized to undergo preoperative RT and surgery (n=63) or surgery alone (n=73). The expression levels of SPARCL1 in normal mucosa (n=29), primary tumor (n=136), and lymph node metastasis (n=35) were determined by immunohistochemistry. Results: Tumors with RT had stronger SPARCL1 expression than tumors without RT (P=.003). In the RT group, strong SPARCL1 expression was related to better survival than weak expression in patients with stage III tumors, independent of sex, age, differentiation, and margin status (P=.022; RR = 18.128; 95% confidence interval, 1.512-217.413). No such relationship was found in the non-RT group (P=.224). Further analysis of interactions among SPARCL1 expression, RT, and survival showed statistical significance (P=.024). In patients with metastases who received RT, strong SPARCL1 expression was related to better survival compared to weak expression (P=.041) but not in the non-RT group (P=.569). Conclusions: SPARCL1 expression increases with RT and is related to better prognosis in rectal cancer patients with RT but not in patients without RT. This result may help us to select the patients best suited for preoperative RT.

  18. Absence of functional TolC protein causes increased stress response gene expression in Sinorhizobium meliloti

    PubMed Central

    2010-01-01

    Background The TolC protein from Sinorhizobium meliloti has previously been demonstrated to be required for establishing successful biological nitrogen fixation symbiosis with Medicago sativa. It is also needed in protein and exopolysaccharide secretion and for protection against osmotic and oxidative stresses. Here, the transcriptional profile of free-living S. meliloti 1021 tolC mutant is described as a step toward understanding its role in the physiology of the cell. Results Comparison of tolC mutant and wild-type strains transcriptomes showed 1177 genes with significantly increased expression while 325 had significantly decreased expression levels. The genes with an increased expression suggest the activation of a cytoplasmic and extracytoplasmic stress responses possibly mediated by the sigma factor RpoH1 and protein homologues of the CpxRA two-component regulatory system of Enterobacteria, respectively. Stress conditions are probably caused by perturbation of the cell envelope. Consistent with gene expression data, biochemical analysis indicates that the tolC mutant suffers from oxidative stress. This is illustrated by the elevated enzyme activity levels detected for catalase, superoxide dismutase and glutathione reductase. The observed increase in the expression of genes encoding products involved in central metabolism and transporters for nutrient uptake suggests a higher metabolic rate of the tolC mutant. We also demonstrated increased swarming motility in the tolC mutant strain. Absence of functional TolC caused decreased expression mainly of genes encoding products involved in nitrogen metabolism and transport. Conclusion This work shows how a mutation in the outer membrane protein TolC, common to many bacterial transport systems, affects expression of a large number of genes that act in concert to restore cell homeostasis. This finding further underlines the fundamental role of this protein in Sinorhizobium meliloti biology. PMID:20573193

  19. Expression of bactericidal/permeability-increasing protein requires C/EBP epsilon.

    PubMed

    Tanaka, Miyuki; Gombart, Adrian F; Koeffler, H Phillip; Shiohara, Masaaki

    2007-05-01

    Bactericidal/permeability-increasing protein (BPI) is a 55-kd cationic protein found mainly in neutrophil primary granules. BPI shows cytotoxicity against Gram-negative bacteria. In this study, we studied the role of a myeloid-specific transcription factor, CCAAT/enhancer binding protein epsilon (C/EBP epsilon), in the regulation of BPI gene expression. A patient with neutrophil-specific granule deficiency with a homozygous inactivating mutation in the CEBP epsilon gene showed severely impaired expression of both BPI messenger RNA (mRNA) and BPI protein. Both U937 and NB4 cells treated with 10-7 M all-trans retinoic acid (ATRA) for 6 days displayed increased levels of BPI protein and accompanying up-regulated C/EBP epsilon expression. Chromatin-immunoprecipitation analysis and electrophoretic mobility shift assays revealed binding of the C/EBP epsilon protein to the C/EBP-binding site in the BPI gene promoter. U937 cells stably transfected with a zinc-inducible C/EBP epsilon expression vector showed a 30-fold increase in BPI mRNA levels compared with cells transfected with control empty vector after culturing for 48 hours with 100 microM ZnSO4. BPI mRNA expression was severely reduced in the bone marrow of C/EBP epsilon-deficient mice compared with wild-type mice. Expression of BPI in human cord blood cells was increased by incubation with 10-7 MATRA for 48 hours. These results demonstrate the requirement for C/EBP epsilon in mediating BPI gene expression in myeloid cells in vitro and in vivo. PMID:17483073

  20. Sulphur limitation and early sulphur deficiency responses in poplar: significance of gene expression, metabolites, and plant hormones

    PubMed Central

    Honsel, Anne; Kojima, Mikiko; Haas, Richard; Frank, Wolfgang; Sakakibara, Hitoshi; Herschbach, Cornelia; Rennenberg, Heinz

    2012-01-01

    The influence of sulphur (S) depletion on the expression of genes related to S metabolism, and on metabolite and plant hormone contents was analysed in young and mature leaves, fine roots, xylem sap, and phloem exudates of poplar (Populus tremula×Populus alba) with special focus on early consequences. S depletion was applied by a gradual decrease of sulphate availability. The observed changes were correlated with sulphate contents. Based on the decrease in sulphate contents, two phases of S depletion could be distinguished that were denominated as ‘S limitation’ and ‘early S deficiency’. S limitation was characterized by improved sulphate uptake (enhanced root-specific sulphate transporter PtaSULTR1;2 expression) and reduction capacities (enhanced adenosine 5′-phosphosulphate (APS) reductase expression) and by enhanced remobilization of sulphate from the vacuole (enhanced putative vacuolar sulphate transporter PtaSULTR4;2 expression). During early S deficiency, whole plant distribution of S was impacted, as indicated by increasing expression of the phloem-localized sulphate transporter PtaSULTR1;1 and by decreasing glutathione contents in fine roots, young leaves, mature leaves, and phloem exudates. Furthermore, at ‘early S deficiency’, expression of microRNA395 (miR395), which targets transcripts of PtaATPS3/4 (ATP sulphurylase) for cleavage, increased. Changes in plant hormone contents were observed at ‘early S deficiency’ only. Thus, S depletion affects S and plant hormone metabolism of poplar during ‘S limitation’ and ‘early S deficiency’ in a time series of events. Despite these consequences, the impact of S depletion on growth of poplar plants appears to be less severe than in Brassicaceae such as Arabidopsis thaliana or Brassica sp. PMID:22162873

  1. Campylobacter jejuni Increases Flagellar Expression and Adhesion of Noninvasive Escherichia coli: Effects on Enterocytic Toll-Like Receptor 4 and CXCL-8 Expression

    PubMed Central

    Reti, Kristen L.; Tymensen, Lisa D.; Davis, Shevaun P.; Amrein, Matthias W.

    2015-01-01

    Campylobacter jejuni is the most common cause of bacterium-induced gastroenteritis, and while typically self-limiting, C. jejuni infections are associated with postinfectious intestinal disorders, including flares in patients with inflammatory bowel disease and postinfectious irritable bowel syndrome (PI-IBS), via mechanisms that remain obscure. Based on the hypothesis that acute campylobacteriosis may cause pathogenic microbiota dysbiosis, we investigated whether C. jejuni may activate dormant virulence genes in noninvasive Escherichia coli and examined the epithelial pathophysiological consequences of these alterations. Microarray and quantitative real-time PCR analyses revealed that E. coli adhesin, flagellum, and hemolysin gene expression were increased when E. coli was exposed to C. jejuni-conditioned medium. Increased development of bacterial flagella upon exposure to live C. jejuni or C. jejuni-conditioned medium was observed under transmission electron microscopy. Atomic force microscopy demonstrated that the forces of bacterial adhesion to colonic T84 enterocytes, and the work required to rupture this adhesion, were significantly increased in E. coli exposed to C. jejuni-conditioned media. Finally, C. jejuni-modified E. coli disrupted TLR4 gene expression and induced proinflammatory CXCL-8 gene expression in colonic enterocytes. Together, these data suggest that exposure to live C. jejuni, and/or to its secretory-excretory products, may activate latent virulence genes in noninvasive E. coli and that these alterations may directly trigger proinflammatory signaling in intestinal epithelia. These observations shed new light on mechanisms that may contribute, at least in part, to postcampylobacteriosis inflammatory disorders. PMID:26371123

  2. Campylobacter jejuni increases flagellar expression and adhesion of noninvasive Escherichia coli: effects on enterocytic Toll-like receptor 4 and CXCL-8 expression.

    PubMed

    Reti, Kristen L; Tymensen, Lisa D; Davis, Shevaun P; Amrein, Matthias W; Buret, Andre G

    2015-12-01

    Campylobacter jejuni is the most common cause of bacterium-induced gastroenteritis, and while typically self-limiting, C. jejuni infections are associated with postinfectious intestinal disorders, including flares in patients with inflammatory bowel disease and postinfectious irritable bowel syndrome (PI-IBS), via mechanisms that remain obscure. Based on the hypothesis that acute campylobacteriosis may cause pathogenic microbiota dysbiosis, we investigated whether C. jejuni may activate dormant virulence genes in noninvasive Escherichia coli and examined the epithelial pathophysiological consequences of these alterations. Microarray and quantitative real-time PCR analyses revealed that E. coli adhesin, flagellum, and hemolysin gene expression were increased when E. coli was exposed to C. jejuni-conditioned medium. Increased development of bacterial flagella upon exposure to live C. jejuni or C. jejuni-conditioned medium was observed under transmission electron microscopy. Atomic force microscopy demonstrated that the forces of bacterial adhesion to colonic T84 enterocytes, and the work required to rupture this adhesion, were significantly increased in E. coli exposed to C. jejuni-conditioned media. Finally, C. jejuni-modified E. coli disrupted TLR4 gene expression and induced proinflammatory CXCL-8 gene expression in colonic enterocytes. Together, these data suggest that exposure to live C. jejuni, and/or to its secretory-excretory products, may activate latent virulence genes in noninvasive E. coli and that these alterations may directly trigger proinflammatory signaling in intestinal epithelia. These observations shed new light on mechanisms that may contribute, at least in part, to postcampylobacteriosis inflammatory disorders. PMID:26371123

  3. Reduced LIMK2 expression in colorectal cancer reflects its role in limiting stem cell proliferation

    PubMed Central

    Lourenço, Filipe C; Munro, June; Brown, Jennifer; Cordero, Julia; Stefanatos, Rhoda; Strathdee, Karen; Orange, Clare; Feller, Stephan M; Sansom, Owen J; Vidal, Marcos; Murray, Graeme I; Olson, Michael F

    2014-01-01

    Objective Colorectal cancer (CRC) is a major contributor to cancer mortality and morbidity. LIM kinase 2 (LIMK2) promotes tumour cell invasion and metastasis. The objectives of this study were to determine how LIMK2 expression is associated with CRC progression and patient outcome, and to use genetically modified Drosophila and mice to determine how LIMK2 deletion affects gastrointestinal stem cell regulation and tumour development. Design LIMK2 expression and activity were measured by immunostaining tumours from CRC-prone mice, human CRC cell lines and 650 human tumours. LIMK knockdown in Drosophila or Limk2 deletion in mice allowed for assessment of their contributions to gastrointestinal stem cell homeostasis and tumour development. Results LIMK2 expression was reduced in intestinal tumours of cancer-prone mice, as well as in human CRC cell lines and tumours. Reduced LIMK2 expression and substrate phosphorylation were associated with shorter patient survival. Genetic analysis in Drosophila midgut and intestinal epithelial cells isolated from genetically modified mice revealed a conserved role for LIMK2 in constraining gastrointestinal stem cell proliferation. Limk2 deletion increased colon tumour size in a colitis-associated colorectal mouse cancer model. Conclusions This study revealed that LIMK2 expression and activity progressively decrease with advancing stage, and supports the hypothesis that there is selective pressure for reduced LIMK2 expression in CRC to relieve negative constraints imposed upon gastrointestinal stem cells. PMID:23585469

  4. Increase of Calcium Sensing Receptor Expression Is Related to Compensatory Insulin Secretion during Aging in Mice

    PubMed Central

    Oh, Yoon Sin; Seo, Eun-Hui; Lee, Young-Sun; Cho, Sung Chun; Jung, Hye Seung; Park, Sang Chul; Jun, Hee-Sook

    2016-01-01

    Type 2 diabetes is caused by both insulin resistance and relative insulin deficiency. To investigate age-related changes in glucose metabolism and development of type 2 diabetes, we compared glucose homeostasis in different groups of C57BL/6J mice ranging in age from 4 months to 20 months (4, 8, 12, 16 and 20 months). Interestingly, we observed that non-fasting glucose levels were not significantly changed, but glucose tolerance gradually increased by 20 months of age, whereas insulin sensitivity declined with age. We found that the size of islets and glucose-stimulated insulin secretion increased with aging. However, mRNA expression of pancreatic and duodenal homeobox 1 and granuphilin was decreased in islets of older mice compared with that of 4-month-old mice. Serum calcium (Ca2+) levels were significantly decreased at 12, 20 and 28 months of age compared with 4 months and calcium sensing receptor (CaSR) mRNA expression in the islets significantly increased with age. An extracellular calcium depletion agent upregulated CaSR mRNA expression and consequently enhanced insulin secretion in INS-1 cells and mouse islets. In conclusion, we suggest that decreased Ca2+ levels and increased CaSR expression might be involved in increased insulin secretion to compensate for insulin resistance in aged mice. PMID:27441644

  5. Tributyltin increases the expression of apoptosis- and adipogenesis-related genes in rat ovaries

    PubMed Central

    Lee, Hyojin; Lim, Sojeong; Yun, Sujin; Yoon, Ayoung; Park, Gayoung

    2012-01-01

    Objective Tributyltin (TBT), an endocrine disrupting chemical, has been reported to decrease ovarian function by causing apoptosis in the ovary, but the mechanism is not fully understood. Therefore, we examined whether TBT increases the expression of adipogenesis-related genes in the ovary and the increased expression of these genes is associated with apoptosis induction. Methods Three-week-old Sprague-Dawley rats were orally administered TBT (1 or 10 mg/kg body weight) or sesame oil as a control for 7 days. The ovaries were obtained and weighed on day 8, and then they were fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or frozen for RNA extraction. Using the total RNA of the ovaries, adipogenesis- and apoptosis-related genes were analyzed by real-time polymerase chain reaction (PCR). Results The ovarian weight was significantly decreased in rats administered 10 mg/kg TBT compared to that in control rats. As determined by the TUNEL assay, the number of apoptotic follicles in ovary was significantly increased in rats administered 10 mg/kg TBT. The real-time PCR results showed that the expression of adipogenesis-related genes such as PPARγ, aP2, CD36, and PEPCK was increased after TBT administration. In addition, apoptosis-related genes such as TNFα and TNFR1 were expressed more in the TBT-administered rats compared with the control rats. Conclusion The present study demonstrates that TBT induces the expression of adipogenesis- and apoptosis-related genes in the ovary leading to apoptosis in the ovarian follicles. These results suggest that the increased expression of adipogenesis-related genes in the ovary by TBT exposure might induce apoptosis resulting in a loss of ovarian function. PMID:22563546

  6. Role of membrane depolarization and extracellular calcium in increased complement receptor expression during neutrophil (PMN) activation

    SciTech Connect

    Berger, M.; Wetzler, E.; Birx, D.L.

    1986-03-05

    During PMN activation the surface expression of receptors (R) for C3b and C3bi increases rapidly. This is necessary for optimal cell adhesion, migration, and phagocytosis. Following stimulation with fMLP or LTB-4, the increased expression of C3bR depends only on the Ca/sup + +/ released from intracellular stores and is not inhibited by 5mM EDTA, while the increase in C3biR also requires extracellular Ca/sup + +/. CR expression also increases when the PMN are depolarized with 140 mM K/sup +/, but with this stimulus, EDTA inhibits C3bR by 67% and C3biR 100%, suggesting that intracellular Ca/sup + +/ stores may not be released. Pertussis toxin caused dose-dependent inhibition of both CR responses to fMLP and also inhibited the increases in both CR induced by K/sup +/. Membrane depolarization (monitored by di-O-C5 fluorescence) due to fMLP was similarly inhibited by toxin but the depolarization due to K/sup +/ was not. The dose of phorbol myristate acetate that maximally increased CR expression, 0.1 ng/ml, did not depolarize the membrane. These results suggest that membrane depolarization is neither necessary nor sufficient for increased CR expression. A Ca/sup + +/ and GTP binding protein-dependent enzyme such as phospholipase C is necessary to the amplify initial signals generated either by release of Ca/sup + +/ stores or by opening voltage dependent Ca/sup + +/ channels following membrane depolarization.

  7. Chemoresistance of CD133{sup +} colon cancer may be related with increased survivin expression

    SciTech Connect

    Lee, Mi-Ra; Ji, Sun-Young; Mia-Jan, Khalilullah; Cho, Mee-Yon

    2015-07-31

    CD133, putative cancer stem cell marker, deemed to aid chemoresistance. However, this claim has been challenged recently and we previously reported that patients with CD133{sup +} colon cancer have benefit from 5-fluorouracil (5-FU) chemotherapy incontrast to no benefit in patients with CD133{sup −} cancer. To elucidate the role of CD133 expression in chemoresistance, we silenced the CD133 expression in a colon cancer cell line and determined its effect on the biological characteristics downstream. We comparatively analyzed the sequential changes of MDR1, ABCG2, AKT1 and survivin expression and the result of proliferation assay (WST-1 assay) with 5-FU treatment in CD133{sup +} and siRNA-induced CD133{sup −} cells, derived from Caco-2 colon cancer cell line. 5-FU treatment induced significantly increase of the mRNA expression of MDR1, ABCG2 and AKT1genes, but not protein level. CD133 had little to no effect on the mRNA and protein expression of these genes. However, survivin expression at mRNA and protein level were significantly increased in CD133{sup +} cells compared with siRNA-induced CD133-cells and Mock (not sorted CD133{sup +} cells) at 96 h after siRNA transfection. The cytotoxicity assay demonstrated notable increase of chemoresistance to 5-FU treatment (10 μM) in CD133{sup +} cells at 96 h after siRNA transfection. From this study, we conclude that CD133{sup +} cells may have chemoresistance to 5-FU through the mechanism which is related with survivin expression, instead of MDR1, ABCG2 and AKT1 expression. Therefore a survivin inhibitor can be a new target for effective treatment of CD133{sup +} colon cancer. - Highlights: • We evaluate the role of CD133 in chemoresistance of colon cancer. • We compared the chemoresistance of CD133{sup +} cells and siRNA-induced CD133{sup −} cells. • CD133 had little to no effect on MDR1, ABCG2 and AKT1 expression. • Survivin expression and chemoresistance were increased in CD133{sup +} colon cancer cells.

  8. Adipocyte-derived lipids increase angiotensin-converting enzyme (ACE) expression and modulate macrophage phenotype.

    PubMed

    Kohlstedt, Karin; Trouvain, Caroline; Namgaladze, Dmitry; Fleming, Ingrid

    2011-03-01

    Human monocytes/macrophages express the angiotensin-converting enzyme (ACE) but nothing is known about its role under physiological conditions. As adipose tissue contains resident macrophages that have been implicated in the generation of insulin resistance in expanding fat mass, we determined whether adipocytes release factors that affect ACE expression and function in monocytes. Incubation of human monocyte-derived macrophages with conditioned medium from freshly isolated human adipocytes (BMI = 25.4 ± 0.96) resulted in a 4-fold increase in ACE expression. The effect was insensitive to denaturation and different proteases but abolished after lipid extraction. mRNA levels of the major histocompatibility complex class II protein increased in parallel with ACE, whereas the expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-6, and cyclooxygenase-2 decreased. As a consequence of the reduction in MCP-1, monocyte recruitment was also attenuated. Moreover, adipocyte-conditioned medium prevented the interferon (IFN)-γ induced formation of TNF-α, IL-6, and MCP-1, all markers of classically-activated (M1 type) macrophages. The decrease in cytokine expression in adipocyte-conditioned medium-treated macrophages was sensitive to ACE silencing by small interfering RNA (siRNA). Accordingly, ACE overexpression in THP-1 cells mimicked the effect of adipocyte-conditioned medium. In both cell types, ACE inhibition failed to affect the changes induced by adipocyte conditioned-medium treatment and ACE overexpression. Thus, the modulation of macrophage polarization by ACE appears to be mediated independently of enzyme activity, probably via intracellular signaling. Interestingly, human macrophage ACE expression was also upregulated by IL-4 and IL-13, which promote the "alternative" activation of macrophages and decreased by LPS and IFN-γ. Mechanistically, adipocyte-conditioned medium stimulated the phosphorylation of

  9. Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity.

    PubMed Central

    Hirschi, K D

    1999-01-01

    Calcium (Ca(2)+) efflux from the cytosol modulates Ca(2+) concentrations in the cytosol, loads Ca(2+) into intracellular compartments, and supplies Ca(2+) to organelles to support biochemical functions. The Ca(2+)/H(+) antiporter CAX1 (for CALCIUM EXCHANGER 1) of Arabidopsis is thought to be a key mediator of these processes. To clarify the regulation of CAX1, we examined CAX1 RNA expression in response to various stimuli. CAX1 was highly expressed in response to exogenous Ca(2+). Transgenic tobacco plants expressing CAX1 displayed symptoms of Ca(2+) deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium concentrations, and to cold shock, but increasing the Ca(2+) in the media abrogated these sensitivities. Tobacco plants expressing CAX1 also demonstrated increased Ca(2+) accumulation and altered activity of the tonoplast-enriched Ca(2+)/H(+) antiporter. These results emphasize that regulated expression of Ca(2+)/H(+) antiport activity is critical for normal growth and adaptation to certain stresses. PMID:10559438

  10. Exercise-Mediated Increase in Nigral Tyrosine Hydroxylase Is Accompanied by Increased Nigral GFR-α1 and EAAC1 Expression in Aging Rats

    PubMed Central

    Arnold, Jennifer C.; Salvatore, Michael F.

    2016-01-01

    Exercise may alleviate locomotor impairment in Parkinson's disease (PD) or aging. Identifying molecular responses immediately engaged by exercise in the nigrostriatal pathway and allied tissue may reveal critical targets associated with its long-term benefits. In aging, there is loss of tyrosine hydroxylase (TH) and the glial cell line-derived neurotrophic factor (GDNF) receptor, GFR-α1, in the substantia nigra (SN). Exercise can increase GDNF expression, but its effect on GFR-α1 expression is unknown. Infusion of GDNF into striatum or GFR-α1 in SN, respectively, can increase locomotor activity and TH function in SN but not striatum in aged rats. GDNF may also increase glutamate transporter expression, which attenuates TH loss in PD models. We utilized a footshock-free treadmill exercise regimen to determine the immediate impact of short-term exercise on GFR-α1 expression, dopamine regulation, glutamate transporter expression, and glutamate uptake in 18 month old male Brown-Norway/Fischer 344 F1 hybrid rats. GFR-α1 and TH expression significantly increased in SN but not striatum. This exercise regimen did not affect glutamate uptake or glutamate transporter expression in striatum. However, EAAC1 expression increased in SN. These results indicate that nigral GFR-α1 and EAAC1 expression increased in conjunction with increased nigral TH expression following short-term exercise. PMID:26599339

  11. Myotonic Dystrophy: Increased expression of the normal allele in CDM infants muscle

    SciTech Connect

    Radvanyi, H.H.; Gourdon, G.; Junien, C. |

    1994-09-01

    Myotonic dystrophy (DM) is an autosomal dominant multisystemic disorder characterized by a highly variable clinical phenotype. The mutation has been identified as an unstable trinucleotide CTG repeat in the 3{prime} untranslated region of the myotonin-protein kinase (MT-PK) gene. Congenital myotonic dystrophy (CDM), which represents the most severe phenotype, is exclusively maternally inherited. Recent studies, analysis by Northern blots and RT-PCR provided apparently conflicting results on the mutated allele expression in samples from congenitally affected children. The level of expression of the mutant allele depends on the extent of the repeat in the adult form and is no longer expressed when over 800-1300 repeats, whether in adult forms or in CDM. Could this decrease account for the late onset forms? However, the differences between the two phenotypes cannot be explained by the same mechanism. Alternatively, these differences could be due to differences in expression of the normal allele. We analyzed by quantitative RT-PCR the expression of the MT-PK gene in muscle samples from four CDM infants and two aged-matched normal controls. In two of these, the mutant allele (3.3 and 8 kb) was undetectable on Northern blots. We observed an increased expression of the MT-PK gene (10- to 20-fold) in tissues of severely affected congenital patients which can be attributed to the normal allele. Since expression of the normal allele is either normal or slightly decreased in the adult form, the dramatic increase in the congenital form could reflect a disturbance in muscle differentiation. Expression studies of MT-PK at different stages of development and, especially after the 20th week, are therefore required.

  12. Expression of the peptide hormone hepcidin increases in cardiomyocytes under myocarditis and myocardial infarction.

    PubMed

    Isoda, Manabu; Hanawa, Haruo; Watanabe, Ritsuo; Yoshida, Tsuyoshi; Toba, Ken; Yoshida, Kaori; Kojima, Mayuko; Otaki, Keita; Hao, Kazuhisa; Ding, Limin; Tanaka, Komei; Takayama, Tsugumi; Kato, Kiminori; Okura, Yuji; Kodama, Makoto; Ota, Yoshimi; Hayashi, Junichi; Aizawa, Yoshifusa

    2010-08-01

    The micronutrient iron is an essential component that plays a role in many crucial metabolic reactions. The peptide hormone hepcidin is thought to play a central role in iron homeostasis and its expression is induced by iron overloading and inflammation. Recently, hepcidin has been reported to be expressed also in the heart; however, the kinetics of altered hepcidin expression in diseases of the heart remain unknown. In this study, we examined cardiac expression of hepcidin in rat experimental autoimmune myocarditis (EAM), human myocarditis and rat acute myocardial infarction (AMI). In rat EAM and AMI hearts, hepcidin was expressed in cardiomyocytes; ferroportin, which is a cellular iron exporter bound by hepcidin, was also expressed in various cells. Analysis of the time course of the hepcidin to cytochrome oxidase subunit 6a (Cox6a)2 expression ratio showed that it abruptly increased more than 100-fold in hearts in the very early phase of EAM and in infarcted areas 1 day after MI. The hepcidin/Cox6a2 expression ratio correlated significantly with that of interleukin-6/gamma-actin in both EAM and AMI hearts (r=0.781, P<.0001 and r=0.563, P=.0003). In human hearts with histological myocarditis, the ratio was significantly higher than in those without myocarditis (0.0400+/-0.0195 versus 0.0032+/-0.0017, P=.0045). Hepcidin is strongly induced in cardiomyocytes under myocarditis and MI, conditions in which inflammatory cytokine levels increase and may play an important role in iron homeostasis and free radical generation. PMID:19615879

  13. Long-Term Increased Carnitine Palmitoyltransferase 1A Expression in Ventromedial Hypotalamus Causes Hyperphagia and Alters the Hypothalamic Lipidomic Profile

    PubMed Central

    Fabriàs, Gemma; Casas, Josefina; Costa, Ana S. H.; Malandrino, Maria Ida; Fernández-López, José-Antonio; Remesar, Xavier; Gao, Su; Chohnan, Shigeru; Rodríguez-Peña, Maria Sol; Petry, Harald; Asins, Guillermina; Hegardt, Fausto G.; Herrero, Laura; Serra, Dolors

    2014-01-01

    Lipid metabolism in the ventromedial hypothalamus (VMH) has emerged as a crucial pathway in the regulation of feeding and energy homeostasis. Carnitine palmitoyltransferase (CPT) 1A is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation and it has been proposed as a crucial mediator of fasting and ghrelin orexigenic signalling. However, the relationship between changes in CPT1A activity and the intracellular downstream effectors in the VMH that contribute to appetite modulation is not fully understood. To this end, we examined the effect of long-term expression of a permanently activated CPT1A isoform by using an adeno-associated viral vector injected into the VMH of rats. Peripherally, this procedure provoked hyperghrelinemia and hyperphagia, which led to overweight, hyperglycemia and insulin resistance. In the mediobasal hypothalamus (MBH), long-term CPT1AM expression in the VMH did not modify acyl-CoA or malonyl-CoA levels. However, it altered the MBH lipidomic profile since ceramides and sphingolipids increased and phospholipids decreased. Furthermore, we detected increased vesicular γ-aminobutyric acid transporter (VGAT) and reduced vesicular glutamate transporter 2 (VGLUT2) expressions, both transporters involved in this orexigenic signal. Taken together, these observations indicate that CPT1A contributes to the regulation of feeding by modulating the expression of neurotransmitter transporters and lipid components that influence the orexigenic pathways in VMH. PMID:24819600

  14. Long-term increased carnitine palmitoyltransferase 1A expression in ventromedial hypotalamus causes hyperphagia and alters the hypothalamic lipidomic profile.

    PubMed

    Mera, Paula; Mir, Joan Francesc; Fabriàs, Gemma; Casas, Josefina; Costa, Ana S H; Malandrino, Maria Ida; Fernández-López, José-Antonio; Remesar, Xavier; Gao, Su; Chohnan, Shigeru; Rodríguez-Peña, Maria Sol; Petry, Harald; Asins, Guillermina; Hegardt, Fausto G; Herrero, Laura; Serra, Dolors

    2014-01-01

    Lipid metabolism in the ventromedial hypothalamus (VMH) has emerged as a crucial pathway in the regulation of feeding and energy homeostasis. Carnitine palmitoyltransferase (CPT) 1A is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation and it has been proposed as a crucial mediator of fasting and ghrelin orexigenic signalling. However, the relationship between changes in CPT1A activity and the intracellular downstream effectors in the VMH that contribute to appetite modulation is not fully understood. To this end, we examined the effect of long-term expression of a permanently activated CPT1A isoform by using an adeno-associated viral vector injected into the VMH of rats. Peripherally, this procedure provoked hyperghrelinemia and hyperphagia, which led to overweight, hyperglycemia and insulin resistance. In the mediobasal hypothalamus (MBH), long-term CPT1AM expression in the VMH did not modify acyl-CoA or malonyl-CoA levels. However, it altered the MBH lipidomic profile since ceramides and sphingolipids increased and phospholipids decreased. Furthermore, we detected increased vesicular γ-aminobutyric acid transporter (VGAT) and reduced vesicular glutamate transporter 2 (VGLUT2) expressions, both transporters involved in this orexigenic signal. Taken together, these observations indicate that CPT1A contributes to the regulation of feeding by modulating the expression of neurotransmitter transporters and lipid components that influence the orexigenic pathways in VMH. PMID:24819600

  15. Increased Expression of Intranuclear Matrix Metalloproteinase 9 in Atrophic Renal Tubules Is Associated with Renal Fibrosis

    PubMed Central

    Tsai, Jen-Pi; Liou, Jia-Hung; Kao, Wei-Tse; Wang, Shao-Chung; Lian, Jong-Da; Chang, Horng-Rong

    2012-01-01

    Background Reduced turnover of extracellular matrix has a role in renal fibrosis. Matrix metalloproteinases (MMPs) is associated with many glomerular diseases, but the histological association of MMPs and human renal fibrosis is unclear. Methods This is a retrospective study. Institutional Review Board approval was obtained for the review of patients’ medical records, data analysis and pathological specimens staining with waiver of informed consents. Specimens of forty-six patients were examined by immunohistochemical stain of MMP-9 in nephrectomized kidneys, and the association of renal expression of MMP-9 and renal fibrosis was determined. MMP-9 expression in individual renal components and fibrosis was graded as high or low based on MMP-9 staining and fibrotic scores. Results Patients with high interstitial fibrosis scores (IFS) and glomerular fibrosis scores (GFS) had significantly higher serum creatinine, lower estimated glomerular filtration rate (eGFR), and were more likely to have chronic kidney disease (CKD) and urothelial cell carcinoma. Univariate analysis showed that IFS and GFS were negatively associated with normal and atrophic tubular cytoplasmic MMP-9 expression and IFS was positively correlated with atrophic tubular nuclear MMP-9 expression. Multivariate stepwise regression indicated that MMP-9 expression in atrophic tubular nuclei (r = 0.4, p = 0.002) was an independent predictor of IFS, and that MMP-9 expression in normal tubular cytoplasm (r = −0.465, p<0.001) was an independent predictor of GFS. Conclusions Interstitial fibrosis correlated with MMP-9 expression in the atrophic tubular nuclei. Our results indicate that renal fibrosis is associated with a decline of MMP-9 expression in the cytoplasm of normal tubular cells and increased expression of MMP-9 in the nuclei of tubular atrophic renal tubules. PMID:23110201

  16. Increased expression of the antiapoptotic protein MCL1 in canine mast cell tumors.

    PubMed

    Amagai, Yosuke; Tanaka, Akane; Matsuda, Akira; Oida, Kumiko; Jung, Kyungsook; Nishikawa, Sho; Jang, Hyosun; Ishizaka, Saori; Matsuda, Hiroshi

    2013-07-31

    Myeloid cell leukemia sequence 1 (MCL1) is a potent antiapoptotic protein that plays a critical role in cell survival and drug resistance in various cancers. However, to the best of our knowledge, the role of MCL1 in mast cell tumors (MCTs) has not been investigated in dogs. Here, we detected increased MCL1 expression in MCT cell lines, regardless of the presence of a c-kit mutation. MCL1 expression increased when the cells were exposed to specific inhibitors of mitogen-activated protein kinase or Janus kinase-signaling pathways, thus protecting the cells from apoptosis, but not when KIT or phosphatidylinositol-3 kinase signaling cascades were inhibited. These results indicate that MCL1 expression may contribute to MCT survival and confer drug resistance. PMID:23428776

  17. Increased IMP dehydrogenase gene expression in solid tumor tissues and tumor cell lines

    SciTech Connect

    Collart, F.R.; Chubb, C.B.; Mirkin, B.L.; Huberman, E.

    1992-07-10

    IMP dehydrogenase, a regulatory enzyme of guanine nucleotide biosynthesis, may play a role in cell proliferation and malignancy. To assess this possibility, we examined IMP dehydrogenase expression in a series of human solid tumor tissues and tumor cell lines in comparison with their normal counterparts. Increased IMP dehydrogenase gene expression was observed in brain tumors relative to normal brain tissue and in sarcoma cells relative to normal fibroblasts. Similarly, in several B- and T-lymphoid leukemia cell lines, elevated levels of IMP dehydrogenase mRNA and cellular enzyme were observed in comparison with the levels in peripheral blood lymphocytes. These results are consistent with an association between increased IMP dehydrogenase expression and either enhanced cell proliferation or malignant transformation.

  18. Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression.

    PubMed

    Liu, Xin-Hua; Yao, Shen; Qiao, Rui-Fang; Levine, Alice C; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Bauman, William A; Cardozo, Christopher P

    2011-10-14

    Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy. PMID:21945932

  19. Cholesteryl ester hydroperoxides increase macrophage CD36 gene expression via PPAR{alpha}

    SciTech Connect

    Jedidi, Iness; Couturier, Martine; Therond, Patrice; Gardes-Albert, Monique; Legrand, Alain; Barouki, Robert; Bonnefont-Rousselot, Dominique; Aggerbeck, Martine . E-mail: Martine.Aggerbeck@univ-paris5.fr

    2006-12-22

    The uptake of oxidized LDL by macrophages is a key event in the development of atherosclerosis. The scavenger receptor CD36 is one major receptor that internalizes oxidized LDL. In differentiated human macrophages, we compared the regulation of CD36 expression by copper-oxidized LDL or their products. Only oxidized derivatives of cholesteryl ester (CEOOH) increased the amount of CD36 mRNA (2.5-fold). Both oxidized LDL and CEOOH treatment increased two to fourfold the transcription of promoters containing peroxisome-proliferator-activated-receptor responsive elements (PPRE) in the presence of PPAR{alpha} or {gamma}. Electrophoretic-mobility-shift-assays with nuclear extracts prepared from macrophages treated by either oxidized LDL or CEOOH showed increased binding of PPAR{alpha} to the CD36 gene promoter PPRE. In conclusion, CEOOH present in oxidized LDL increase CD36 gene expression in a pathway involving PPAR{alpha}.

  20. Nav1.7 expression is increased in painful human dental pulp

    PubMed Central

    Luo, Songjiang; Perry, Griffin M; Levinson, S Rock; Henry, Michael A

    2008-01-01

    Background Animal studies and a few human studies have shown a change in sodium channel (NaCh) expression after inflammatory lesions, and this change is implicated in the generation of pain states. We are using the extracted human tooth as a model system to study peripheral pain mechanisms and here examine the expression of the Nav1.7 NaCh isoform in normal and painful samples. Pulpal sections were labeled with antibodies against: 1) Nav1.7, N52 and PGP9.5, and 2) Nav1.7, caspr (a paranodal protein used to identify nodes of Ranvier), and myelin basic protein (MBP), and a z-series of optically-sectioned images were obtained with the confocal microscope. Nav1.7-immunofluorescence was quantified in N52/PGP9.5-identified nerve fibers with NIH ImageJ software, while Nav1.7 expression in myelinated fibers at caspr-identified nodal sites was evaluated and further characterized as either typical or atypical as based on caspr-relationships. Results Results show a significant increase in nerve area with Nav1.7 expression within coronal and radicular fiber bundles and increased expression at typical and atypical caspr-identified nodal sites in painful samples. Painful samples also showed an augmentation of Nav1.7 within localized areas that lacked MBP, including those associated with atypical caspr-identified sites, thus identifying NaCh remodeling within demyelinating axons as the basis for a possible pulpal pain mechanism. Conclusion This study identifies the increased axonal expression and augmentation of Nav1.7 at intact and remodeling/demyelinating nodes within the painful human dental pulp where these changes may contribute to constant, increased evoked and spontaneous pain responses that characterize the pain associated with toothache. PMID:18426592

  1. Increased Cx32 expression in spinal cord TrkB oligodendrocytes following peripheral axon injury.

    PubMed

    Coulibaly, Aminata P; Isaacson, Lori G

    2016-08-01

    Following injury to motor axons in the periphery, retrograde influences from the injury site lead to glial cell plasticity in the vicinity of the injured neurons. Following the transection of peripherally located preganglionic axons of the cervical sympathetic trunk (CST), a population of oligodendrocyte (OL) lineage cells expressing full length TrkB, the cognate receptor for brain derived neurotrophic factor (BDNF), is significantly increased in number in the spinal cord. Such robust plasticity in OL lineage cells in the spinal cord following peripheral axon transection led to the hypothesis that the gap junction communication protein connexin 32 (Cx32), which is specific to OL lineage cells, was influenced by the injury. Following CST transection, Cx32 expression in the spinal cord intermediolateral cell column (IML), the location of the parent cell bodies, was significantly increased. The increased Cx32 expression was localized specifically to TrkB OLs in the IML, rather than other cell types in the OL cell lineage, with the population of Cx32/TrkB cells increased by 59%. Cx32 expression in association with OPCs was significantly decreased at one week following the injury. The results of this study provide evidence that peripheral axon injury can differentially affect the gap junction protein expression in OL lineage cells in the adult rat spinal cord. We conclude that the retrograde influences originating from the peripheral injury site elicit dramatic changes in the CNS expression of Cx32, which in turn may mediate the plasticity of OL lineage cells observed in the spinal cord following peripheral axon injury. PMID:27246301

  2. Increased expression of estrogen-related receptor β during adaptation of adult cardiomyocytes to sustained hypoxia

    PubMed Central

    Cunningham, Kathryn F; Beeson, Gyda C; Beeson, Craig C; McDermott, Paul J

    2016-01-01

    Estrogen-related Receptors (ERR) are members of the steroid hormone receptor superfamily of transcription factors that regulate expression of genes required for energy metabolism including mitochondrial biogenesis, fatty acid oxidation and oxidative phosphorylation. While ERRα and EPPγ isoforms are known to share a wide array of target genes in the adult myocardium, the function of ERRβ has not been characterized in cardiomyocytes. The purpose of this study was to determine the role of ERRβ in regulating energy metabolism in adult cardiomyocytes in primary culture. Adult feline cardiomyocytes were electrically stimulated to contract in either hypoxia (0.5% O2) or normoxia (21% O2). As compared to baseline values measured in normoxia, ERRβ mRNA levels increased significantly after 8 hours of hypoxia and remained elevated over 24 h. Conversely, ERRβ mRNA decreased to normoxic levels after 4 hours of reoxygenation. Hypoxia increased expression of the α and β isoforms of Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1) mRNA by 6-fold and 3-fold, respectively. Knockdown of ERRβ expression via adenoviral-mediated delivery of ERRβ shRNA blocked hypoxia-induced increases in PGC-1β mRNA, but not PGC-1α mRNA. Loss of ERRβ had no effect on mtDNA content as measured after 24 h of hypoxia. To determine whether loss of ERRβ affected mitochondrial function, oxygen consumption rates (OCR) were measured in contracting versus quiescent cardiomyocytes in normoxia. OCR was significantly lower in contracting cardiomyocytes expressing ERRβ shRNA than scrambled shRNA controls. Maximal OCR also was reduced by ERRβ knockdown. In conclusion: 1) hypoxia increases in ERRβ mRNA expression in contracting cardiomyocytes; 2) ERRβ is required for induction of the PGC-1β isoform in response to hypoxia; 3) ERRβ expression is required to sustain OCR in normoxic conditions. PMID:27335690

  3. Tumor Necrosis Factor B (TNFB) Genetic Variants and Its Increased Expression Are Associated with Vitiligo Susceptibility

    PubMed Central

    Laddha, Naresh C.; Dwivedi, Mitesh; Gani, Amina R.; Mansuri, Mohmmad Shoab; Begum, Rasheedunnisa

    2013-01-01

    Genetic polymorphisms in TNFB are involved in the regulation of its expression and are found to be associated with various autoimmune diseases. The aim of the present study was to determine whether TNFB +252A/G (rs909253) and exon 3 C/A (rs1041981) polymorphisms are associated with vitiligo susceptibility, and expression of TNFB and ICAM1 affects the disease onset and progression. We have earlier reported the role of TNFA in autoimmune pathogenesis of vitiligo, and we now show the involvement of TNFB in vitiligo pathogenesis. The two polymorphisms investigated in the TNFB were in strong linkage disequilibrium and significantly associated with vitiligo. TNFB and ICAM1 transcripts were significantly increased in patients compared to controls. Active vitiligo patients showed significant increase in TNFB transcripts compared to stable vitiligo. The genotype-phenotype analysis revealed that TNFB expression levels were higher in patients with GG and AA genotypes as compared to controls. Patients with the early age of onset and female patients showed higher TNFB and ICAM1 expression. Overall, our findings suggest that the increased TNFB transcript levels in vitiligo patients could result, at least in part, from variations at the genetic level which in turn leads to increased ICAM1 expression. For the first time, we show that TNFB +252A/G and exon 3 C/A polymorphisms are associated with vitiligo susceptibility and influence the TNFB and ICAM1 expression. Moreover, the study also emphasizes influence of TNFB and ICAM1 on the disease progression, onset and gender bias for developing vitiligo. PMID:24312346

  4. 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

  5. An Analysis of Naturalistic Interventions for Increasing Spontaneous Expressive Language in Children with Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Lane, Justin D.; Lieberman-Betz, Rebecca; Gast, David L.

    2016-01-01

    The purpose of this review was to identify naturalistic language interventions for increasing spontaneous expressive language (defined in this review as absence of verbal prompt or other verbalization from adults or peers) in young children with autism spectrum disorder. Also, the methodological rigor and effectiveness of each study were evaluated…

  6. Methotrexate increases skeletal muscle GLUT4 expression and improves metabolic control in experimental diabetes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Long-term administration of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) mimics the effects of endurance exercise by activating AMP kinase and by increasing skeletal muscle expression of GLUT4 glucose transporter. AICAR is an intermediate in the purine de novo synthesis, and its tissue conc...

  7. Use of CYP52A2A promoter to increase gene expression in yeast

    DOEpatents

    Craft, David L.; Wilson, C. Ron; Eirich, Dudley; Zhang, Yeyan

    2004-01-06

    A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

  8. Increased hepatic CD36 expression contributes to dyslipidemia associated with diet-induced obesity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The etiology of type 2 diabetes often involves diet-induced obesity (DIO), which is associated with elevated plasma fatty acids and lipoprotein associated triglycerides. Since aberrant hepatic fatty acid uptake may contribute to this, we investigated whether increased expression of a fatty acid tran...

  9. Natural variations in expression of regulatory and detoxification related genes under limiting phosphate and arsenate stress in Arabidopsis thaliana

    PubMed Central

    Shukla, Tapsi; Kumar, Smita; Khare, Ria; Tripathi, Rudra D.; Trivedi, Prabodh K.

    2015-01-01

    Abiotic stress including nutrient deficiency and heavy metal toxicity severely affects plant growth, development, and productivity. Genetic variations within and in between species are one of the important factors in establishing interactions and responses of plants with the environment. In the recent past, natural variations in Arabidopsis thaliana have been used to understand plant development and response toward different stresses at genetic level. Phosphorus deficiency negatively affects plant growth and metabolism and modulates expression of the genes involved in Pi homeostasis. Arsenate, As(V), a chemical analog of Pi, is taken up by the plants via phosphate transport system. Studies suggest that during Pi deficiency, enhanced As(V) uptake leads to increased toxicity in plants. Here, the natural variations in Arabidopsis have been utilized to study the As(V) stress response under limiting Pi condition. The primary root length was compared to identify differential response of three Arabidopsis accessions (Col-0, Sij-1, and Slavi-1) under limiting Pi and As(V) stress. To study the molecular mechanisms responsible for the differential response, comprehensive expression profiling of the genes involved in uptake, detoxification, and regulatory mechanisms was carried out. Analysis suggests genetic variation-dependent regulatory mechanisms may affect differential response of Arabidopsis natural variants toward As(V) stress under limiting Pi condition. Therefore, it is hypothesized that detailed analysis of the natural variations under multiple stress conditions might help in the better understanding of the biological processes involved in stress tolerance and adaptation. PMID:26557133

  10. The Expression of LIGHT Was Increased and the Expression of HVEM and BTLA Were Decreased in the T Cells of Patients with Rheumatoid Arthritis

    PubMed Central

    Zhang, Junlong; Liao, Yun; Li, Linhui; Liu, Xinle; Wu, Zhiqiang; Cai, Bei; Bai, Yangjuan; Wang, Lanlan

    2016-01-01

    Background Currently, the pathogenesis of rheumatoid arthritis (RA) is not clearly understood. The LIGHT/HVEM/BTLA co-signaling pathway may be involved in the pathogenesis of RA, although reports on the expression levels of LIGHT, HVEM and BTLA in T lymphocytes from RA patients are limited. Method In this study, we recruited 30 healthy controls and 21 RA patients. Clinical characteristics were collected for RA patients. The levels of LIGHT, HVEM and BTLA expressed on the surface of circulating T cells of RA patients and healthy controls were measured by flow cytometry. Result The percentages of CD3+, CD4+ and CD8+ T lymphocytes that expressed BTLA from RA patients were all higher than those of the controls (all p < 0.05), while the percentages of CD3+, CD4+ and CD8+ T lymphocytes that expressed HVEM and LIGHT were all lower than those of the controls (all p < 0.05). The rheumatoid factor and the percentage of HVEM+CD4+ T lymphocytes showed a statistically significant negative correlation in RA patients (r = -0.453, p = 0.039), as did the swollen joint count and the percentage of BTLA+CD8+ T lymphocytes (r = -0.501, p = 0.021). Conclusion Here, we provide the first report on the increased expression of BTLA in T lymphocytes and on the decreased expression of HVEM and LIGHT in RA patients. BTLA, HVEM and LIGHT might be involved in the pathogenesis of RA and have the potential to be new clinically useful characteristics of RA. PMID:27183113

  11. Dexamethasone increases aquaporin-2 protein expression in ex vivo inner medullary collecting duct suspensions

    PubMed Central

    Chen, Minguang; Cai, Hui; Klein, Janet D.; Laur, Oskar; Chen, Guangping

    2015-01-01

    Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and plays an important role in the maintenance of body water homeostasis. Excessive glucocorticoid as often seen in Cushing's syndrome causes water retention. However, whether and how glucocorticoid regulates AQP2 remains unclear. In this study, we examined the direct effect of dexamethasone on AQP2 protein expression and activity. Dexamethasone increased AQP2 protein abundance in rat inner medullary collecting duct (IMCD) suspensions. This was confirmed in HEK293 cells transfected with AQP2 cDNA. Cell surface protein biotinylation showed an increase of dexamethasone-induced cell membrane AQP2 expression and this effect was blocked by glucocorticoid receptor antagonist RU486. Functionally, dexamethasone treatment of oocytes injected with an AQP2 cRNA increased water transport activity as judged by cell rupture time in a hypo-osmotic solution (66 ± 13 s in dexamethasone vs. 101 ± 11 s in control, n = 15). We further found that dexamethasone treatment reduced AQP2 protein degradation, which could result in an increase of AQP2 protein. Interestingly, dexamethasone promoted cell membrane AQP2 moving to less buoyant lipid raft submicrodomains. Taken together, our data demonstrate that dexamethasone promotes AQP2 protein expression and increases water permeability mainly via inhibition of AQP2 protein degradation. The increase in AQP2 activity promotes water reabsorption, which may contribute to glucocorticoid-induced water retention and hypertension. PMID:26578982

  12. Dexamethasone increases aquaporin-2 protein expression in ex vivo inner medullary collecting duct suspensions.

    PubMed

    Chen, Minguang; Cai, Hui; Klein, Janet D; Laur, Oskar; Chen, Guangping

    2015-01-01

    Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and plays an important role in the maintenance of body water homeostasis. Excessive glucocorticoid as often seen in Cushing's syndrome causes water retention. However, whether and how glucocorticoid regulates AQP2 remains unclear. In this study, we examined the direct effect of dexamethasone on AQP2 protein expression and activity. Dexamethasone increased AQP2 protein abundance in rat inner medullary collecting duct (IMCD) suspensions. This was confirmed in HEK293 cells transfected with AQP2 cDNA. Cell surface protein biotinylation showed an increase of dexamethasone-induced cell membrane AQP2 expression and this effect was blocked by glucocorticoid receptor antagonist RU486. Functionally, dexamethasone treatment of oocytes injected with an AQP2 cRNA increased water transport activity as judged by cell rupture time in a hypo-osmotic solution (66 ± 13 s in dexamethasone vs. 101 ± 11 s in control, n = 15). We further found that dexamethasone treatment reduced AQP2 protein degradation, which could result in an increase of AQP2 protein. Interestingly, dexamethasone promoted cell membrane AQP2 moving to less buoyant lipid raft submicrodomains. Taken together, our data demonstrate that dexamethasone promotes AQP2 protein expression and increases water permeability mainly via inhibition of AQP2 protein degradation. The increase in AQP2 activity promotes water reabsorption, which may contribute to glucocorticoid-induced water retention and hypertension. PMID:26578982

  13. Increased expression of the prolactin receptor is associated with malignant laryngeal tumors

    PubMed Central

    GONZÁLEZ-LUCANO, LUIS R.; MUÑOZ-VALLE, JOSÉ F.; ASCENCIO-CEDILLO, RAFAEL; DOMÍNGUEZ-ROSALES, JOSÉ A.; LÓPEZ-RINCÓN, GONZALO; DEL TORO-ARREOLA, SUSANA; BUENO-TOPETE, MIRIAM; DANERI-NAVARRO, ADRIÁN; ESTRADA-CHÁVEZ, CIRO; PEREIRA-SUÁREZ, ANA L.

    2012-01-01

    The altered expression of the prolactin receptor (PRLR) has been associated with the development of various types of cancer, particularly breast, prostate and endometrial cancer. However, in laryngeal tumors, the expression of PRLR has not yet been documented. The aim of this study was to determine the expression and localization of PRLR in laryngeal cancer (LC) in comparison with recurrent respiratory papillomatosis (RRP). PRLR expression was analyzed in 48 paraffin-embedded tissues (18 RRP and 30 laryngeal cancer tissues) by immunoperoxidase staining. Furthermore, PRLR expression was evaluated in ten samples from each group by Western blot analysis and quantitative real-time PCR. PRLR was observed in all laryngeal tumors at different intensities. PRLR overexpression was significantly associated (P<0.005) with LC. The staining pattern was homogeneous, mainly cytoplasmic, and confined to the tumor area. We found increased expression of different isoforms in LC in comparison with RRP. Our results suggest a possible role of PRL/PRLR in the development of LC. PRLR may be useful as a target for further investigations in laryngeal tissues. PMID:22969936

  14. Lhx4 Deficiency: Increased Cyclin-Dependent Kinase Inhibitor Expression and Pituitary Hypoplasia

    PubMed Central

    Gergics, Peter; Brinkmeier, Michelle L.

    2015-01-01

    Defects in the Lhx4, Lhx3, and Pitx2 genes can cause combined pituitary hormone deficiency and pituitary hypoplasia in both humans and mice. Not much is known about the mechanism underlying hypoplasia in these mutants beyond generally increased cell death and poorly maintained proliferation. We identified both common and unique abnormalities in developmental regulation of key cell cycle regulator gene expression in each of these three mutants. All three mutants exhibit reduced expression of the proliferative marker Ki67 and the transitional marker p57. We discovered that expression of the cyclin-dependent kinase inhibitor 1a (Cdkn1a or p21) is expanded dorsally in the pituitary primordium of both Lhx3 and Lhx4 mutants. Uniquely, Lhx4 mutants exhibit reduced cyclin D1 expression and have auxiliary pouch-like structures. We show evidence for indirect and direct effects of LHX4 on p21 expression in αT3-1 pituitary cells. In summary, Lhx4 is necessary for efficient pituitary progenitor cell proliferation and restriction of p21 expression. PMID:25668206

  15. Deletion of Rictor in brain and fat alters peripheral clock gene expression and increases blood pressure.

    PubMed

    Drägert, Katja; Bhattacharya, Indranil; Pellegrini, Giovanni; Seebeck, Petra; Azzi, Abdelhalim; Brown, Steven A; Georgiopoulou, Stavroula; Held, Ulrike; Blyszczuk, Przemyslaw; Arras, Margarete; Humar, Rok; Hall, Michael N; Battegay, Edouard; Haas, Elvira

    2015-08-01

    The mammalian target of rapamycin complex 2 (mTORC2) contains the essential protein RICTOR and is activated by growth factors. mTORC2 in adipose tissue contributes to the regulation of glucose and lipid metabolism. In the perivascular adipose tissue, mTORC2 ensures normal vascular reactivity by controlling expression of inflammatory molecules. To assess whether RICTOR/mTORC2 contributes to blood pressure regulation, we applied a radiotelemetry approach in control and Rictor knockout (Rictor(aP2KO)) mice generated using adipocyte protein-2 gene promoter-driven CRE recombinase expression to delete Rictor. The 24-hour mean arterial pressure was increased in Rictor(aP2KO) mice, and the physiological decline in mean arterial pressure during the dark period was impaired. In parallel, heart rate and locomotor activity were elevated during the dark period with a pattern similar to blood pressure changes. This phenotype was associated with mild cardiomyocyte hypertrophy, decreased cardiac natriuretic peptides, and their receptor expression in adipocytes. Moreover, clock gene expression was reduced or phase-shifted in perivascular adipose tissue. No differences in clock gene expression were observed in the master clock suprachiasmatic nucleus, although Rictor gene expression was also lower in brain of Rictor(aP2KO) mice. Thus, this study highlights the importance of RICTOR/mTORC2 for interactions between vasculature, adipocytes, and brain to tune physiological outcomes, such as blood pressure and locomotor activity. PMID:26101345

  16. Brain SERT Expression of Male Rats Is Reduced by Aging and Increased by Testosterone Restitution

    PubMed Central

    Herrera-Pérez, José Jaime; Fernández-Guasti, Alonso; Martínez-Mota, Lucía

    2013-01-01

    In preclinical and clinical studies aging has been associated with a deteriorated response to antidepressant treatment. We hypothesize that such impairment is explained by an age-related decrease in brain serotonin transporter (SERT) expression associated with low testosterone (T) levels. The objectives of this study were to establish (1) if brain SERT expression is reduced by aging and (2) if the SERT expression in middle-aged rats is increased by T-restitution. Intact young rats (3–5 months) and gonad-intact middle-aged rats with or without T-restitution were used. The identification of the brain SERT expression was done by immunofluorescence in prefrontal cortex, lateral septum, hippocampus, and raphe nuclei. An age-dependent reduction of SERT expression was observed in all brain regions examined, while T-restitution recovered the SERT expression only in the dorsal raphe of middle-aged rats. This last action seems relevant since dorsal raphe plays an important role in the antidepressant action of selective serotonin reuptake inhibitors. All data suggest that this mechanism accounts for the T-replacement usefulness to improve the response to antidepressants in the aged population. PMID:26317087

  17. The expression of marker for endometrial stem cell and fibrosis was increased in intrauterine adhesious

    PubMed Central

    Hu, Jianguo; Zeng, Biao; Jiang, Xingwei; Hu, lina; Meng, Ying; Zhu, Yi; Mao, Min

    2015-01-01

    Objectives: The objective of the present study was to evaluate whether fibrotic markers and endometrial stem cell markers were abnormal expressed in endometrium of intrauterine adhesions and a female mouse model for intrauterine adhesions. Methods: We revaluated endometrial fibrosis using Masson’s stain. We detected the expression of endometrium stem cell markers (CD146 and CD140b) and fibrosis markers (TGF-Beta, CTGF, collagen protein I and collagen protein III) in endometrial tissue with intrauterine adhesions using real-time PCR and S-P (Streptavidin-Peroxidase) immunohistochemistry. We create a female mouse model for intrauterine adhesions using mechanical injury, and then revalue the expression of endometrial stem cell markers and fibrosis markers in endometrial tissue of mouse model for intrauterine adhesions. Results: The ratio of the area with endometrial fibrosis to total endometrial area in intrauterine adhesious significantly increased compared with the normal endometrial tissue (P < 0.05); The expression levels of fibrotic markers and endometrial stem cell markers were higher in the endometrial tissue with intrauterine adhesious compared to normal endometrial tissue (P < 0.05). The animal experiments showed that the ratio of the area with endometrial fibrosis to total endometrial area significantly increased compared with the control group (P < 0.05); The expression levels of fibrotic markers and endometrial stem cell markers were higher in the endometrial tissue compared to the control group (P < 0.05). Conclusion: Aberrant activation of fibrosis may be involved in the pathology of intrauterine adhesious. PMID:25973037

  18. Age-related increase of VGF-expression in T lymphocytes

    PubMed Central

    Micheel, Justus; Dobrowolny, Henrik; Mawrin, Christian; Krause, Tim J.; Adamaszek, Michael; Bogerts, Bernhard; Bommhardt, Ursula; Hartig, Roland; Busse, Mandy

    2014-01-01

    VGF is a protein expressed by neurons and processed into several peptides. It plays a role in energy homeostasis and promotes growth and survival. Recently, VGF mRNA was detected in peripheral leukocytes. Since it is known that aging is associated with a decrease in the development and function of neuronal as well as immune cells, we addressed the question whether a peripheral expression of VGF by CD3+ T cells and CD56+ NK cells is correlated with age. Therefore, the frequency of VGF+CD3+ and VGF+CD56+ cells was determined in mentally healthy volunteers aged between 22 and 88. We found an age-dependent increase in the number of VGF+CD3+ T cells that correlated with HbA1c and the body mass index (BMI). VGF-expression by NK cells was age-independent. Blockade of VGF reduced proliferation and secretion of cytokines such as IL-2, IL-17A, IL-1β, IL-10 and TNF by CD3+ T cells and PBMCs. Rapamycin-mediated T cell blockade significantly reduced the frequency of VGF-expressing T cells. We conclude that VGF contributes to survival and function of peripheral T cells. The age-dependent increase in VGF-expression could serve as mechanism that counterregulates the decrease in functionality of T lymphocytes. PMID:25013207

  19. AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44.

    PubMed Central

    Lamb, R F; Hennigan, R F; Turnbull, K; Katsanakis, K D; MacKenzie, E D; Birnie, G D; Ozanne, B W

    1997-01-01

    Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in tumor cell invasion and metastasis. In contrast to normal 208F rat fibroblasts, Fos-transformed 208F fibroblasts are growth factor independent for invasion. We demonstrate that invasion of v-Fos- or epidermal growth factor (EGF)-transformed cells requires AP-1 activity. v-Fos-transformed cell invasion is inhibited by c-jun antisense oligonucleotides and by expression of a c-jun dominant negative mutant, TAM-67. EGF-induced invasion is inhibited by both c-fos and c-jun antisense oligonucleotides. CD44s, the standard form of a transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. We demonstrate that increased expression of CD44 in Fos- and EGF-transformed cells is dependent upon AP-1. CD44 antisense oligonucleotides reduce expression of CD44 in v-Fos- or EGF-transformed cells and inhibit invasion but not migration. Expression of a fusion protein between human CD44s and Aequorea victoria green fluorescent protein (GFP) in 208F cells complements the inhibition of invasion by the rat-specific CD44 antisense oligonucleotide. We further show that both v-Fos and EGF transformations result in a concentration of endogenous CD44 or exogenous CD44-GFP at the ends of pseudopodial cell extensions. These results support the hypothesis that one role of AP-1 in transformation is to activate a multigenic invasion program. PMID:9001250

  20. 76 FR 30035 - Fisheries of the Northeastern United States; Northeast Multispecies Fishery; Trip Limit Increase...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-24

    ... (13,607.8 kg) per trip. However, the final rule implementing FW 45 (79 FR 23042; May 1, 2011... further approach the common pool sub-annual catch limit (sub-ACL). DATES: Effective May 19, 2011, through... pool sub-ACLs. For FY 2011, the common pool sub-ACL for GB cod is 218,528 lb (99 mt). The current...

  1. Increased expressions of ADAMTS-13 and apoptosis contribute to neuropathology during Toxoplasma gondii encephalitis in mice.

    PubMed

    Dincel, Gungor Cagdas; Atmaca, Hasan Tarik

    2016-06-01

    Toxoplasma gondii (T. gondii) is a protozoan parasite with the potential of causing severe encephalitis among immunocompromised humans and animals. Our previous study showed that T. gondii induces high nitric oxide (NO) production, high glial activation (GFAP) and neurofilament expressions, leading to severe neurodegeneration in toxoplasma encephalitis (TE) in the central nervous system (CNS). The aim of this experimental study was to investigate ADAMTS-13 expression and apoptosis in CNS and to identify whether they have any correlation with toxoplasmosis neuropathology and neurodegeneration. Mice were infected with ME49 strain T. gondii and the levels of ADAMTS-13, caspase 3, caspase 8, caspase 9, TNFR1 and Bcl-xL expressions were examined in brain tissues by immunohistochemistry, during the development and establishment of chronic infections at 10, 30 and 60 days post-infection. Results of the study revealed that the levels of ADAMTS-13 (P < 0.005), caspase 3 (P < 0.05), caspase 8 (P < 0.05), caspase 9 (P < 0.005) and TNFR1 (P < 0.05) expressions in the brain markedly increased while Bcl-xL expression decreased (P < 0.005). The most prominent finding from our study was that 10, 30 and 60 days post-infection ADAMTS-13 increased significantly and this may play an important role in the regulation and protection of the blood-brain barrier integrity and CNS microenvironment in TE. These results also suggest that T. gondii-mediated apoptosis might play a pivotal role and a different type of role in the mechanism of neurodegeneration and neuropathology in the process of TE. Furthermore, expression of ADAMTS-13 might give an idea of the progress and is critical for diagnosis of this disease. To the best of the authors' knowledge, this is the first report on ADAMTS-13 expression in the CNS of T. gondii-infected mice. PMID:26542631

  2. Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

    SciTech Connect

    Feng, Shan-Li; Sun, Ming-Rui; Li, Ting-Ting; Yin, Xin; Xu, Chang-Qing; Sun, Yi-Hua

    2011-03-11

    Research highlights: {yields} Calcium-sensing receptor (CaR) activation stimulates TRP channels. {yields} CaR promoted transient receptor potential C3 (TRPC3) expression. {yields} Adult rat ventricular myocytes display capacitative calcium entry (CCE), which was operated by TRPCs. {yields} TRPC channels activation induced by CaR activator sustained the increased [Ca{sup 2+}]{sub i} to evoke cardiomyocytes apoptosis. -- Abstract: Transient receptor potential (TRP) channels are expressed in cardiomyocytes, which gate a type of influx of extracellular calcium, the capacitative calcium entry. TRP channels play a role in mediating Ca{sup 2+} overload in the heart. Calcium-sensing receptors (CaR) are also expressed in rat cardiac tissue and promote the apoptosis of cardiomyocytes by Ca{sup 2+} overload. However, data about the link between CaR and TRP channels in rat heart are few. In this study, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of the TRP canonical proteins TRPC1 and TRPC3 in adult and neonatal rat cardiomyocytes. Laser scan confocal microscopy was used to detect intracellular [Ca{sup 2+}]{sub i} levels in isolated adult rat ventricular myocytes. The results showed that, in adult rat cardiomyocytes, the depletion of Ca{sup 2+} stores in the endoplasmic/sarcoplasmic reticulum (ER/SR) by thapsigargin induced a transient increase in [Ca{sup 2+}]{sub i} in the absence of [Ca{sup 2+}]{sub o} and the subsequent restoration of [Ca{sup 2+}]{sub o} sustained the increased [Ca{sup 2+}]{sub i} for a few minutes, whereas, the persisting elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of the TRPC inhibitor SKF96365. The stimulation of CaR by its activator gadolinium chloride (GdCl{sub 3}) or spermine also resulted in the same effect and the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of [Ca{sup 2+}]{sub o}. In adult and neonatal rat cardiomyocytes, GdCl{sub 3

  3. Aqueous Tear Deficiency Increases Conjunctival Interferon-γ (IFN-γ) Expression and Goblet Cell Loss

    PubMed Central

    Pflugfelder, Stephen C.; De Paiva, Cintia S.; Moore, Quianta L.; Volpe, Eugene A.; Li, De-Quan; Gumus, Koray; Zaheer, Mahira L.; Corrales, Rosa M.

    2015-01-01

    Purpose To investigate the hypothesis that increased interferon-γ (IFN-γ) expression is associated with conjunctival goblet cell loss in subjects with tear dysfunction. Methods Goblet cell density (GCD) was measured in impression cytology from the temporal bulbar conjunctiva, and gene expression was measured in cytology samples from the nasal bulbar conjunctiva obtained from 68 subjects, including normal control, meibomian gland disease (MGD), non-Sjögren syndrome (non-SSATD)-, and Sjögren syndrome (SSATD)-associated aqueous tear deficiency. Gene expression was evaluated by real-time PCR. Tear meniscus height (TMH) was measured by optical coherence tomography. Fluorescein and lissamine green dye staining evaluated corneal and conjunctival disease, respectively. Between-group mean differences and correlation coefficients were calculated. Results Compared to control, IFN-γ expression was significantly higher in both ATD groups, and its receptor was higher in SSATD. Expression of IL-13 and its receptor was similar in all groups. Goblet cell density was lower in the SSATD group; expression of MUC5AC mucin was lower and cornified envelope precursor small proline-rich region (SPRR)-2G higher in both ATD groups. Interferon-γ transcript number was inversely correlated with GCD (r = −0.37, P < 0.04) and TMH (r = −0.37, P = 0.02), and directly correlated with lissamine green staining (r = 0.51, P < 0.001) and SPRR-2G expression (r = 0.32, P < 0.05). Conclusions Interferon-γ expression in the conjunctiva was higher in aqueous deficiency and correlated with goblet cell loss and severity of conjunctival disease. These results support findings of animal and culture studies showing that IFN-γ reduces conjunctival goblet cell number and mucin production. PMID:26618646

  4. Increased oxidative stress and antioxidant expression in mouse keratinocytes following exposure to paraquat

    SciTech Connect

    Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.; Laskin, Debra L. Heck, Diane E.; Laskin, Jeffrey D.

    2008-09-15

    Paraquat (1,1'-dimethyl-4,4'-bipyridinium) is a widely used herbicide known to induce skin toxicity. This is thought to be due to oxidative stress resulting from the generation of cytotoxic reactive oxygen intermediates (ROI) during paraquat redox cycling. The skin contains a diverse array of antioxidant enzymes which protect against oxidative stress including superoxide dismutase (SOD), catalase, glutathione peroxidase-1 (GPx-1), heme oxygenase-1 (HO-1), metallothionein-2 (MT-2), and glutathione-S-transferases (GST). In the present studies we compared paraquat redox cycling in primary cultures of undifferentiated and differentiated mouse keratinocytes and determined if this was associated with oxidative stress and altered expression of antioxidant enzymes. We found that paraquat readily undergoes redox cycling in both undifferentiated and differentiated keratinocytes, generating superoxide anion and hydrogen peroxide as well as increased protein oxidation which was greater in differentiated cells. Paraquat treatment also resulted in increased expression of HO-1, Cu,Zn-SOD, catalase, GSTP1, GSTA3 and GSTA4. However, no major differences in expression of these enzymes were evident between undifferentiated and differentiated cells. In contrast, expression of GSTA1-2 was significantly greater in differentiated relative to undifferentiated cells after paraquat treatment. No changes in expression of MT-2, Mn-SOD, GPx-1, GSTM1 or the microsomal GST's mGST1, mGST2 and mGST3, were observed in response to paraquat. These data demonstrate that paraquat induces oxidative stress in keratinocytes leading to increased expression of antioxidant genes. These intracellular proteins may be important in protecting the skin from paraquat-mediated cytotoxicity.

  5. Prominin-2 expression increases protrusions, decreases caveolae and inhibits Cdc42 dependent fluid phase endocytosis

    SciTech Connect

    Singh, Raman Deep Schroeder, Andreas S.; Scheffer, Luana; Holicky, Eileen L.; Wheatley, Christine L.; Marks, David L. Pagano, Richard E.

    2013-05-10

    Highlights: •Prominin-2 expression induced protrusions that co-localized with lipid raft markers. •Prominin-2 expression decreased caveolae, caveolar endocytosis and increased pCav1. •Prominin-2 expression inhibited fluid phase endocytosis by inactivation of Cdc42. •These endocytic effects can be reversed by adding exogenous cholesterol. •Caveolin1 knockdown restored fluid phase endocytosis in Prominin2 expressing cells. -- Abstract: Background: Membrane protrusions play important roles in biological processes such as cell adhesion, wound healing, migration, and sensing of the external environment. Cell protrusions are a subtype of membrane microdomains composed of cholesterol and sphingolipids, and can be disrupted by cholesterol depletion. Prominins are pentaspan membrane proteins that bind cholesterol and localize to plasma membrane (PM) protrusions. Prominin-1 is of great interest as a marker for stem and cancer cells, while Prominin-2 (Prom2) is reportedly restricted to epithelial cells. Aim: To characterize the effects of Prom-2 expression on PM microdomain organization. Methods: Prom2-fluorescent protein was transfected in human skin fibroblasts (HSF) and Chinese hamster ovary (CHO) cells for PM raft and endocytic studies. Caveolae at PM were visualized using transmission electron microscopy. Cdc42 activation was measured and caveolin-1 knockdown was performed using siRNAs. Results: Prom2 expression in HSF and CHO cells caused extensive Prom2-positive protrusions that co-localized with lipid raft markers. Prom2 expression significantly decreased caveolae at the PM, reduced caveolar endocytosis and increased caveolin-1 phosphorylation. Prom2 expression also inhibited Cdc42-dependent fluid phase endocytosis via decreased Cdc42 activation. Effects on endocytosis were reversed by addition of cholesterol. Knockdown of caveolin-1 by siRNA restored Cdc42 dependent fluid phase endocytosis in Prom2-expressing cells. Conclusions: Prom2 protrusions primarily

  6. Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

    SciTech Connect

    Yu, Wei; Chai, Hongyan; Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue; Yang, Guifang; Cai, Xiaojun; Falck, John R.; Yang, Jing

    2012-10-01

    Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ► CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ► The pro-angiogenic effects of CYP4Z1 have

  7. Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells

    NASA Technical Reports Server (NTRS)

    Chen, J.; Fabry, B.; Schiffrin, E. L.; Wang, N.; Ingber, D. E. (Principal Investigator)

    2001-01-01

    A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

  8. Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

    SciTech Connect

    Kim, Chae E.; Lee, Seung J.; Seo, Kyo W.; Park, Hye M.; Yun, Jung W.; Bae, Jin U.; Bae, Sun S.; Kim, Chi D.

    2010-05-15

    Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B{sub 4} (LTB{sub 4}) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB{sub 4} production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK among these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB{sub 4}. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB{sub 4}, subsequent MMP-9 production and plaque rupture.

  9. Vacuum-assisted closure increases ICAM-1, MIF, VEGF and collagen I expression in wound therapy

    PubMed Central

    WANG, WEIYANG; PAN, ZHENYU; HU, XIANG; LI, ZONGHUAN; ZHAO, YONG; YU, AI-XI

    2014-01-01

    Severe traumatic wounds are challenging to manage during surgery. The introduction of vacuum-assisted closure (VAC) is a breakthrough in wound management. The aim of the present study was to investigate the effect of VAC on cytokines in wounds during the management of severe traumatic wounds following initial debridement. VAC and conventional wound care (CWC) were independently applied to severe traumatic wounds on pigs. The expression levels of intercellular adhesion molecule-1 (ICAM-1), migration inhibitory factor (MIF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor, collagen I and human fibroblast collagenase 1 were detected by quantitative polymerase chain reaction and western blotting. VAC significantly increased the expression of ICAM-1, MIF, VEGF and collagen I compared with that induced by CWC at the protein and mRNA levels. Therefore, the results of the present study indicate that VAC therapy is an effective method for treating severe traumatic wounds, as it increases the expression of cytokines in wounds. VAC significantly increases the expression of ICAM-1, MIF, VEGF and collagen I to manage severe traumatic wounds. PMID:24940415

  10. Trimetazidine protects against hypoxia-reperfusion-induced cardiomyocyte apoptosis by increasing microRNA-21 expression

    PubMed Central

    Yang, Qiong; Yang, Kan; Li, An-Ying

    2015-01-01

    Myocardial tissue injury caused by ischemia and hypoxia is a major cause of fatal diseases, including coronary atherosclerosis resulting from myocardial infarction and stroke. Trimetazidine (TMZ), as an anti-ischemic and antioxidant agent, has been demonstrated to preventing ischemia/reperfusion-induced cardiomyocyte apoptosis. However, the anti-apoptosis mechanism of TMZ has not been fully elucidated. The present study demonstrated that miR-21 involved trimetazidine-induced anti-apoptosis during H/R injury in H9C2 cell. In this study, TMZ increased miR-21 expression which further upregulated the Akt signaling activity via suppressing the expression of phosphatase and tensin homolog (PTEN) in H/R H9C2 cell. The increased activity of Akt signaling decreased the ratio of Bax/Bcl-2 and the expression of caspase-3 and inhibited H/R induced apoptosis. In conclusion, this study revealed the mechanism that TMZ up-regulated miR-21 expression, then miR-21 targeted PTEN increasing the PI3K pathway and finally the activation of this pathway counteracted the apoptotic effect of hypoxia/reperfusion. PMID:26097555

  11. Trimetazidine protects against hypoxia-reperfusion-induced cardiomyocyte apoptosis by increasing microRNA-21 expression.

    PubMed

    Yang, Qiong; Yang, Kan; Li, An-Ying

    2015-01-01

    Myocardial tissue injury caused by ischemia and hypoxia is a major cause of fatal diseases, including coronary atherosclerosis resulting from myocardial infarction and stroke. Trimetazidine (TMZ), as an anti-ischemic and antioxidant agent, has been demonstrated to preventing ischemia/reperfusion-induced cardiomyocyte apoptosis. However, the anti-apoptosis mechanism of TMZ has not been fully elucidated. The present study demonstrated that miR-21 involved trimetazidine-induced anti-apoptosis during H/R injury in H9C2 cell. In this study, TMZ increased miR-21 expression which further upregulated the Akt signaling activity via suppressing the expression of phosphatase and tensin homolog (PTEN) in H/R H9C2 cell. The increased activity of Akt signaling decreased the ratio of Bax/Bcl-2 and the expression of caspase-3 and inhibited H/R induced apoptosis. In conclusion, this study revealed the mechanism that TMZ up-regulated miR-21 expression, then miR-21 targeted PTEN increasing the PI3K pathway and finally the activation of this pathway counteracted the apoptotic effect of hypoxia/reperfusion. PMID:26097555

  12. Nandrolone reduces activation of Notch signaling in denervated muscle associated with increased Numb expression

    SciTech Connect

    Liu, Xin-Hua; Yao, Shen; Qiao, Rui-Fang; Levine, Alice C.; Kirschenbaum, Alexander; Pan, Jiangping; Wu, Yong; Qin, Weiping; Bauman, William A.; Cardozo, Christopher P.

    2011-10-14

    Highlights: {yields} Nerve transection increased Notch signaling in paralyzed muscle. {yields} Nandrolone prevented denervation-induced Notch signaling. {yields} Nandrolone induced the expression of an inhibitor of the Notch signaling, Numb. {yields} Reduction of denervation-induced Notch signaling by nandrolone is likely through upregulation of Numb. -- Abstract: Nandrolone, an anabolic steroid, slows denervation-atrophy in rat muscle. The molecular mechanisms responsible for this effect are not well understood. Androgens and anabolic steroids activate Notch signaling in animal models of aging and thereby mitigate sarcopenia. To explore the molecular mechanisms by which nandrolone prevents denervation-atrophy, we investigated the effects of nandrolone on Notch signaling in denervated rat gastrocnemius muscle. Denervation significantly increased Notch activity reflected by elevated levels of nuclear Notch intracellular domain (NICD) and expression of Hey1 (a Notch target gene). Activation was greatest at 7 and 35 days after denervation but remained present at 56 days after denervation. Activation of Notch in denervated muscle was prevented by nandrolone associated with upregulated expression of Numb mRNA and protein. These data demonstrate that denervation activates Notch signaling, and that nandrolone abrogates this response associated with increased expression of Numb, suggesting a potential mechanism by which nandrolone reduces denervation-atrophy.

  13. Trichostatin A increases SMN expression and survival in a mouse model of spinal muscular atrophy

    PubMed Central

    Avila, Amy M.; Burnett, Barrington G.; Taye, Addis A.; Gabanella, Francesca; Knight, Melanie A.; Hartenstein, Parvana; Cizman, Ziga; Di Prospero, Nicholas A.; Pellizzoni, Livio; Fischbeck, Kenneth H.; Sumner, Charlotte J.

    2007-01-01

    The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by mutation of the telomeric survival motor neuron 1 (SMN1) gene with retention of the centromeric SMN2 gene. We sought to establish whether the potent and specific hydroxamic acid class of histone deacetylase (HDAC) inhibitors activates SMN2 gene expression in vivo and modulates the SMA disease phenotype when delivered after disease onset. Single intraperitoneal doses of 10 mg/kg trichostatin A (TSA) in nontransgenic and SMA model mice resulted in increased levels of acetylated H3 and H4 histones and modest increases in SMN gene expression. Repeated daily doses of TSA caused increases in both SMN2-derived transcript and SMN protein levels in neural tissues and muscle, which were associated with an improvement in small nuclear ribonucleoprotein (snRNP) assembly. When TSA was delivered daily beginning on P5, after the onset of weight loss and motor deficit, there was improved survival, attenuated weight loss, and enhanced motor behavior. Pathological analysis showed increased myofiber size and number and increased anterior horn cell size. These results indicate that the hydroxamic acid class of HDAC inhibitors activates SMN2 gene expression in vivo and has an ameliorating effect on the SMA disease phenotype when administered after disease onset. PMID:17318264

  14. Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli

    SciTech Connect

    Martinez, A.; York, S.W.; Yomano, L.P.; Pineda, V.L.; Davis, F.C.; Shelton, J.C.; Ingram, L.O.

    1999-10-01

    Previous studies have shown an unexpectedly high nutrient requirement for efficient ethanol production by ethanologenic recombinants of Escherichia coli B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc, adhB) encoding the ethanol pathway. The basis for this requirement has been identified as a media-dependent effect on the expression of the Z. mobilis genes rather than a nutritional limitation. Ethanol production was substantially increased without additional nutrients simply by increasing the level of pyruvate decarboxylase activity. This was accomplished by adding a multicopy plasmid containing pdc alone (but not adhB alone) to strain LY01, and by adding multicopy plasmids which express pdc and adhB from strong promoters. New strong promoters were isolated from random fragments of Z. mobilis DNA and characterized but were not used to construct integrated biocatalysts. These promoters contained regions resembling recognition sites for 3 different E. coli sigma factors: {sigma}{sup 70}, {sigma}{sup 38}, and {sigma}{sup 28}. The most effective plasmid-based promoters for fermentation were recognized by multiple sigma factors, expressed both pdc and adhB at high levels, and produced ethanol efficiently while allowing up to 80% reduction in complex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageous to maintain the high levels of PDC and ADH needed for efficient ethanol production throughout batch fermentation.

  15. Adenovirus expressing β2-microglobulin recovers HLA class I expression and antitumor immunity by increasing T-cell recognition.

    PubMed

    Del Campo, A B; Carretero, J; Muñoz, J A; Zinchenko, S; Ruiz-Cabello, F; González-Aseguinolaza, G; Garrido, F; Aptsiauri, N

    2014-08-01

    Optimal tumor cell surface expression of human leukocyte antigen (HLA) class I molecules is essential for the presentation of tumor-associated peptides to T-lymphocytes. However, a hallmark of many types of tumor is the loss or downregulation of HLA class I expression associated with ineffective tumor antigen presentation to T cells. Frequently, HLA loss can be caused by structural alterations in genes coding for HLA class I complex, including the light chain of the complex, β2-microglobulin (β2m). Its best-characterized function is to interact with HLA heavy chain and stabilize the complex leading to a formation of antigen-binding cleft recognized by T-cell receptor on CD8+ T cells. Our previous study demonstrated that alterations in the β2m gene are frequently associated with cancer immune escape leading to metastatic progression and resistance to immunotherapy. These types of defects require genetic transfer strategies to recover normal expression of HLA genes. Here we characterize a replication-deficient adenoviral vector carrying human β2m gene, which is efficient in recovering proper tumor cell surface HLA class I expression in β2m-negative tumor cells without compromising the antigen presentation machinery. Tumor cells transduced with β2m induced strong activation of T cells in a peptide-specific HLA-restricted manner. Gene therapy using recombinant adenoviral vectors encoding HLA genes increases tumor antigen presentation and represents a powerful tool for modulation of tumor cell immunogenicity by restoration of missing or altered HLA genes. It should be considered as part of cancer treatment in combination with immunotherapy. PMID:24971583

  16. Phosphorus limitation strategy to increase propionic acid flux towards 3-hydroxyvaleric acid monomers in Cupriavidus necator.

    PubMed

    Grousseau, Estelle; Blanchet, Elise; Déléris, Stéphane; Albuquerque, Maria G E; Paul, Etienne; Uribelarrea, Jean-Louis

    2014-02-01

    Properties of polyhydroxybutyrate-co-hydroxyvalerate (P(3HB-co-3HV)) depend on their 3HV content. 3HV can be produced by Cupriavidus necator from propionic acid. Few studies explored carbon distribution and dynamics of 3HV and 3HB monomers production, and none of them have been done with phosphorus as limiting nutrient. In this study, fed-batch cultures of C. necator with propionic acid, as sole carbon source or mixed with butyric acid, were performed. Phosphorus deficiency allowed sustaining 3HV production rate and decreasing 3HB production rate, leading to an instant production of up to 100% of 3HV. When a residual growth is sustained by a phosphorus feeding, the maximum 3HV percentage produced from propionic acid is limited to 33% (Mole.Mole(-1)). The association of a second carbon source like butyric acid lead to higher conversion of propionic acid into 3HV. This study showed the importance of the limiting nutrient and of the culture strategy to get the appropriate product. PMID:24365742

  17. ALX1 promotes migration and invasion of lung cancer cells through increasing snail expression

    PubMed Central

    Yao, Wei; Liu, Yong; Zhang, Zhuo; Li, Guoquan; Xu, Xiaoying; Zou, Kun; Xu, Yinghui; Zou, Lijuan

    2015-01-01

    Lung cancer is one of the main causes in cancer-related death. Here we reported a novel functional role of Aristaless-like homeobox1 (ALX1) in lung carcinogenesis. Analysis of ALX1 in lung cancer specimens confirms upregulation of ALX1 in lung cancer, especially these with distant metastasis. Moreover, higher level of ALX1 expression is associated with poorer prognosis of lung cancer patients. Ectopic expression of ALX1 significantly promotes lung cancer cell proliferation, migration and invasion, while ALX1 silencing by siRNA significantly inhibits these abilities of lung cancer cells. The functional role of ALX1 is dependent on increasing Snail expression and knockdown of Snail could restrain the role of ALX1. Collectively, we identify critical roles of ALX1 in lung cancer development and progression. These findings may serve as a framework for future investigations designed to more comprehensive determination of ALX1 as a potential therapeutic target. PMID:26722397

  18. Increased atypical PKC expression and activity in the phrenic motor nucleus following cervical spinal injury

    PubMed Central

    Guenther, C.H.; Windelborn, J.A.; Tubon, T.C.; Yin, J.C.P.; Mitchell, G.S.

    2012-01-01

    Atypical protein kinase C (aPKC) isoforms are expressed in phrenic motor neurons, a group of motor neurons critical for breathing. Following C2 cervical hemisection (C2HS), spontaneous plasticity occurs in crossed-spinal synaptic pathways to phrenic motor neurons, at least partially restoring inspiratory phrenic activity below the injury. Since aPKCs are necessary for synaptic plasticity in other systems, we tested the hypothesis that C2HS increases aPKC expression and activity in spinal regions associated with the phrenic motor nucleus. C2 laminectomy (sham) or C2HS was performed on adult, male Lewis rats. Ventral spinal segments C3–5 were harvested 1, 3 or 28 days post-surgery, and prepared for aPKC enzyme activity assays and immunoblots. Ventral cervical aPKC activity was elevated 1 and 28, but not 3, days post-C2HS (1 day: 63% vs sham ipsilateral to injury; p<0.05; 28 day: 426% vs sham; p<0.05; no difference in ipsilateral vs contralateral response). Total PKCζ/ι protein expression was unchanged by C2HS, but total and phosphorylated PKMζ (constitutively active PKCζ isoform) increased ipsilateral to injury 28 days post-C2HS (p<0.05). Ipsilateral aPKC activity and expression were strongly correlated (r2=0.675, p<0.001). In a distinct group of rats, immunohistochemistry confirmed that aPKCs are expressed in neurons 28 days post-C2HS, including large, presumptive phrenic motor neurons; aPKCs were not detected in adjacent microglia (OX-42 positive cells) or astrocytes (GFAP positive cells). Changes in aPKC expression in the phrenic motor nucleus following C2HS suggests that aPKCs may contribute to functional recovery following cervical spinal injury. PMID:22329943

  19. Over-Expression of the LH Receptor Increases Distant Metastases in an Endometrial Cancer Mouse Model

    PubMed Central

    Pillozzi, Serena; Fortunato, Angelo; De Lorenzo, Emanuele; Borrani, Elena; Giachi, Massimo; Scarselli, Gianfranco; Arcangeli, Annarosa; Noci, Ivo

    2013-01-01

    Objective: The aim of the present study was to define the role of luteinizing hormone receptor (LH-R) expression in endometrial cancer (EC), using preclinical mouse models, to further transfer these data to the clinical setting. Materials and Methods: The role of LH-R over-expression was studied using EC cells (Hec1A, e.g., cells with low endogenous LH-R expression) transfected with the LH-R (Hec1A-LH-R). In vitro cell proliferation was measured through the WST-1 assay, whereas cell invasion was measured trough the matrigel assay. The effects of LH-R over-expression in vivo were analyzed in an appropriately developed preclinical mouse model of EC, which mimicked postmenopausal conditions. The model consisted in an orthotopic xenograft of Hec1A cells into immunodeficient mice treated daily with recombinant LH, to assure high levels of LH. Results: In vitro data indicated that LH-R over-expression increased Hec1A invasiveness. In vivo results showed that tumors arising from Hec1A-LH-R cells injection displayed a higher local invasion and a higher number of distant metastases, mainly in the lung, compared to tumors obtained from the injection of Hec1A cells. LH withdrawal strongly inhibited local and distant metastatic spread of tumors, especially those arising from Hec1A-LH-R cells. Conclusion: The over-expression of the LH-R increases the ability of EC cells to undergo local invasion and metastatic spread. This occurs in the presence of high LH serum concentrations. PMID:24312898

  20. GNMT expression increases hepatic folate contents and folate-dependent methionine synthase-mediated homocysteine remethylation.

    PubMed

    Wang, Yi-Cheng; Chen, Yi-Ming; Lin, Yan-Jun; Liu, Shih-Ping; Chiang, En-Pei Isabel

    2011-01-01

    Glycine N-methyltransferase (GNMT) is a major hepatic enzyme that converts S-adenosylmethionine to S-adenosylhomocysteine while generating sarcosine from glycine, hence it can regulate mediating methyl group availability in mammalian cells. GNMT is also a major hepatic folate binding protein that binds to, and, subsequently, may be inhibited by 5-methyltetrafolate. GNMT is commonly diminished in human hepatoma; yet its role in cellular folate metabolism, in tumorigenesis and antifolate therapies, is not understood completely. In the present study, we investigated the impacts of GNMT expression on cell growth, folate status, methylfolate-dependent reactions and antifolate cytotoxicity. GNMT-diminished hepatoma cell lines transfected with GNMT were cultured under folate abundance or restriction. Folate-dependent homocysteine remethylation fluxes were investigated using stable isotopic tracers and gas chromatography/mass spectrometry. Folate status was compared between wild-type (WT), GNMT transgenic (GNMT(tg)) and GNMT knockout (GNMT(ko)) mice. In the cell model, GNMT expression increased folate concentration, induced folate-dependent homocysteine remethylation, and reduced antifolate methotrexate cytotoxicity. In the mouse models, GNMT(tg) had increased hepatic folate significantly, whereas GNMT(ko) had reduced folate. Liver folate levels correlated well with GNMT expressions (r = 0.53, P = 0.002); and methionine synthase expression was reduced significantly in GNMT(ko), demonstrating impaired methylfolate-dependent metabolism by GNMT deletion. In conclusion, we demonstrated novel findings that restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Studies on how GNMT expression impacts the distribution of different folate cofactors and the regulation of specific folate dependent reactions are underway. PMID:21210071

  1. Increased expression of formin-like 3 contributes to metastasis and poor prognosis in colorectal carcinoma.

    PubMed

    Zeng, Yuan-Feng; Xiao, Yi-Sheng; Lu, Ming-Zhi; Luo, Xiao-Jiang; Hu, Guo-Zhu; Deng, Ke-Yu; Wu, Xiao-Mu; Xin, Hong-Bo

    2015-04-01

    Formin-like 3 (FMNL3), a member of diaphanous-related formins subfamily, plays an important role in cytoskeleton reorganization, cell adhesion and cancer cell invasion in vitro. This study aimed to explore the expression of FMNL3 in colorectal carcinoma (CRC) cell-lines and tissues, and further evaluate its prognostic value and correlation with the clinicopathological parameters, and also investigate the effects of FMNL3 gene silencing on the growth and metastasis of CRC in vivo. Immunohistochemical analysis showed that FMNL3 protein was distributed in a punctuate aggregation pattern and located mainly in the cytoplasm of glandular cavity side, close to the nucleus of CRC cells. The positive rate of FMNL3 expression was 87.5% (84/96) in CRC, which was significantly higher than that in adjacent normal mucosa (30%, 9/30). Moreover, FMNL3 protein expressed far more in primary CRC with metastasis and corresponding lymph nodes metastatic CRC than in primary CRC without metastasis. Increased expression of FMNL3 was closely correlated with tumor size, differentiation, serosal invasion, and both lymph node metastasis and distant metastasis. However, it was not correlated with patients' age and gender. According to Kaplan-Meier survival analyses, patients with FMNL3 high expression level had lower overall survival rate than that with FMNL3 low expression level. Univariate and multivariate analyses revealed that high FMNL3 expression was a significant and independent prognostic predictor of patients with CRC. In addition, FMNL3 mRNA and protein levels were substantially up-regulated in CRC-metastasis-derived cell lines, as compared to those in primary-CRC-derived ones. FMNL3 gene silencing suppressed the growth and metastasis of CRC in vivo. In conclusion, FMNL3 plays an important role in the progression and metastasis of CRC and may be a novel potential prognostic predictor and therapeutic target for patients with CRC. PMID:25758200

  2. IDO expression in brain tumors increases the recruitment of regulatory T cells and negatively impacts survival

    PubMed Central

    Wainwright, Derek A.; Balyasnikova, Irina V.; Chang, Alan L.; Ahmed, Atique U.; Moon, Kyung-Sub; Auffinger, Brenda; Tobias, Alex L.; Han, Yu; Lesniak, Maciej S.

    2012-01-01

    Purpose Glioblastoma multiforme (GBM) is an aggressive adult brain tumor with a poor prognosis. One hallmark of GBM is the accumulation of immunosuppressive and tumor-promoting CD4+FoxP3+GITR+ regulatory T cells (Tregs). Here, we investigated the role of indoleamine 2,3 dioxygenase (IDO) in brain tumors and the impact on Treg recruitment. Experimental Design To determine the clinical relevance of IDO expression in brain tumors, we first correlated patient survival to the level of IDO expression from resected glioma specimens. We also used novel orthotopic and transgenic models of glioma to study how IDO affects Tregs. The impact of tumor-derived and peripheral IDO expression on Treg recruitment, GITR expression and long-term survival was determined. Results Downregulated IDO expression in glioma predicted a significantly better prognosis in patients. Co-incidently, both IDO -competent and -deficient mice showed a survival advantage bearing IDO-deficient brain tumors, when compared to IDO-competent brain tumors. Moreover, IDO-deficiency was associated with a significant decrease in brain-resident Tregs, both in orthotopic and transgenic mouse glioma models. IDO-deficiency was also associated with lower GITR expression levels on Tregs. Interestingly, the long-term survival advantage conferred by IDO-deficiency was lost in T cell-deficient mice. Conclusions These clinical and pre-clinical data confirm that IDO expression increases the recruitment of immunosuppressive Tregs which leads to tumor outgrowth. In contrast, IDO deficiency decreases Treg recruitment and enhances T cell-mediated tumor rejection. Thus, the data suggest a critical role for IDO-mediated immunosuppression in glioma and supports the continued investigation of IDO-Treg interactions in the context of brain tumors. PMID:22932670

  3. GW8510 Increases Insulin Expression in Pancreatic Alpha Cells through Activation of p53 Transcriptional Activity

    PubMed Central

    Fomina-Yadlin, Dina; Kubicek, Stefan; Vetere, Amedeo; He, Kaihui Hu; Schreiber, Stuart L.; Wagner, Bridget K.

    2012-01-01

    Background Expression of insulin in terminally differentiated non-beta cell types in the pancreas could be important to treating type-1 diabetes. Previous findings led us to hypothesize involvement of kinase inhibition in induction of insulin expression in pancreatic alpha cells. Methodology/Principal Findings Alpha (αTC1.6) cells and human islets were treated with GW8510 and other small-molecule inhibitors for up to 5 days. Alpha cells were assessed for gene- and protein-expression levels, cell-cycle status, promoter occupancy status by chromatin immunoprecipitation (ChIP), and p53-dependent transcriptional activity. GW8510, a putative CDK2 inhibitor, up-regulated insulin expression in mouse alpha cells and enhanced insulin secretion in dissociated human islets. Gene-expression profiling and gene-set enrichment analysis of GW8510-treated alpha cells suggested up-regulation of the p53 pathway. Accordingly, the compound increased p53 transcriptional activity and expression levels of p53 transcriptional targets. A predicted p53 response element in the promoter region of the mouse Ins2 gene was verified by chromatin immunoprecipitation (ChIP). Further, inhibition of Jun N-terminal kinase (JNK) and p38 kinase activities suppressed insulin induction by GW8510. Conclusions/Significance The induction of Ins2 by GW8510 occurred through p53 in a JNK- and p38-dependent manner. These results implicate p53 activity in modulation of Ins2 expression levels in pancreatic alpha cells, and point to a potential approach toward using small molecules to generate insulin in an alternative cell type. PMID:22242153

  4. FGF23 is elevated in multiple myeloma and increases heparanase expression by tumor cells

    PubMed Central

    Suvannasankha, Attaya; Tompkins, Douglas R.; Edwards, Daniel F.; Petyaykina, Katarina V.; Crean, Colin D.; Fournier, Pierrick G.; Parker, Jamie M.; Sandusky, George E.; Ichikawa, Shoji; Imel, Erik A.; Chirgwin, John M.

    2015-01-01

    Multiply myeloma (MM) grows in and destroys bone, where osteocytes secrete FGF23, a hormone which affects phosphate homeostasis and aging. We report that multiple myeloma (MM) cells express receptors for and respond to FGF23. FGF23 increased mRNA for EGR1 and its target heparanase, a pro-osteolytic factor in MM. FGF23 signals through a complex of klotho and a classical FGF receptor (FGFR); both were expressed by MM cell lines and patient samples. Bone marrow plasma cells from 42 MM patients stained positively for klotho, while plasma cells from 8 patients with monoclonal gammopathy of undetermined significance (MGUS) and 6 controls were negative. Intact, active FGF23 was increased 2.9X in sera of MM patients compared to controls. FGF23 was not expressed by human MM cells, but co-culture with mouse bone increased its mRNA. The FGFR inhibitor NVP-BGJ398 blocked the heparanase response to FGF23. NVP-BGJ398 did not inhibit 8226 growth in vitro but significantly suppressed growth in bone and induction of the osteoclast regulator RANK ligand, while decreasing heparanase mRNA. The bone microenvironment provides resistance to some anti-tumor drugs but increased the activity of NVP-BGJ398 against 8226 cells. The FGF23/klotho/heparanase signaling axis may offer targets for treatment of MM in bone. PMID:25944690

  5. FGF23 is elevated in multiple myeloma and increases heparanase expression by tumor cells.

    PubMed

    Suvannasankha, Attaya; Tompkins, Douglas R; Edwards, Daniel F; Petyaykina, Katarina V; Crean, Colin D; Fournier, Pierrick G; Parker, Jamie M; Sandusky, George E; Ichikawa, Shoji; Imel, Erik A; Chirgwin, John M

    2015-08-14

    Multiply myeloma (MM) grows in and destroys bone, where osteocytes secrete FGF23, a hormone which affects phosphate homeostasis and aging. We report that multiple myeloma (MM) cells express receptors for and respond to FGF23. FGF23 increased mRNA for EGR1 and its target heparanase, a pro-osteolytic factor in MM. FGF23 signals through a complex of klotho and a classical FGF receptor (FGFR); both were expressed by MM cell lines and patient samples. Bone marrow plasma cells from 42 MM patients stained positively for klotho, while plasma cells from 8 patients with monoclonal gammopathy of undetermined significance (MGUS) and 6 controls were negative. Intact, active FGF23 was increased 2.9X in sera of MM patients compared to controls. FGF23 was not expressed by human MM cells, but co-culture with mouse bone increased its mRNA. The FGFR inhibitor NVP-BGJ398 blocked the heparanase response to FGF23. NVP-BGJ398 did not inhibit 8226 growth in vitro but significantly suppressed growth in bone and induction of the osteoclast regulator RANK ligand, while decreasing heparanase mRNA. The bone microenvironment provides resistance to some anti-tumor drugs but increased the activity of NVP-BGJ398 against 8226 cells. The FGF23/klotho/heparanase signaling axis may offer targets for treatment of MM in bone. PMID:25944690

  6. Ovarian Steroids Increase PSD-95 Expression and Dendritic Spines in the Dorsal Raphe of Ovariectomized Macaques

    PubMed Central

    Rivera, Heidi M.; Bethea, Cynthia L.

    2014-01-01

    Estradiol (E) and progesterone (P) promote spinogenesis in several brain areas. Intracellular signaling cascades that promote spinogenesis involve RhoGTPases, glutamate signaling and synapse assembly. We found that in serotonin neurons, E±P administration increases (a) gene and protein expression of RhoGTPases, (b) gene expression of glutamate receptors (c) gene expression of pivotal synapse assembly proteins. Therefore, in this study we determined whether structural changes in dendritic spines in the dorsal raphe follow the observed changes in gene and protein expression. Dendritic spines were examined with immunogold silver staining of a spine marker protein, postsynaptic density-95 (PSD-95) and with Golgi staining. In the PSD-95 study, adult Ovx monkeys received placebo, E, P, or E+P for 1 month (n=3/group). Sections were immunostained for PSD-95 and the number of PSD-95-positive puncta was determined with stereology. E, P and E+P treatment significantly increased the total number of PSD-95-positive puncta (ANOVA, P=0.04). In the Golgi study, adult Ovx monkeys received placebo, E or E+P for 1 month (n=3–4) and the midbrain was Golgi-stained. A total of 80 neurons were analyzed with Neurolucida software. There was a significant difference in spine density that depended on branch order (two-way ANOVA). E+P treatment significantly increased spine density in higher-order (3–5°) dendritic branches relative to Ovx group (Bonferroni, P<0.05). In summary, E+P leads to the elaboration of dendritic spines on dorsal raphe neurons. The ability of E to induce PSD-95, but not actual spines, suggests either a sampling or time lag issue. Increased spinogenesis on serotonin dendrites would facilitate excitatory glutamatergic input and, in turn, increase serotonin neurotransmission throughout the brain. PMID:23959764

  7. 12 CFR 226.55 - Limitations on increasing annual percentage rates, fees, and charges.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...) Workout and temporary hardship arrangement exception. A card issuer may increase an annual percentage rate... to the consumer's completion of a workout or temporary hardship arrangement or the consumer's...

  8. Epstein-Barr Virus MicroRNA Expression Increases Aggressiveness of Solid Malignancies

    PubMed Central

    Pandya, Deep; Mariani, Marisa; He, Shiquan; Andreoli, Mirko; Spennato, Manuela; Dowell-Martino, Candice; Fiedler, Paul; Ferlini, Cristiano

    2015-01-01

    The Cancer Genome Atlas (TCGA) microRNA (miRNA) initiative has revealed a pivotal role for miRNAs in cancer. Utilizing the TCGA raw data, we performed the first mapping of viral miRNA sequences within cancer and adjacent normal tissues. Results were integrated with TCGA RNA-seq to link the expression of viral miRNAs to the phenotype. Using clinical data and viral miRNA mapping results we also performed outcome analysis. Three lines of evidence lend credence to an active role of viral miRNAs in solid malignancies. First, expression of viral miRNA is consistently higher in cancerous compared to adjacent noncancerous tissues. Second, viral miRNA expression is associated with significantly worse clinical outcome among patients with early stage malignancy. These patients are also featured by increased expression of PD1/PD-L1, a pathway implicated in tumors escaping immune destruction. Finally, a particular cluster of EBV-miRNA (miR-BART2, miR-BART4, miR-BART5, miR-BART18, and miR-BART22) is associated with expression of cytokines known to inhibit host response to cancer. Quantification of specific viral miRNAs may help identify patients who are at risk of poor outcome. These patients may be candidates for novel therapeutic strategies incorporating antiviral agents and/or inhibitors of the PD-1/PD-L1 pathway. PMID:26375401

  9. Increased endometrial expression of CC-chemokine receptor-1 in women with adenomyosis.

    PubMed

    Xu, Hong; Yang, Yanfeng; Zhou, Caiyun; Huang, Xiufeng; Lin, Jun; Zhang, Xinmei

    2014-09-01

    Abnormal endometrial expression of CC-chemokine receptor-1 (CCR1) may play a role in the pathogenesis of endometriosis. Adenomyosis, also called endometriosis interna, occurs when the endometrium invades the myometrium. The objective of this study was to determine CCR1 expression in endometrium in women with adenomyosis as compared to women without adenomyosis. We evaluated endometrial mRNA and protein expression in women with and without adenomyosis using quantitative polymerase chain reaction (PCR), immunohistochemical staining and western blot analysis, respectively. We detected CCR1-immunoreactive expression in endometrium in all women with and without adenomyosis. CCR1-immunoreactive staining in endometrial cells was significantly higher in women with adenomyosis (4.89±1.06) compared to those without adenomyosis (2.21±1.16, P<0.001). Women with adenomyosis had higher levels of CCR1 mRNA in endometrium compared to women without adenomyosis (P<0.05). CCR1 protein levels in endometrium were significantly higher in women with adenomyosis (1.66±0.79) compared to women without adenomyosis (0.56±0.13, P<0.001), and positively correlated with the severity of dysmenorrhea (r=0.87, P<0.001). These results suggest that increased CC-chemokine receptor expression may play a role in the pathogenesis of adenomyosis. PMID:24599574

  10. Ectopic expression of new alternative splice variant of Smac/DIABLO increases mammospheres formation

    PubMed Central

    Martinez-Ruiz, Gustavo U; Victoria-Acosta, Georgina; Vazquez-Santillan, Karla I; Jimenez-Hernandez, Luis; Muñoz-Galindo, Laura; Ceballos-Cancino, Gisela; Maldonado, Vilma; Melendez-Zajgla, Jorge

    2014-01-01

    Smac-α is a mitochondrial protein that, during apoptosis, is translocated to the cytoplasm, where it negatively regulates members of the inhibitor of apoptosis (IAP) family via the IAP-binding motif (IBM) contained within its amino-terminus. Here, we describe a new alternative splice variant from Smac gene, which we have named Smac-ε. Smac-ε lacks both an IBM and a mitochondrial-targeting signal (MTS) element. Smac-ε mRNA exhibits a tissue-specific expression pattern in healthy human tissues as well as in several cancer cell lines. The steady-state levels of endogenous Smac-ε protein is regulated by the proteasomal pathway. When ectopically expressed, this isoform presents a cytosolic localization and is unable to associate with or to regulate the expression of X-linked Inhibitor of apoptosis protein, the best-studied member of IAP family. Nevertheless, over-expression of Smac-ε increases mammosphere formation. Whole genome expression analyses from these mammospheres show activation of several pro-survival and growth pathways, including Estrogen-Receptor signaling. In conclusion, our results support the functionality of this new Smac isoform. PMID:25337193

  11. Different Resistance-Training Regimens Evoked a Similar Increase in Myostatin Inhibitors Expression.

    PubMed

    Santos, A R; Lamas, L; Ugrinowitsch, C; Tricoli, V; Miyabara, E H; Soares, A G; Aoki, M S

    2015-08-01

    The aim of the present study was to investigate the effect of different resistance-training regimens (S or P) on the expression of genes related to the MSTN signaling pathway in physically-active men. 29 male subjects with at least 2 years of experience in strength training were assigned to either a strength-training group (S; n=11) or a power-training group (P; n=11). The control group (C; n=7) was composed of healthy physically-active males. The S and the P groups performed high- and low-intensity squats, respectively, 3 times per week, for 8 weeks. Muscle biopsies from the vastus lateralis muscle were collected before and after the training period. No change was observed in MSTN, ACTIIB, GASP-1 and FOXO-3 A gene expression after the training period. A similar increase in the gene expression of the inhibitory proteins of the MSTN signaling pathway, FLST (S: 4.2 fold induction and P: 3.7 fold induction, p<0.01) and FL-3 (S: 5.6 fold induction and P: 5.6 fold induction, p<0.01), was detected after the training period. SMAD-7 gene expression was similarly augmented after both training protocols (S: 2.5 fold induction; P: 2.8 fold induction; p<0.05). In conclusion, the resistance-training regimens (S and P) activated the expression of inhibitors of the MSTN signaling pathway in a similar manner. PMID:25822941

  12. Increased expression of CSP and CYP genes in adult silkworm females exposed to avermectins.

    PubMed

    Xuan, Ning; Guo, Xia; Xie, Hong-Yan; Lou, Qi-Nian; Lu, Xing-Bo; Liu, Guo-Xia; Picimbon, Jean-François

    2015-04-01

    We analyzed 20 chemosensory protein (CSP) genes of the silkworm Bombyx mori. We found a high number of retrotransposons inserted in introns. We then analyzed expression of the 20 BmorCSP genes across tissues using quantitative real-time polymerase chain reaction (PCR). Relatively low expression levels of BmorCSPs were found in the gut and fat body tissues. We thus tested the effects of endectocyte insecticide abamectin (B1a and B1b avermectins) on BmorCSP gene expression. Quantitative real-time PCR experiments showed that a single brief exposure to insecticide abamectin increased dramatically CSP expression not only in the antennae but in most tissues, including gut and fat body. Furthermore, our study showed coordinate expression of CSPs and metabolic cytochrome P450 enzymes in a tissue-dependent manner in response to the insecticide. The function of CSPs remains unknown. Based on our results, we suggest a role in detecting xenobiotics that are then detoxified by cytochrome P450 anti-xenobiotic enzymes. PMID:24677614

  13. Overgeneralizing belonging: limited exposure to baby-faced targets increases the feeling of social belonging.

    PubMed

    Sacco, Donald F; Wilson, John Paul; Hugenberg, Kurt; Wirth, James H

    2014-01-01

    We tested the hypothesis that exposure to babyish faces can serve a social surrogacy function, such that even limited exposure to babyish faces can fulfill social belongingness needs. We manipulated the sex and facial maturity of a target face seen in an imagined social interaction, on a between-participants basis. Regardless of target sex, individuals indicated greater satisfaction of social belongingness needs following an imagined interaction with a babyish face, compared to a mature adult face. These results indicate that brief exposure to babyish (relative to mature) faces, even without an extensive interaction, can lead to the satisfaction of social belongingness needs. PMID:25154111

  14. Increased extrasynaptic GluN2B expression is involved in cognitive impairment after isoflurane anesthesia

    PubMed Central

    LI, LUNXU; LI, ZHENGQIAN; CAO, YIYUN; FAN, DONGSHENG; CHUI, DEHUA; GUO, XIANGYANG

    2016-01-01

    There is increasing concern regarding the postoperative cognitive dysfunction (POCD) in the aging population, and general anesthetics are believed to be involved. Isoflurane exposure induced increased N-methyl-D-aspartic acid receptor (NMDAR) GluN2B subunit expression following anesthesia, which was accompanied by alteration of the cognitive function. However, whether isoflurane affects this expression in different subcellular compartments, and is involved in the development of POCD remains to be elucidated. The aims of the study were to investigate the effects of isoflurane on the expression of the synaptic and extrasynaptic NMDAR subunits, GluN2A and GluN2B, as well as the associated alteration of cognitive function in aged rats. The GluN2B antagonist, Ro25–6981, was given to rats exposed to isoflurane to determine the role of GluN2B in the isoflurane-induced alteration of cognitive function. The results showed that spatial learning and memory tested in the Morris water maze (MWM) was impaired at least 7 days after isoflurane exposure, and was returned to control levels 30 days thereafter. Ro25-6981 treatment can alleviate this impairment. Extrasynaptic GluN2B protein expression, but not synaptic GluN2B or GluN2A, increased significantly after isoflurane exposure compared to non-isoflurane exposure, and returned to control levels approximately 30 days thereafter. The results of the present study indicated that isoflurane induced the prolonged upregulation of extrasynaptic GluN2B expression after anesthesia and is involved in reversible cognitive impairment. PMID:27347033

  15. Increased G Protein-Coupled Receptor Kinase (GRK) Expression in the Anterior Cingulate Cortex in Schizophrenia

    PubMed Central

    Funk, Adam J.; Haroutunian, Vahram; Meador-Woodruff, James H.; McCullumsmith, Robert E.

    2014-01-01

    Background Current pharmacological treatments for schizophrenia target G protein-coupled receptors (GPCRs), including dopamine receptors. Ligand bound GPCRs are regulated by a family of G protein-coupled receptor kinases (GRKs), members of which uncouple the receptor from heterotrimeric G proteins, desensitize the receptor, and induce receptor internalization via the arrestin family of scaffolding and signaling molecules. GRKs initiate the activation of downstream signaling pathways, can regulate receptors and signaling molecules independent of GPCR phosphorylation, and modulate epigenetic regulators like histone deacetylases (HDACs). We hypothesize that expression of GRK proteins are altered in schizophrenia, consistent with previous findings of alterations up and downstream from this family of molecules that facilitate intracellular signaling processes. Methods In this study we measured protein expression via Western blot analysis for GRKs 2, 3, 5, and 6 in the anterior cingulate cortex of patients with schizophrenia (N = 36) and a comparison group (N = 33). To control for antipsychotic treatment we measured these same targets in haloperidol treated vs. untreated rats (N = 10 for both). Results We found increased levels of GRK5 in schizophrenia. No changes were detected in GRK protein expression in rats treated with haloperidol decanoate for 9 months. Conclusion These data suggest that increased GRK5 expression may contribute the the pathophysiology of schizophrenia via abnormal regulation of the cytoskeleton, endocytosis, signaling, GPCRs, and histone modification. PMID:25153362

  16. Increased liver pathology in hepatitis C virus transgenic mice expressing the hepatitis B virus X protein

    SciTech Connect

    Keasler, Victor V.; Lerat, Herve; Madden, Charles R.; Finegold, Milton J.; McGarvey, Michael J.; Mohammed, Essam M.A.; Forbes, Stuart J.; Lemon, Stanley M.; Hadsell, Darryl L.; Grona, Shala J.; Hollinger, F. Blaine; Slagle, Betty L. . E-mail: bslagle@bcm.edu

    2006-04-10

    Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis.

  17. Interleukin-18 Increases TLR4 and Mannose Receptor Expression and Modulates Cytokine Production in Human Monocytes

    PubMed Central

    Dias-Melicio, Luciane Alarcão; Fernandes, Reginaldo Keller; Rodrigues, Daniela Ramos; Golim, Marjorie Assis; Soares, Angela Maria Victoriano Campos

    2015-01-01

    Interleukin-18 is a proinflammatory cytokine belonging to the interleukin-1 family of cytokines. This cytokine exerts many unique biological and immunological effects. To explore the role of IL-18 in inflammatory innate immune responses, we investigated its impact on expression of two toll-like receptors (TLR2 and TLR4) and mannose receptor (MR) by human peripheral blood monocytes and its effect on TNF-α, IL-12, IL-15, and IL-10 production. Monocytes from healthy donors were stimulated or not with IL-18 for 18 h, and then the TLR2, TLR4, and MR expression and intracellular TNF-α, IL-12, and IL-10 production were assessed by flow cytometry and the levels of TNF-α, IL-12, IL-15, and IL-10 in culture supernatants were measured by ELISA. IL-18 treatment was able to increase TLR4 and MR expression by monocytes. The production of TNF-α and IL-10 was also increased by cytokine treatment. However, IL-18 was unable to induce neither IL-12 nor IL-15 production by these cells. Taken together, these results show an important role of IL-18 on the early phase of inflammatory response by promoting the expression of some pattern recognition receptors (PRRs) that are important during the microbe recognition phase and by inducing some important cytokines such as TNF-α and IL-10. PMID:25873755

  18. Mechanisms of increased Trichodesmium fitness under iron and phosphorus co-limitation in the present and future ocean.

    PubMed

    Walworth, Nathan G; Fu, Fei-Xue; Webb, Eric A; Saito, Mak A; Moran, Dawn; Mcllvin, Matthew R; Lee, Michael D; Hutchins, David A

    2016-01-01

    Nitrogen fixation by cyanobacteria supplies critical bioavailable nitrogen to marine ecosystems worldwide; however, field and lab data have demonstrated it to be limited by iron, phosphorus and/or CO2. To address unknown future interactions among these factors, we grew the nitrogen-fixing cyanobacterium Trichodesmium for 1 year under Fe/P co-limitation following 7 years of both low and high CO2 selection. Fe/P co-limited cell lines demonstrated a complex cellular response including increased growth rates, broad proteome restructuring and cell size reductions relative to steady-state growth limited by either Fe or P alone. Fe/P co-limitation increased abundance of a protein containing a conserved domain previously implicated in cell size regulation, suggesting a similar role in Trichodesmium. Increased CO2 further induced nutrient-limited proteome shifts in widespread core metabolisms. Our results thus suggest that N2-fixing microbes may be significantly impacted by interactions between elevated CO2 and nutrient limitation, with broad implications for global biogeochemical cycles in the future ocean. PMID:27346420

  19. Mechanisms of increased Trichodesmium fitness under iron and phosphorus co-limitation in the present and future ocean

    PubMed Central

    Walworth, Nathan G.; Fu, Fei-Xue; Webb, Eric A.; Saito, Mak A.; Moran, Dawn; Mcllvin, Matthew R.; Lee, Michael D.; Hutchins, David A.

    2016-01-01

    Nitrogen fixation by cyanobacteria supplies critical bioavailable nitrogen to marine ecosystems worldwide; however, field and lab data have demonstrated it to be limited by iron, phosphorus and/or CO2. To address unknown future interactions among these factors, we grew the nitrogen-fixing cyanobacterium Trichodesmium for 1 year under Fe/P co-limitation following 7 years of both low and high CO2 selection. Fe/P co-limited cell lines demonstrated a complex cellular response including increased growth rates, broad proteome restructuring and cell size reductions relative to steady-state growth limited by either Fe or P alone. Fe/P co-limitation increased abundance of a protein containing a conserved domain previously implicated in cell size regulation, suggesting a similar role in Trichodesmium. Increased CO2 further induced nutrient-limited proteome shifts in widespread core metabolisms. Our results thus suggest that N2-fixing microbes may be significantly impacted by interactions between elevated CO2 and nutrient limitation, with broad implications for global biogeochemical cycles in the future ocean. PMID:27346420

  20. Skeletal muscle amino acid transporter expression is increased in young and older adults following resistance exercise

    PubMed Central

    Fry, Christopher S.; Glynn, Erin L.; Timmerman, Kyle L.; Dickinson, Jared M.; Walker, Dillon K.; Gundermann, David M.; Volpi, Elena; Rasmussen, Blake B.

    2011-01-01

    Amino acid transporters and mammalian target of rapamycin complex 1 (mTORC1) signaling are important contributors to muscle protein anabolism. Aging is associated with reduced mTORC1 signaling following resistance exercise, but the role of amino acid transporters is unknown. Young (n = 13; 28 ± 2 yr) and older (n = 13; 68 ± 2 yr) subjects performed a bout of resistance exercise. Skeletal muscle biopsies (vastus lateralis) were obtained at basal and 3, 6, and 24 h postexercise and were analyzed for amino acid transporter mRNA and protein expression and regulators of amino acid transporter transcription utilizing real-time PCR and Western blotting. We found that basal amino acid transporter expression was similar in young and older adults (P > 0.05). Exercise increased L-type amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, sodium-coupled neutral amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, and cationic amino acid transporter 1/SLC7A1 mRNA expression in both young and older adults (P < 0.05). L-type amino acid transporter 1 and CD98 protein increased only in younger adults (P < 0.05). eukaryotic initiation factor 2 α-subunit (S52) increased similarly in young and older adults postexercise (P < 0.05). Ribosomal protein S6 (S240/244) and activating transcription factor 4 nuclear protein expression tended to be higher in the young, while nuclear signal transducer and activator of transcription 3 (STAT3) (Y705) was higher in the older subjects postexercise (P < 0.05). These results suggest that the rapid upregulation of amino acid transporter expression following resistance exercise may be regulated differently between the age groups, but involves a combination of mTORC1, activating transcription factor 4, eukaryotic initiation factor 2 α-subunit, and STAT3. We propose an increase in amino acid transporter expression may contribute to enhanced amino acid sensitivity following exercise in young and older

  1. Lung CD8+ T cells in COPD have increased expression of bacterial TLRs

    PubMed Central

    2013-01-01

    Background Toll-like receptors (TLRs) on T cells can modulate their responses, however, the extent and significance of TLR expression by lung T cells, NK cells, or NKT cells in chronic obstructive pulmonary disease (COPD) is unknown. Methods Lung tissue collected from clinically-indicated resections (n = 34) was used either: (a) to compare the expression of TLR1, TLR2, TLR2/1, TLR3, TLR4, TLR5, TLR6 and TLR9 on lung CD8+ T cells, CD4+ T cells, NK cells and NKT cells from smokers with or without COPD; or (b) to isolate CD8+ T cells for culture with anti-CD3ε without or with various TLR ligands. We measured protein expression of IFN-γ, TNF-α, IL-13, perforin, granzyme A, granzyme B, soluble FasL, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL9 in supernatants. Results All the lung subsets analyzed demonstrated low levels of specific TLR expression, but the percentage of CD8+ T cells expressing TLR1, TLR2, TLR4, TLR6 and TLR2/1 was significantly increased in COPD subjects relative to those without COPD. In contrast, from the same subjects, only TLR2/1 and TLR2 on lung CD4+ T cells and CD8+ NKT cells, respectively, showed a significant increase in COPD and there was no difference in TLR expression on lung CD56+ NK cells. Production of the Tc1 cytokines IFN-γ and TNF-α by lung CD8+ T cells were significantly increased via co-stimulation by Pam3CSK4, a specific TLR2/1 ligand, but not by other agonists. Furthermore, this increase in cytokine production was specific to lung CD8+ T cells from patients with COPD as compared to lung CD8+ T cells from smokers without COPD. Conclusions These data suggest that as lung function worsens in COPD, the auto-aggressive behavior of lung CD8+ T cells could increase in response to microbial TLR ligands, specifically ligands against TLR2/1. PMID:23374856

  2. Shuganjieyu capsule increases neurotrophic factor expression in a rat model of depression

    PubMed Central

    Fu, Jinhua; Zhang, Yingjin; Wu, Renrong; Zheng, Yingjun; Zhang, Xianghui; Yang, Mei; Zhao, Jingping; Liu, Yong

    2014-01-01

    Shuganjieyu capsule has been approved for clinical treatment by the State Food and Drug Ad-ministration of China since 2008. In the clinic, Shuganjieyu capsule is often used to treat mild to moderate depression. In the rat model of depression established in this study, Shuganjieyu capsule was administered intragastrically daily before stress. Behavioral results confirmed that depressive symptoms lessened after treatment with high-dose (150 mg/kg) Shuganjieyu capsule. Immunohistochemistry results showed that high-dose Shuganjieyu capsule significantly increased phosphorylation levels of phosphorylation cyclic adenosine monophosphate response element binding protein and brain-derived neurotrophic factor expression in the medial prefrontal cortex and hippocampal CA3 area. Overall, our results suggest that in rats, Shuganjieyu capsule effec-tively reverses depressive-like behaviors by increasing expression levels of neurotrophic factors in the brain. PMID:25206843

  3. Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells.

    PubMed

    Denning, G M; Wollenweber, L A; Railsback, M A; Cox, C D; Stoll, L L; Britigan, B E

    1998-12-01

    Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease. PMID:9826354

  4. Pseudomonas Pyocyanin Increases Interleukin-8 Expression by Human Airway Epithelial Cells

    PubMed Central

    Denning, Gerene M.; Wollenweber, Laura A.; Railsback, Michelle A.; Cox, Charles D.; Stoll, Lynn L.; Britigan, Bradley E.

    1998-01-01

    Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1α. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease. PMID:9826354

  5. Increased PADI4 expression in blood and tissues of patients with malignant tumors

    PubMed Central

    2009-01-01

    Background Peptidylarginine deiminase type 4 (PAD4/PADI4) post-translationally converts peptidylarginine to citrulline. Recent studies suggest that PADI4 represses expression of p53-regulated genes via citrullination of histones at gene promoters. Methods Expression of PADI4 was investigated in various tumors and non-tumor tissues (n = 1673) as well as in A549, SKOV3 and U937 tumor cell lines by immunohistochemistry, real-time PCR, and western blot. Levels of PADI4 and citrullinated antithrombin (cAT) were investigated in the blood of patients with various tumors by ELISA (n = 1121). Results Immunohistochemistry detected significant PADI4 expression in various malignancies including breast carcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cancer cells, colorectal adenocarcinomas, renal cancer cells, ovarian adenocarcinomas, endometrial carcinomas, uterine adenocarcinomas, bladder carcinomas, chondromas, as well as other metastatic carcinomas. However, PADI4 expression was not observed in benign leiomyomas of stomach, uterine myomas, endometrial hyperplasias, cervical polyps, teratomas, hydatidiform moles, trophoblastic cell hyperplasias, hyroid adenomas, hemangiomas, lymph hyperplasias, schwannomas, neurofibromas, lipomas, and cavernous hemangiomas of the liver. Additionally, PADI4 expression was not detected in non-tumor tissues including cholecystitis, cervicitis and synovitis of osteoarthritis, except in certain acutely inflamed tissues such as in gastritis and appendicitis. Quantitative PCR and western blot analysis showed higher PADI4 expression in gastric adenocarcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cell cancers and breast cancers (n = 5 for each disease) than in the surrounding healthy tissues. Furthermore, western blot analysis detected PADI4 expression in cultured tumor cell lines. ELISA detected increased PADI4 and cAT levels in the blood of patients with various malignant tumors

  6. High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

    PubMed Central

    Massot, Capucine; Bancel, Doriane; Lopez Lauri, Félicie; Truffault, Vincent; Baldet, Pierre; Stevens, Rebecca; Gautier, Hélène

    2013-01-01

    Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m-2 s-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling. PMID:24367665

  7. 12 CFR 1026.55 - Limitations on increasing annual percentage rates, fees, and charges.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... PROTECTION TRUTH IN LENDING (REGULATION Z) Special Rules Applicable to Credit Card Accounts and Open-End...)(2)(iii), or (b)(2)(xii) on a credit card account under an open-end (not home-secured) consumer credit plan. (b) Exceptions. A card issuer may increase an annual percentage rate or a fee or...

  8. 12 CFR 1026.55 - Limitations on increasing annual percentage rates, fees, and charges.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... PROTECTION TRUTH IN LENDING (REGULATION Z) Special Rules Applicable to Credit Card Accounts and Open-End...)(2)(iii), or (b)(2)(xii) on a credit card account under an open-end (not home-secured) consumer credit plan. (b) Exceptions. A card issuer may increase an annual percentage rate or a fee or...

  9. 12 CFR 226.55 - Limitations on increasing annual percentage rates, fees, and charges.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Rules Applicable to Credit Card Accounts and Open-End Credit Offered to College Students § 226.55... required to be disclosed under § 226.6(b)(2)(ii), (b)(2)(iii), or (b)(2)(xii) on a credit card account under an open-end (not home-secured) consumer credit plan. (b) Exceptions. A card issuer may increase...

  10. 12 CFR 1026.55 - Limitations on increasing annual percentage rates, fees, and charges.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... PROTECTION TRUTH IN LENDING (REGULATION Z) Special Rules Applicable to Credit Card Accounts and Open-End...)(2)(iii), or (b)(2)(xii) on a credit card account under an open-end (not home-secured) consumer credit plan. (b) Exceptions. A card issuer may increase an annual percentage rate or a fee or...

  11. The Increasing Availability of Official Datasets: Methods, Limitations and Opportunities for Studies of Education

    ERIC Educational Resources Information Center

    Gorard, Stephen

    2012-01-01

    The re-use of existing and official data has a very long and largely honourable history in education and social science. The principal change in the 60 years since the first issue of the "British Journal of Educational Studies" has been the increasing range, availability and quality of existing numeric datasets. New and valuable fields of…

  12. Nerve Demyelination Increases Metabotropic Glutamate Receptor Subtype 5 Expression in Peripheral Painful Mononeuropathy

    PubMed Central

    Ko, Miau-Hwa; Hsieh, Yu-Lin; Hsieh, Sung-Tsang; Tseng, To-Jung

    2015-01-01

    Wallerian degeneration or nerve demyelination, arising from spinal nerve compression, is thought to bring on chronic neuropathic pain. The widely distributed metabotropic glutamate receptor subtype 5 (mGluR5) is involved in modulating nociceptive transmission. The purpose of this study was to investigate the potential effects of mGluR5 on peripheral hypersensitivities after chronic constriction injury (CCI). Sprague-Dawley rats were operated on with four loose ligatures around the sciatic nerve to induce thermal hyperalgesia and mechanical allodynia. Primary afferents in dermis after CCI exhibited progressive decreases, defined as partial cutaneous denervation; importantly, mGluR5 expressions in primary afferents were statistically increased. CCI-induced neuropathic pain behaviors through the intraplantar injections of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a selective mGluR5 antagonist, were dose-dependently attenuated. Furthermore, the most increased mGluR5 expressions in primary afferents surrounded by reactive Schwann cells were observed at the distal CCI stumps of sciatic nerves. In conclusion, these results suggest that nerve demyelination results in the increases of mGluR5 expression in injured primary afferents after CCI; and further suggest that mGluR5 represents a main therapeutic target in developing pharmacological strategies to prevent peripheral hypersensitivities. PMID:25739080

  13. High-glucose environment increased thrombospondin-1 expression in keratinocytes via DNA hypomethylation.

    PubMed

    Lan, Cheng-Che E; Huang, Shu-Mei; Wu, Ching-Shuang; Wu, Chin-Han; Chen, Gwo-Shing

    2016-03-01

    Diabetes is an important health issue because of its increasing prevalence and association with impaired wound healing. Epidermal keratinocytes with overexpressed antiangiogenic molecule thrombospondin-1 (TSP1) have been shown to impair proper wound healing. This study examined the potential involvement of keratinocyte-derived TSP1 on diabetic wound healing. Cultured human keratinocytes and diabetic rat model were used to evaluate the effect of high-glucose environment on TSP1 expression in epidermal keratinocytes, and the molecular mechanisms involved in the process were also studied. We demonstrated that high-glucose environment increased TSP1 expression in keratinocytes. In addition, increased oxidative stress induced DNA hypomethylation at the TSP1 promoter region in keratinocytes exposed to high-glucose environment. Similar findings were found in our diabetic rat model. Early antioxidant administration normalized TSP1 expression and global DNA methylation status in diabetic rat skin and improved wound healing in vivo. Because oxidative stress contributed to TSP1 DNA hypomethylation, early recognition of diabetic condition and timely administration of antioxidant are logical approaches to reduce complications associated with diabetes as alterations in epigenome may not be reversible by controlling glucose levels during the later stages of disease course. PMID:26678678

  14. The Increased Expression of Connexin and VEGF in Mouse Ovarian Tissue Vitrification by Follicle Stimulating Hormone

    PubMed Central

    Yang, Yanzhou; Chen, Jie; Wu, Hao; Pei, Xiuying; Chang, Qing; Ma, Wenzhi; Ma, Huiming; Hei, Changchun; Zheng, Xiaomin; Cai, Yufang; Zhao, Chengjun; Yu, Jia; Wang, Yanrong

    2015-01-01

    Ovarian follicular damages were caused by cryoinjury during the process of ovarian vitrification and ischemia/reperfusion during the process of ovarian transplantation. And appropriate FSH plays an important role in antiapoptosis during ovarian follicle development. Therefore, in this study, 0.3 IU/mL FSH was administered into medium during mouse ovarian cryopreservation by vitrification to ascertain the function of FSH on ovarian vitrification and avascular transplantation. The results suggested that the expressions of Cx37, Cx43, apoptotic molecular caspase-3, and angiogenesis molecular VEGF were confirmed using immunohistochemistry, western blotting, and real-time PCR, and the results suggested that the treatment with FSH remarkably increased the number of morphologically normal follicles in vitrified/warmed ovaries by upregulating the expression of Cx37, Cx43, VEGF, and VEGF receptor 2, but downregulating the expression of caspase-3. In addition, the vitrified/warmed ovaries were transplanted, and the related fertility was analyzed, and the results suggested that the fertility, neoangiogenesis, and follicle reserve were remarkably increased in the FSH administrated group. Taken together, administration of 0.3 IU/mL FSH during ovarian cryopreservation by vitrification can maintain ovarian survival during ovarian vitrification and increases the blood supply with avascular transplantation via upregulation of Cx43, Cx37, and VEGF/VEGFR2, as well as through its antiapoptotic effects. PMID:26539488

  15. Prebiotic consumption in pregnant and lactating women increases IL-27 expression in human milk.

    PubMed

    Kubota, Takayuki; Shimojo, Naoki; Nonaka, Ken; Yamashita, Masakatsu; Ohara, Osamu; Igoshi, Yuka; Ozawa, Naoko; Nakano, Taiji; Morita, Yoshinori; Inoue, Yuzaburo; Arima, Takayasu; Chiba, Kohki; Nakamura, Yoshitaka; Ikegami, Shuji; Masuda, Kentaro; Suzuki, Shuichi; Kohno, Yoichi

    2014-02-01

    The consumption of probiotics by pregnant and lactating women may prevent the onset of allergic disorders in their children by increasing the concentrations of immunoactive agents such as cytokines in breast milk. Prebiotics such as fructo-oligosaccharides (FOS) increase the number of beneficial organisms such as bifidobacteria. Thus, prebiotics may have an effect similar to that of probiotics. The objective of the present study was to carry out a comprehensive analysis of mRNA expression in human milk cells to identify changes in the concentrations of cytokines in breast milk after the consumption of FOS (4 g × 2 times/d) by pregnant and lactating women. The microarray analysis of human milk cells demonstrated that the expression levels of five genes in colostrum samples and fourteen genes in 1-month breast milk samples differed more than 3-fold between the FOS and control groups (sucrose group). The mRNA expression level of IL-27, a cytokine associated with immunoregulatory function, was significantly higher in 1-month breast milk samples obtained from the FOS group than in those obtained from the control group. In addition, the protein concentrations of IL-27 in colostrum and 1-month breast milk samples were significantly higher in the FOS group than in the control group. In conclusion, the consumption of FOS by pregnant and lactating women increases the production of IL-27 in breast milk. Future studies will address the association of this phenomenon with the onset of allergic disorders in children. PMID:24073873

  16. Limited effects of preterm birth and the first enteral nutrition on cerebellum morphology and gene expression in piglets.

    PubMed

    Bergström, Anders; Kaalund, Sanne S; Skovgaard, Kerstin; Andersen, Anders D; Pakkenberg, Bente; Rosenørn, Ann; van Elburg, Ruurd M; Thymann, Thomas; Greisen, Gorm O; Sanglid, Per T

    2016-07-01

    Preterm pigs show many signs of immaturity that are characteristic of preterm infants. In preterm infants, the cerebellum grows particularly rapid and hypoplasia and cellular lesions are associated with motor dysfunction and cognitive deficits. We hypothesized that functional brain delays observed in preterm pigs would be paralleled by both structural and molecular differences in the cerebellum relative to term born piglets. Cerebella were collected from term (n = 56) and preterm (90% gestation, n = 112) pigs at 0, 5, and 26 days after birth for stereological volume estimations, large-scale qPCR gene expression analyses (selected neurodevelopmental genes) and western blot protein expression analysis (Sonic Hedgehog pathway). Memory and learning was tested using a T-maze, documenting that preterm pigs showed delayed learning. Preterm pigs also showed reduced volume of both white and gray matter at all three ages but the proportion of white matter increased postnatally, relative to term pigs. Early initiation of enteral nutrition had limited structural or molecular effects. The Sonic Hedgehog pathway was unaffected by preterm birth. Few differences in expression of the selected genes were found, except consistently higher mRNA levels of Midkine, p75, and Neurotrophic factor 3 in the preterm cerebellum postnatally, probably reflecting an adaptive response to preterm birth. Pig cerebellar development appears more affected by postconceptional age than by environmental factors at birth or postnatally. Compensatory mechanisms following preterm birth may include faster white matter growth and increased expression of selected genes for neurotrophic factors and regulation of angiogenesis. While the pig cerebellum is immature in 90% gestation preterm pigs, it appears relatively mature and resilient toward environmental factors. PMID:27462071

  17. Cotinine Exposure Increases Fallopian Tube PROKR1 Expression via Nicotinic AChRα-7

    PubMed Central

    Shaw, Julie L.V.; Oliver, Elizabeth; Lee, Kai-Fai; Entrican, Gary; Jabbour, Henry N.; Critchley, Hilary O.D.; Horne, Andrew W.

    2010-01-01

    Tubal ectopic pregnancy (EP) is the most common cause of maternal mortality in the first trimester of pregnancy; however, its etiology is uncertain. In EP, embryo retention within the Fallopian tube (FT) is thought to be due to impaired smooth muscle contractility (SMC) and alterations in the tubal microenvironment. Smoking is a major risk factor for EP. FTs from women with EP exhibit altered prokineticin receptor-1 (PROKR1) expression, the receptor for prokineticins (PROK). PROK1 is angiogenic, regulates SMC, and is involved in intrauterine implantation. We hypothesized that smoking predisposes women to EP by altering tubal PROKR1 expression. Sera/FT were collected at hysterectomy (n = 21). Serum levels of the smoking metabolite, cotinine, were measured by enzyme-linked immunosorbent assay. FTs were analyzed by q-RT-PCR, immunohistochemistry, and Western blotting for expression of PROKR1 and the predicted cotinine receptor, nicotinic acetylcholine receptor α-7 (AChRα−7). FT explants (n = 4) and oviductal epithelial cells (cell line OE-E6/E7) were treated with cotinine and an nAChRα−7 antagonist. PROKR1 transcription was higher in FTs from smokers (P < 0.01). nAChRα−7 expression was demonstrated in FT epithelium. Cotinine treatment of FT explants and OE-E6/E7 cells increased PROKR1 expression (P < 0.05), which was negated by cotreatment with nAChRα−7 antagonist. Smoking targets human FTs via nAChRα−7 to increase tubal PROKR1, leading to alterations in the tubal microenvironment that could predispose to EP. PMID:20864676

  18. ZBTB2 increases PDK4 expression by transcriptional repression of RelA/p65

    PubMed Central

    Kim, Min-Young; Koh, Dong-In; Choi, Won-Il; Jeon, Bu-Nam; Jeong, Deok-yoon; Kim, Kyung-Sup; Kim, Kunhong; Kim, Se-Hoon; Hur, Man-Wook

    2015-01-01

    The NF-κB is found in almost all animal cell types and is involved in a myriad of cellular responses. Aberrant expression of NF-κB has been linked to cancer, inflammatory diseases and improper development. Little is known about transcriptional regulation of the NF-κB family member gene RelA/p65. Sp1 plays a key role in the expression of the RelA/p65 gene. ZBTB2 represses transcription of the gene by inhibiting Sp1 binding to a Sp1-binding GC-box in the RelA/p65 proximal promoter (bp, −31 to −21). Moreover, recent studies revealed that RelA/p65 directly binds to the peroxisome proliferator-activated receptor-γ coactivator1α (PGC1α) to decrease transcriptional activation of the PGC1α target gene PDK4, whose gene product inhibits pyruvate dehydrogenase (PDH), a key regulator of TCA cycle flux. Accordingly, we observed that RelA/p65 repression by ZBTB2 indirectly results in increased PDK4 expression, which inhibits PDH. Consequently, in cells with ectopic ZBTB2, the concentrations of pyruvate and lactate were higher than those in normal cells, indicating changes in glucose metabolism flux favoring glycolysis over the TCA cycle. Knockdown of ZBTB2 in mouse xenografts decreased tumor growth. ZBTB2 may increase cell proliferation by reprogramming glucose metabolic pathways to favor glycolysis by upregulating PDK4 expression via repression of RelA/p65 expression. PMID:25609694

  19. Melatonin overcomes resistance to clofarabine in two leukemic cell lines by increased expression of deoxycytidine kinase.

    PubMed

    Yamanishi, Miho; Narazaki, Hidehiko; Asano, Takeshi

    2015-03-01

    Drug resistance remains a serious problem in leukemia therapy. Among newly developed nucleoside antimetabolites, clofarabine has broad cytotoxic activity showing therapeutic promise and is currently approved for relapsed acute lymphoblastic leukemia. To investigate the mechanisms responsible for clofarabine resistance, we established two clofarabine-resistant lymphoblastic leukemia cell lines from parental lines. To elucidate the mechanisms against clofarabine resistance in two newly established clofarabine-resistant cell lines, we measured the expression of export pumps multidrug resistance protein 1, multidrug resistance-associated protein 1, and ATP-binding cassette subfamily G member 2. There were no differences in the expression between clofarabine-sensitive and -resistant cell lines. Next, we determined expression of deoxycytidine kinase (dCK), which phosphorylates clofarabine to exert cytotoxicity, in clofarabine-sensitive and -resistant cells. Clofarabine-resistant cells showed significantly decreased expression of dCK RNA when compared with sensitive cells. To elucidate the mechanisms of decreased dCK expression in clofarabine-resistant cells, we analyzed the methylation status of CpG islands of the dCK promoter and found no differences in methylation status between clofarabine-sensitive and -resistant cells. Next, we measured the acetylation status of histone and found that total histone acetylation, and histone H3 and H4 acetylation on chromatin immunoprecipitation assay were significantly decreased in resistant cells. Melatonin is an indolamine that functions in the regulation of chronobiological rhythms to exert cytotoxic effects. We examined the effects of melatonin in clofarabine-resistant cells and found that melatonin treatment led to significantly increased cytotoxicity with clofarabine in resistant cells via increased acetylation. Melatonin may be a useful candidate for overcoming clofarabine resistance in two newly established clofarabine

  20. Mechanical stretch increases CCN2/CTGF expression in anterior cruciate ligament-derived cells

    SciTech Connect

    Miyake, Yoshiaki; Furumatsu, Takayuki; Kubota, Satoshi; Kawata, Kazumi; Ozaki, Toshifumi; Takigawa, Masaharu

    2011-06-03

    Highlights: {yields} CCN2/CTGF localizes to the ligament-to-bone interface, but is not to the midsubstance region of human anterior cruciate ligament (ACL). {yields} Mechanical stretch induces higher increase of CCN2/CTGF gene expression and protein secretion in ACL interface cells compared with ACL midsubstance cells. {yields} CCN2/CTGF treatment stimulates the proliferation of ACL interface cells. -- Abstract: Anterior cruciate ligament (ACL)-to-bone interface serves to minimize the stress concentrations that would arise between two different tissues. Mechanical stretch plays an important role in maintaining cell-specific features by inducing CCN family 2/connective tissue growth factor (CCN2/CTGF). We previously reported that cyclic tensile strain (CTS) stimulates {alpha}1(I) collagen (COL1A1) expression in human ACL-derived cells. However, the biological function and stress-related response of CCN2/CTGF were still unclear in ACL fibroblasts. In the present study, CCN2/CTGF was observed in ACL-to-bone interface, but was not in the midsubstance region by immunohistochemical analyses. CTS treatments induced higher increase of CCN2/CTGF expression and secretion in interface cells compared with midsubstance cells. COL1A1 expression was not influenced by CCN2/CTGF treatment in interface cells despite CCN2/CTGF stimulated COL1A1 expression in midsubstance cells. However, CCN2/CTGF stimulated the proliferation of interface cells. Our results suggest that distinct biological function of stretch-induced CCN2/CTGF might regulate region-specific phenotypes of ACL-derived cells.

  1. Effects of increased milking frequency on gene expression in the bovine mammary gland

    PubMed Central

    Connor, Erin E; Siferd, Stephen; Elsasser, Theodore H; Evock-Clover, Christina M; Van Tassell, Curtis P; Sonstegard, Tad S; Fernandes, Violet M; Capuco, Anthony V

    2008-01-01

    Background Previous research has demonstrated that increased milking frequency of dairy cattle during the first few weeks of lactation enhances milk yield, and that the effect persists throughout the entire lactation period. The specific mechanisms controlling this increase in milk production are unknown, but suggested pathways include increased mammary epithelial cell number, secretory capacity, and sensitivity to lactogenic hormones. We used serial analysis of gene expression (SAGE) and microarray analysis to identify changes in gene expression in the bovine mammary gland in response to 4× daily milking beginning at d 4 of lactation (IMF4) relative to glands milked 2× daily (Control) to gain insight into physiological changes occurring within the gland during more frequent milking. Results Results indicated changes in gene expression related to cell proliferation and differentiation, extracellular matrix (ECM) remodeling, metabolism, nutrient transport, and immune function in IMF4 versus Control cows. In addition, pathways expected to promote neovascularization within the gland appeared to be up regulated in IMF4 cows. To validate this finding, immunolocalization of Von Willebrandt's factor (VWF), an endothelial cell marker, and its co-localization with the nuclear proliferation antigen Ki67 were evaluated in mammary tissue sections at approximately d 7 and d 14 of lactation in cows milked 4× daily versus Controls to estimate endothelial cell abundance and proliferation within the gland. Consistent with expression of genes related to neovascularization, both abundance of VWF and its co-localization with Ki67 appeared to be elevated in cows milked 4× daily, suggesting persistent increased milk yield in response to increased milking frequency may be mediated or complemented by enhanced mammary ECM remodeling and neovascularization within the gland. Conclusion Additional study is needed to determine whether changes in ECM remodeling and neovascularization of the

  2. Increased Expression of Serglycin in Specific Carcinomas and Aggressive Cancer Cell Lines

    PubMed Central

    Korpetinou, Angeliki; Papachristou, Dionysios J.; Lampropoulou, Angeliki; Bouris, Panagiotis; Labropoulou, Vassiliki T.; Noulas, Argyrios; Karamanos, Nikos K.; Theocharis, Achilleas D.

    2015-01-01

    In the present pilot study, we examined the presence of serglycin in lung, breast, prostate, and colon cancer and evaluated its expression in cell lines and tissues. We found that serglycin was expressed and constitutively secreted in culture medium in high levels in more aggressive cancer cells. It is worth noticing that aggressive cancer cells that harbor KRAS or EGFR mutations secreted serglycin constitutively in elevated levels. Furthermore, we detected the transcription of an alternative splice variant of serglycin lacking exon 2 in specific cell lines. In a limited number of tissue samples analyzed, serglycin was detected in normal epithelium but was also expressed in higher levels in advanced grade tumors as shown by immunohistochemistry. Serglycin staining was diffuse, granular, and mainly cytoplasmic. In some cancer cells serglycin also exhibited membrane and/or nuclear immunolocalization. Interestingly, the stromal cells of the reactive tumor stroma were positive for serglycin, suggesting an enhanced biosynthesis for this proteoglycan in activated tumor microenvironment. Our study investigated for first time the distribution of serglycin in normal epithelial and cancerous lesions in most common cancer types. The elevated levels of serglycin in aggressive cancer and stromal cells may suggest a key role for serglycin in disease progression. PMID:26581653

  3. Copper-GHK increases integrin expression and p63 positivity by keratinocytes.

    PubMed

    Kang, Youn-A; Choi, Hye-Ryung; Na, Jung-Im; Huh, Chang-Hun; Kim, Min-Ji; Youn, Sang-Woong; Kim, Kyu-Han; Park, Kyoung-Chan

    2009-04-01

    Glycyl-L-histidyl-L-lysyl (GHK) possesses a high affinity for copper(II) ions, with which it spontaneously forms a complex (copper-GHK). It is well known that copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. This study was conducted to investigate the effects of copper-GHK on keratinocytes. Proliferative effects were analyzed and hematoxylin and eosin staining and immunohistochemistry were conducted to evaluate the effects of copper-GHK in skin equivalent (SE) models. In addition, western blotting was performed. In monolayer cultured keratinocytes, copper-GHK increased the proliferation of keratinocytes. When the SE models were evaluated, basal cells became cuboidal when copper-GHK was added. Immunohistochemical analysis revealed that copper-GHK increased proliferating cell nuclear antigen (PCNA) and p63 positivity. Furthermore, the expression of integrin alpha6 and beta1 increased in SE models, and these results were confirmed by Western blotting. The results of this study indicate that treatment with copper-GHK may increase the proliferative potential of basal keratinocytes by modulating the expression of integrins, p63 and PCNA. In addition, increased levels of p63, a putative stem cell marker of the skin, suggests that copper-GHK promotes the survival of basal stem cells in the skin. PMID:19319546

  4. Hypertension produced by placental ischemia in pregnant rats is associated with increased soluble endoglin expression.

    PubMed

    Gilbert, Jeffrey S; Gilbert, Sara A B; Arany, Marietta; Granger, Joey P

    2009-02-01

    Recent clinical studies indicate that an excess of angiostatic factors, such as soluble endoglin (sEng), is related to the occurrence of preeclampsia. Although recent clinical studies report that sEng is increased in preeclamptic women, the mechanisms underlying its overexpression remain unclear. Evidence suggests that hypoxia and induction of heme oxygenase-1 have opposing effects on sEng expression, the former stimulatory and the latter inhibitory. Hence, we hypothesized that placental ischemia because of reduced uterine perfusion pressure (RUPP) in the pregnant rat would increase sEng expression and decrease heme oxygenase-1. Mean arterial pressure was obtained via arterial catheter, and serum and placental proteins were measured by Western blot. Mean arterial pressure was increased (132+/-3 mm Hg versus 102+/-2 mm Hg; P<0.001), and fetal (2.35+/-0.05 g versus 1.76+/-0.08 g; P<0.001) and placental weight were decreased (0.47+/-0.04 g versus 0.58+/-0.03 g; P<0.01) in the RUPP compared with normal pregnant controls. Serum sEng (0.10+/-0.02 arbitrary pixel units [apu] versus 0.05+/-0.01 apu; P<0.05) and placental endoglin (4.7+/-2.3 apu versus 1.45+/-0.42 apu; P<0.05) were increased along with placental hypoxia inducible factor-1 alpha (1.42+/-0.25 apu versus 0.68+/-0.09 apu; P<0.05) expression in the RUPP versus the normal pregnant dams. Placental HO-1 (1.4+/-0.3 apu versus 2.5+/-0.1 apu; P<0.05) expression decreased in the RUPP compared with normal pregnant dams. The present findings support our hypothesis that placental ischemia because of RUPP increases the expression of sEng and shifts the balance of angiogenic factors in the maternal circulation toward an angiostatic state. The present study provides further evidence that placental ischemia is a strong in vivo stimulus of angiostatic factors during pregnancy. PMID:19075097

  5. Increased MFG-E8 expression and its implications in the vascular pathophysiology of cocaine abuse

    PubMed Central

    Kimura-Kojima, Haruka; Unuma, Kana; Funakoshi, Takeshi; Kato, Chizuru; Komatsu, Ayumi; Aki, Toshihiko; Uemura, Koichi

    2016-01-01

    The aim of this study was to examine the possible involvement of smooth muscle cell remodeling and the induction of MFG-E8 (milk fat globule protein epidermal growth factor-VIII) in vascular pathophysiology during cocaine administration in cultured cells and rats. Cocaine exerts bifurcate effects on vascular cells; it stimulates vasoconstriction through enhancement of catecholamine release at low doses, while it suppresses cardiovascular functions through inhibition of ion channels at high doses. Short-term exposure to a high concentration of cocaine (3 mM, 24 hr) resulted in cell death of A7r5 rat aorta-derived smooth muscle cells. On the other hand, long-term exposure of the same cells to a low concentration (0.3 mM, ~7 days) resulted in a transient increase in MFG-E8 expression followed by an increased tendency toward cyclin D1, PCNA (proliferating cell nuclear antigen), and CDK4 (cyclin-dependent protein kinase-4) expression. Interestingly, autophagy was not induced, but rather was impaired, in cocaine-treated cells. Increased expressions of MFG-E8, PCNA, and CDK4 were also observed in the aortic vascular cells of rats administered cocaine (50 mg/kg, 2 days, i.v.), confirming that cocaine induced MFG-E8 expression in vivo. Taken together, the results show that MFG-E8 is induced in vascular cells exposed to cocaine, and that this induction is likely to be involved in the vascular toxicity elicited by cocaine abuse. PMID:27182119

  6. Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in Escherichia coli

    PubMed Central

    Zhang, Lu; Alfano, James R.

    2014-01-01

    The oxidation of l-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ΔkatG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity. PMID:25384482

  7. Cell- and isoform-specific increases in arginase expression in acute silica-induced pulmonary inflammation.

    PubMed

    Poljakovic, Mirjana; Porter, Dale W; Millecchia, Lyndell; Kepka-Lenhart, Diane; Beighley, Christopher; Wolfarth, Michael G; Castranova, Vincent; Morris, Sidney M

    2007-01-15

    Arginase induction was reported in several inflammatory lung diseases, suggesting that this may be a common feature underlying the pathophysiology of such diseases. As little is known regarding arginase expression in silicosis, the induction and cellular localization of arginase were elucidated in lungs of Sprague-Dawley rats 24 h following exposure to varying doses of silica by intratracheal instillation. Arginase expression was evaluated by activity assay, quantification of arginase I and arginase II mRNA levels using real-time polymerase chain reaction (PCR), and immunohistochemistry. Analyses of cells and fluid obtained by bronchoalveolar lavage (BAL) showed that markers of pulmonary inflammation, tissue damage, activation of alveolar macrophages (AM) and NO production were significantly increased by all silica doses. Arginase activity was increased also in AMs isolated from BAL fluid of silica-treated rats. Silica produced two- and three-fold increases in arginase activity of whole lung at doses of 1 and 5 mg/100 g body weight, respectively. Levels of arginase I mRNA, but not of arginase II mRNA, were similarly elevated. In control lungs, arginase I immunoreactivity was observed only in AMs sparsely dispersed throughout the lung; no inducible nitric oxide synthase (iNOS) immunoreactivity was detected. In silica-treated lungs, arginase I and iNOS were co-expressed in most AMs that were abundantly clustered at inflammatory foci. The rapid induction of arginase I expression in inflammatory lung cells, similar to induction of arginase in other inflammatory lung diseases, implicates elevated arginase activity as a factor in the development of lung damage following exposure to silica. PMID:17365572

  8. Cell- and Isoform-specific Increases in Arginase Expression in Acute Silica-induced Pulmonary Inflammation

    PubMed Central

    Poljakovic, Mirjana; Porter, Dale W.; Millecchia, Lyndell; Kepka-Lenhart, Diane; Beighley, Christopher; Wolfarth, Michael G.; Castranova, Vincent; Morris, Sidney M.

    2009-01-01

    Arginase induction was reported in several inflammatory lung diseases, suggesting that this may be a common feature underlying the pathophysiology of such diseases. As little is known regarding arginase expression in silicosis, the induction and cellular localization of arginase was elucidated in lungs of Sprague-Dawley rats 24 hr following exposure to varying doses of silica by intratracheal instillation. Arginase expression was evaluated by activity assay, quantification of arginase I and arginase II mRNA levels using real-time PCR, and immunohistochemistry. Analyses of cells and fluid obtained by bronchoalveolar lavage (BAL) showed that markers of pulmonary inflammation, tissue damage, activation of alveolar macrophages (AM) and NO production were significantly increased by all silica doses. Arginase activity was increased also in AMs isolated from BAL fluid of silica-treated rats. Silica produced 2- and 3-fold increases in arginase activity of whole lung at doses of 1 and 5 mg/100g body weight, respectively. Levels of arginase I mRNA, but not of arginase II mRNA, were similarly elevated. In control lungs, arginase I immunoreactivity was observed only in AMs sparsely dispersed throughout the lung; no iNOS immunoreactivity was detected. In silica-treated lungs, arginase I and iNOS were co-expressed in most AMs that were abundantly clustered at inflammatory foci. The rapid induction of arginase I expression in inflammatory lung cells, similar to induction of arginase in other inflammatory lung diseases, implicates elevated arginase activity as a factor in the development of lung damage following exposure to silica. PMID:17365572

  9. Hepatitis C Virus Increases Occludin Expression via the Upregulation of Adipose Differentiation-Related Protein

    PubMed Central

    Branche, Emilie; Conzelmann, Stéphanie; Parisot, Clotilde; Bedert, Ludmila; Lévy, Pierre L.; Bartosch, Birke

    2016-01-01

    The hepatitis C virus (HCV) life cycle is closely associated with lipid metabolism. In particular, HCV assembly initiates at the surface of lipid droplets. To further understand the role of lipid droplets in HCV life cycle, we assessed the relationship between HCV and the adipose differentiation-related protein (ADRP), a lipid droplet-associated protein. Different steps of HCV life cycle were assessed in HCV-infected human Huh-7 hepatoma cells overexpressing ADRP upon transduction with a lentiviral vector. HCV infection increased ADRP mRNA and protein expression levels by 2- and 1.5-fold, respectively. The overexpression of ADRP led to an increase of (i) the surface of lipid droplets, (ii) the total cellular neutral lipid content (2.5- and 5-fold increase of triglycerides and cholesterol esters, respectively), (iii) the cellular free cholesterol level (5-fold) and (iv) the HCV particle production and infectivity (by 2- and 3.5-fold, respectively). The investigation of different steps of the HCV life cycle indicated that the ADRP overexpression, while not affecting the viral replication, promoted both virion egress and entry (~12-fold), the latter possibly via an increase of its receptor occludin. Moreover, HCV infection induces an increase of both ADRP and occludin expression. In HCV infected cells, the occludin upregulation was fully prevented by the ADRP silencing, suggesting a specific, ADRP-dependent mechanism. Finally, in HCV-infected human livers, occludin and ADRP mRNA expression levels correlated with each other. Alltogether, these findings show that HCV induces ADRP, which in turns appears to confer a favorable environment to viral spread. PMID:26731658

  10. Schisandra polysaccharide increased glucose consumption by up-regulating the expression of GLUT-4.

    PubMed

    Jin, Dun; Zhao, Ting; Feng, Wei-Wei; Mao, Guang-Hua; Zou, Ye; Wang, Wei; Li, Qian; Chen, Yao; Wang, Xin-Tong; Yang, Liu-Qing; Wu, Xiang-Yang

    2016-06-01

    In our previous study, a polysaccharide was extracted from Schisandra Chinensis (Trucz.) Baill and found with anti-diabetic effects. The aim of this study was to investigate the anti-diabetic effects of the low weight molecular polysaccharide (SCPP11) purified from crude Schisandra polysaccharide and illustrate the underlying mechanism in buffalo rat liver cells. The insulin resistance model of BRL cells was established by incubating with insulin solution for 24h. The effects of SCPP11 on regulating related protein and mRNA expression in an insulin and AMPK signal pathway were investigated by western blot and RT-PCR analysis. SCPP11 showed no cytotoxicity to BRL cells and could improve the glucose consumption in BRL cells. SCPP11 increased the protein expression of Akt, p-AMPK and GLUT-4 in BRL cells. Moreover, SCPP11 could enhance the mRNA expression levels of IRS-1, PI3K, Akt, GLUT-4, AMPKα and PPAR-γ in BRL cells at the same time. In conclusion, SCPP11 possessed effects in improving glucose consumption by up-regulating the expression of GLUT-4 which might occur via insulin and AMPK signal pathway and could be a potential functional food to prevent and mitigate the insulin resistance condition. PMID:26993529

  11. Increased invasiveness and aggressiveness in breast epithelia with cytoplasmic p63 expression

    PubMed Central

    Hsiao, Yi-Hsuan; Su, Yan A.; Tsai, Horng-Der; Mason, Jeffrey T.; Chou, Ming-Chih; Man, Yan-gao

    2010-01-01

    Our previous studies revealed that pregnancy associated breast cancer (PABC) had significantly reduced nuclear p63 expression in myoepithelia, while intense cytoplasmic p63 expression in associated epithelia. Our current study assessed these epithelia using immunohistochemistry with a panel of aggressiveness and invasiveness related markers and comparative genomic hybridization (array-CGH) with over 30,000 DNA probes. These epithelia showed several unique alterations, including (1) immunohistochemical and morphological resemblance to invasive cancer, (2) significant gain in copy numbers of DNA coding genes for morphogenesis, angiogenesis, and metastasis, and (3) significant loss in copy numbers of DNA coding genes for tumor suppressors, cell adhesion, and macromolecular complex assembly or intra-cellular trafficking. Detected array-CGH alterations correlated well with in vivo expression of a number of corresponding proteins tested. These findings suggest that aberrant sub-cellular localization of p63 expression in normal or hyperplastic appearing epithelial cells may significant contribute to increased invasiveness and aggressiveness of these cells. PMID:20714441

  12. Increased nuclear ?-catenin expression in oral potentially malignant lesions: A marker of epithelial dysplasia

    PubMed Central

    Rojas-Alcayaga, Gonzalo; Maturana, Andrea; Aitken, Juan-Pablo; Rojas, Carolina; Ortega, Ana-Verónica

    2015-01-01

    Background Deregulation of ?-catenin is associated with malignant transformation; however, its relationship with potentially malignant and malignant oral processes is not fully understood. The aim of this study was to determine and compare the nuclear ?-catenin expression in oral dysplasia and oral squamous cell carcinoma (OSCC). Material and Methods Cross sectional study. Immunodetection of ?-catenin was performed on 72 samples, with the following distribution: 21 mild dysplasia, 12 moderate dysplasia, severe dysplasia 3, 36 OSCC including 19 well differentiated, 15 moderately differentiated and 2 poorly differentiated. Through microscopic observation the number of positive cells per 1000 epithelial cells was counted. For the statistical analysis, the Kruskal Wallis test was used. Results Nuclear expression of ?-catenin was observed in all samples with severe and moderate dysplasia, with a median of 267.5, in comparison to mild dysplasia whose median was 103.75. Only 10 samples (27.7%) with OSCC showed nuclear expression, with statistically significant differences between groups (p < 0.05). Conclusions Our results are consistent with most of the reports which show increased presence of ?-catenin in severe and moderate dysplasia compared to mild dysplasia; however the expression of nuclear ?-catenin decreased after starting the invasive neoplastic process. This suggests a role for this protein in the progression of dysplasia and early malignant transformation to OSCC. Immunodetection of ?-catenin could be a possible immune marker in the detection of oral dysplasia. Key words:Oral squamous cell carcinoma (OSCC), ?-catenin, oral dysplasia. PMID:26241451

  13. Glucose deprivation induces chemoresistance in colorectal cancer cells by increasing ATF4 expression

    PubMed Central

    Hu, Ya-Ling; Yin, Yuan; Liu, He-Yong; Feng, Yu-Yang; Bian, Ze-Hua; Zhou, Le-Yuan; Zhang, Ji-Wei; Fei, Bo-Jian; Wang, Yu-Gang; Huang, Zhao-Hui

    2016-01-01

    AIM: To investigate the role of activating transcription factor 4 (ATF4) in glucose deprivation (GD) induced colorectal cancer (CRC) drug resistance and the mechanism involved. METHODS: Chemosensitivity and apoptosis were measured under the GD condition. Inhibition of ATF4 using short hairpin RNA in CRC cells under the GD condition and in ATF4-overexpressing CRC cells was performed to identify the role of ATF4 in the GD induced chemoresistance. Quantitative real-time RT-PCR and Western blot were used to detect the mRNA and protein expression of drug resistance gene 1 (MDR1), respectively. RESULTS: GD protected CRC cells from drug-induced apoptosis (oxaliplatin and 5-fluorouracil) and induced the expression of ATF4, a key gene of the unfolded protein response. Depletion of ATF4 in CRC cells under the GD condition can induce apoptosis and drug re-sensitization. Similarly, inhibition of ATF4 in the ATF4-overexpressing CRC cells reintroduced therapeutic sensitivity and apoptosis. In addition, increased MDR1 expression was observed in GD-treated CRC cells. CONCLUSION: These data indicate that GD promotes chemoresistance in CRC cells through up-regulating ATF4 expression. PMID:27468213

  14. Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality

    PubMed Central

    Alvarez, Silvia; Díaz, Marcos; Flach, Johanna; Rodriguez-Acebes, Sara; López-Contreras, Andrés J.; Martínez, Dolores; Cañamero, Marta; Fernández-Capetillo, Oscar; Isern, Joan; Passegué, Emmanuelle; Méndez, Juan

    2015-01-01

    Replicative stress during embryonic development influences ageing and predisposition to disease in adults. A protective mechanism against replicative stress is provided by the licensing of thousands of origins in G1 that are not necessarily activated in the subsequent S-phase. These ‘dormant' origins provide a backup in the presence of stalled forks and may confer flexibility to the replication program in specific cell types during differentiation, a role that has remained unexplored. Here we show, using a mouse strain with hypomorphic expression of the origin licensing factor mini-chromosome maintenance (MCM)3 that limiting origin licensing in vivo affects the functionality of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to complete maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full origin licensing ensures their correct differentiation and functionality. PMID:26456157

  15. Inflammatory Eicosanoids Increase Amyloid Precursor Protein Expression via Activation of Multiple Neuronal Receptors

    PubMed Central

    Herbst-Robinson, Katie J.; Liu, Li; James, Michael; Yao, Yuemang; Xie, Sharon X.; Brunden, Kurt R.

    2015-01-01

    Senile plaques comprised of Aβ peptides are a hallmark of Alzheimer’s disease (AD) brain, as are activated glia that release inflammatory molecules, including eicosanoids. Previous studies have demonstrated that amyloid precursor protein (APP) and Aβ levels can be increased through activation of thromboxane A2-prostanoid (TP) receptors on neurons. We demonstrate that TP receptor regulation of APP expression depends on Gαq-signaling and conventional protein kinase C isoforms. Importantly, we discovered that Gαq-linked prostaglandin E2 and leukotriene D4 receptors also regulate APP expression. Prostaglandin E2 and thromboxane A2, as well as total APP levels, were found to be elevated in the brains of aged 5XFAD transgenic mice harboring Aβ plaques and activated glia, suggesting that increased APP expression resulted from eicosanoid binding to Gαq-linked neuronal receptors. Notably, inhibition of eicosanoid synthesis significantly lowered brain APP protein levels in aged 5XFAD mice. These results provide new insights into potential AD therapeutic strategies. PMID:26672557

  16. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism.

    PubMed

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K; Lehtonen, Jukka Y A

    2016-04-20

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3'-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

  17. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

    PubMed Central

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K.; Lehtonen, Jukka Y.A.

    2016-01-01

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

  18. Cyclic stretch of Embryonic Cardiomyocytes Increases Proliferation, Growth, and Expression While Repressing Tgf-β Signaling

    PubMed Central

    Banerjee, Indroneal; Carrion, Katrina; Serrano, Ricardo; Dyo, Jeffrey; Sasik, Roman; Lund, Sean; Willems, Erik; Aceves, Seema; Meili, Rudolph; Mercola, Mark; Chen, Ju; Zambon, Alexander; Hardiman, Gary; Doherty, Taylor A; Lange, Stephan; del Álamo, Juan C.; Nigam, Vishal

    2014-01-01

    Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction. The objective of this study was to expose embryonic mouse cardiomyocytes (EMCM) to cyclic stretch and examine key molecular and phenotypic responses. Analysis of RNA-Sequencing data demonstrated that gene ontology groups associated with myofibril and cardiac development were significantly modulated. Stretch increased EMCM proliferation, size, cardiac gene expression, and myofibril protein levels. Stretch also repressed several components belonging to the Transforming Growth Factor-β (Tgf-β) signaling pathway. EMCMs undergoing cyclic stretch had decreased Tgf-β expression, protein levels, and signaling. Furthermore, treatment of EMCMs with a Tgf-β inhibitor resulted in increased EMCM size. Functionally, Tgf-β signaling repressed EMCM proliferation and contractile function, as assayed via dynamic monolayer force microscopy (DMFM). Taken together, these data support the hypothesis that biomechanical stimuli play a vital role in normal cardiac development and for cardiac pathology, including HLHS. PMID:25446186

  19. Increased hexokinase II expression in the renal glomerulus of mice in response to arsenic

    SciTech Connect

    Pysher, Michele D.; Sollome, James J.; Regan, Suzanne; Cardinal, Trevor R.; Hoying, James B.; Brooks, Heddwen L.; Vaillancourt, Richard R.

    2007-10-01

    Epidemiological studies link arsenic exposure to increased risks of cancers of the skin, kidney, lung, bladder and liver. Additionally, a variety of non-cancerous conditions such as diabetes mellitus, hypertension, and cardiovascular disease have been associated with chronic ingestion of low levels of arsenic. However, the biological and molecular mechanisms by which arsenic exerts its effects remain elusive. Here we report increased renal hexokinase II (HKII) expression in response to arsenic exposure both in vivo and in vitro. In our model, HKII was up-regulated in the renal glomeruli of mice exposed to low levels of arsenic (10 ppb or 50 ppb) via their drinking water for up to 21 days. Additionally, a similar effect was observed in cultured renal mesangial cells exposed to arsenic. This correlation between our in vivo and in vitro data provides further evidence for a direct link between altered renal HKII expression and arsenic exposure. Thus, our data suggest that alterations in renal HKII expression may be involved in arsenic-induced pathological conditions involving the kidney. More importantly, these results were obtained using environmentally relevant arsenic concentrations.

  20. Aromatase Expression Increases the Survival and Malignancy of Estrogen Receptor Positive Breast Cancer Cells

    PubMed Central

    Bandyopadhyay, Abhik; Kirma, Nameer B.; Tekmal, Rajeshwar R.; Wang, Shui; Sun, Lu-Zhe

    2015-01-01

    In postmenopausal women, local estrogen produced by adipose stromal cells in the breast is believed to support estrogen receptor alpha (ERα) positive breast cancer cell survival and growth. This raises the question of how the ERα positive metastatic breast cancer cells survive after they enter blood and lymph circulation, where estrogen level is very low in postmenopausal women. In this study, we show that the aromatase expression increased when ERα positive breast cancer cells were cultured in suspension. Furthermore, treatment with the aromatase substrate, testosterone, inhibited suspension culture-induced apoptosis whereas an aromatase inhibitor attenuated the effect of testosterone suggesting that suspended circulating ERα positive breast cancer cells may up-regulate intracrine estrogen activity for survival. Consistent with this notion, a moderate level of ectopic aromatase expression rendered a non-tumorigenic ERα positive breast cancer cell line not only tumorigenic but also metastatic in female nude mice without exogenous estrogen supplementation. The increased malignant phenotype was confirmed to be due to aromatase expression as the growth of orthotopic tumors regressed with systemic administration of an aromatase inhibitor. Thus, our study provides experimental evidence that aromatase plays an important role in the survival of metastatic ERα breast cancer cells by suppressing anoikis. PMID:25837259

  1. Nitric Oxide Increases Arterial Endotheial Permeability through Mediating VE-Cadherin Expression during Arteriogenesis

    PubMed Central

    Wu, Xiaoqiong; Guan, Yinglu; Zhang, Bin; Cai, Weijun; Schaper, Jutta; Schaper, Wolfgang

    2015-01-01

    Macrophage invasion is an important event during arteriogenesis, but the underlying mechanism is still only partially understood. The present study tested the hypothesis that nitric oxide (NO) and VE-cadherin, two key mediators for vascular permeability, contribute to this event in a rat ischemic hindlimb model. In addition, the effect of NO on expression of VE-caherin and endothelial permeability was also studied in cultured HUVECs. We found that: 1) in normal arteriolar vessels (NAV), eNOS was moderately expressed in endothelial cells (EC) and iNOS was rarely detected. In contrast, in collateral vessels (CVs) induced by simple femoral artery ligation, both eNOS and iNOS were significantly upregulated (P<0.05). Induced iNOS was found mainly in smooth muscle cells, but also in other vascular cells and macrophages; 2) in NAV VE-cadherin was strongly expressed in EC. In CVs, VE-cadherin was significantly downregulated, with a discontinuous and punctate pattern. Administration of nitric oxide donor DETA NONOate (NONOate) further reduced the amounts of Ve-cadherin in CVs, whereas NO synthase inhibitor L-NAME inhibited downregulation of VE-cadherin in CVs; 3) in normal rats Evans blue extravasation (EBE) was low in the musculus gracilis, FITC-dextron leakage was not detected in the vascular wall and few macrophages were observed in perivascular space. In contrast, EBE was significantly increased in femoral artery ligation rats, FITC-dextron leakage and increased amounts of macrophages were detected in CVs, which were further enhanced by administration of NONOate, but inhibited by L-NAME supplement; 4) in vitro experiments confirmed that an increase in NO production reduced VE-cadherin expression, correlated with increases in the permeability of HUVECs. In conclusion, our data for the first time reveal the expression profile of VE-cadherin and alterations of vascular permeability in CVs, suggesting that NO-mediated VE-cadherin pathway may be one important mechanism

  2. Expression of Fungal diacylglycerol acyltransferase2 Genes to Increase Kernel Oil in Maize[OA

    PubMed Central

    Oakes, Janette; Brackenridge, Doug; Colletti, Ron; Daley, Maureen; Hawkins, Deborah J.; Xiong, Hui; Mai, Jennifer; Screen, Steve E.; Val, Dale; Lardizabal, Kathryn; Gruys, Ken; Deikman, Jill

    2011-01-01

    Maize (Zea mays) oil has high value but is only about 4% of the grain by weight. To increase kernel oil content, fungal diacylglycerol acyltransferase2 (DGAT2) genes from Umbelopsis (formerly Mortierella) ramanniana and Neurospora crassa were introduced into maize using an embryo-enhanced promoter. The protein encoded by the N. crassa gene was longer than that of U. ramanniana. It included 353 amino acids that aligned to the U. ramanniana DGAT2A protein and a 243-amino acid sequence at the amino terminus that was unique to the N. crassa DGAT2 protein. Two forms of N. crassa DGAT2 were tested: the predicted full-length protein (L-NcDGAT2) and a shorter form (S-NcDGAT2) that encoded just the sequences that share homology with the U. ramanniana protein. Expression of all three transgenes in maize resulted in small but statistically significant increases in kernel oil. S-NcDGAT2 had the biggest impact on kernel oil, with a 26% (relative) increase in oil in kernels of the best events (inbred). Increases in kernel oil were also obtained in both conventional and high-oil hybrids, and grain yield was not affected by expression of these fungal DGAT2 transgenes. PMID:21245192

  3. SETDB1 mediated FosB expression increases the cell proliferation rate during anticancer drug therapy

    PubMed Central

    Na, Han-Heom; Noh, Hee-Jung; Cheong, Hyang-Min; Kang, Yoonsung; Kim, Keun-Cheol

    2016-01-01

    The efficacy of anticancer drugs depends on a variety of signaling pathways, which can be positively or negatively regulated. In this study, we show that SETDB1 HMTase is down-regulated at the transcriptional level by several anticancer drugs, due to its inherent instability. Using RNA sequence analysis, we identified FosB as being regulated by SETDB1 during anticancer drug therapy. FosB expression was increased by treatment with doxorubicin, taxol and siSETDB1. Moreover, FosB was associated with an increased rate of proliferation. Combinatory transfection of siFosB and siSETDB1 was slightly increased compared to transfection of siFosB. Furthermore, FosB was regulated by multiple kinase pathways. ChIP analysis showed that SETDB1 and H3K9me3 interact with a specific region of the FosB promoter. These results suggest that SETDB1-mediated FosB expression is a common molecular phenomenon, and might be a novel pathway responsible for the increase in cell proliferation that frequently occurs during anticancer drug therapy. [BMB Reports 2016; 49(4): 238-243] PMID:26949019

  4. Early increase in DcR2 expression and late activation of caspases in the platelet storage lesion.

    PubMed

    Plenchette, S; Moutet, M; Benguella, M; N'Gondara, J P; Guigner, F; Coffe, C; Corcos, L; Bettaieb, A; Solary, E

    2001-10-01

    Platelet transfusion is widely used to prevent bleeding in patients with severe thrombocytopenia. The maximal storage duration of platelet concentrates is usually 5 days, due to the platelet storage lesion that impairs their functions when stored for longer times. Some of the morphological and biochemical changes that characterize this storage lesion are reminiscent of cell death by apoptosis. The present study analyzed whether proteins involved in nucleated cell apoptosis could play a role in the platelet storage lesion. Storage of leukocyte-depleted platelets obtained by apheresis is associated with a late and limited activation of caspases, mainly caspase-3. This event correlates with an increased expression of the pro-apoptotic BH3-only protein Bim in the particulate fraction and a slight and late release of the pro-apoptotic mitochondrial protein Diablo/Smac in the cytosol. Platelets do not express the death receptors Fas, DR4 and DR5 on their plasma membrane, while the expression of the decoy receptor DcR2 increases progressively during platelet storage. Addition of low concentrations of the cryoprotector dimethylsulfoxide accelerates platelet caspase activation during storage, an effect that is partially prevented by the caspase inhibitor z-VAD-fmk. Altogether, DcR2 expression on the plasma membrane is an early event while caspase activation is a late event during platelet storage. These observations suggest that caspases are unlikely to account for the platelet storage lesion. As a consequence, addition of caspase inhibitors may not improve the quality of platelet concentrates stored in standard conditions. PMID:11587215

  5. Using organic matter to increase soil fertility in Burundi: potentials and limitations

    NASA Astrophysics Data System (ADS)

    Kaboneka, Salvator

    2015-04-01

    Agriculture production in Burundi is dominated by small scale farmers (0.5 ha/household) who have only very limited access to mineral inputs. In the past, farmers have relied on fallow practices combined with farm yard manures to maintain and improve soil fertility. However, due to the high population growth and high population density (370/km²), fallow practices are nowadays no longer feasible, animal manures cannot be produced in sufficient quantities to maintain soil productivity and food insecurity has become a quasi permanent reality. Most Burundian soils are characterized by 1:1 types of clay minerals (kaolinite) and are acidic in nature. Such soils are of very low cation exchange capacity (CEC). To compare the effect of % clays and % organic matter (% C), correlations tests have been conducted between the two parameters and the CEC. It was found that in high altitude kaolinitic and acidic soils, CEC was highly correlated to % C and less correlated to % clay, suggesting that organic matter could play an important role in improving fertility and productivity of these soils. Based on these findings, additional studies have been conducted to evaluate the fertilizer and soil amendment values of animal manures (cattle, goat, chicken), and leguminous (Calliandra calothyrsus, Gliricidia sepium, Senna simea, Senna spectabilis) and non-leguminous (Tithonia diversifolia) foliar biomass. It was observed that chicken manure significantly reduces Al3+ levels in acidic soils, while Tithonia diversifolia outperforms in nutrient releases compared to the commonly known leguminous agroforestry shrubs and trees indicated above. Although the above mentioned organic sources can contribute to the soil nutrients supply, the quantities potentially available on farm are generally small. The only solution is to supplement these organic sources with other organic sources (compost, organic household waste), chemical fertilizers and mineral amendments (lime) to achieve Integrated Soil

  6. Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs

    PubMed Central

    Fain, John N.; Company, Joseph M.; Booth, Frank W.; Laughlin, M. Harold; Padilla, Jaume; Jenkins, Nathan T.; Bahouth, Suleiman W.; Sacks, Harold S.

    2013-01-01

    Background Exercise training elevates circulating irisin and induces the expression of the FNDC5 gene in skeletal muscles of mice. Our objective was to determine whether exercise training also increases FNDC5 protein or mRNA expression in the skeletal muscles of pigs as well as plasma irisin. Methods Castrated male pigs of the Rapacz familial hypercholesterolemic (FHM) strain and normal (Yucatan miniature) pigs were sacrificed after 16–20 weeks of exercise training. Samples of cardiac muscle, deltoid and triceps brachii muscle, subcutaneous and epicardial fat were obtained and FNDC5 mRNA, along with that of 6 other genes, was measured in all tissues of FHM pigs by reverse transcription polymerase chain reaction. FNDC protein in deltoid and triceps brachii was determined by Western blotting in both FHM and normal pigs. Citrate synthase activity was measured in the muscle samples of all pigs as an index of exercise training. Irisin was measured by an ELISA assay. Results There was no statistically significant effect of exercise training on FNDC5 gene expression in epicardial or subcutaneous fat, deltoid muscle, triceps brachii muscle or heart muscle. Exercise-training elevated circulating levels of irisin in the FHM pigs and citrate synthase activity in deltoid and triceps brachii muscle. A similar increase in citrate synthase activity was seen in muscle extracts of exercise-trained normal pigs but there was no alteration in circulating irisin. Conclusion Exercise training in pigs does not increase FNDC5 mRNA or protein in the deltoid or triceps brachii of FHM or normal pigs while increasing circulating irisin only in the FHM pigs. These data indicate that the response to exercise training in normal pigs is not comparable to that seen in mice. PMID:23831442

  7. Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress

    PubMed Central

    Williams, Robert; Lemaire, Paul; Lewis, Philip; McDonald, Fiona B.; Lucking, Eric; Hogan, Sean; Sheehan, David; Healy, Vincent; O'Halloran, Ken D.

    2015-01-01

    Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation

  8. Increased transforming growth factor beta 1 expression mediates ozone-induced airway fibrosis in mice

    PubMed Central

    Katre, Ashwini; Ballinger, Carol; Akhter, Hasina; Fanucchi, Michelle; Kim, Dae-Kee; Postlethwait, Edward; Liu, Rui-Ming

    2013-01-01

    Ozone (O3), a commonly encountered environmental pollutant, has been shown to induce pulmonary fibrosis in different animal models; the underlying mechanism, however, remains elusive. To investigate the molecular mechanism underlying O3-induced pulmonary fibrosis, 6- to 8-week-old C57BL/6 male mice were exposed to a cyclic O3 exposure protocol consisting of 2 days of filtered air and 5 days of O3 exposure (0.5 ppm, 8 h/day) for 5 and 10 cycles with or without intraperitoneal injection of IN-1233, a specific inhibitor of the type 1 receptor of transforming growth factor beta (TGF-β), the most potent profibrogenic cytokine. The results showed that O3 exposure for 5 or 10 cycles increased the TGF-β protein level in the epithelial lining fluid (ELF), associated with an increase in the expression of plasminogen activator inhibitor 1 (PAI-1), a TGF-β-responsive gene that plays a critical role in the development of fibrosis under various pathological conditions. Cyclic O3 exposure also increased the deposition of collagens and alpha smooth muscle actin (α-SMA) in airway walls. However, these fibrotic changes were not overt until after 10 cycles of O3 exposure. Importantly, blockage of the TGF-β signaling pathway with IN-1233 suppressed O3-induced Smad2/3 phosphorylation, PAI-1 expression, as well as collagens and α-SMA deposition in the lung. Our data demonstrate for the first time that O3 exposure increases TGF-β expression and activates TGF-β signaling pathways, which mediates O3-induced lung fibrotic responses in vivo. PMID:21689010

  9. Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice.

    PubMed

    Kesby, James P; Hubbard, David T; Markou, Athina; Semenova, Svetlana

    2014-07-01

    Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population. PMID

  10. Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice

    PubMed Central

    Kesby, James P.; Hubbard, David T.; Markou, Athina; Semenova, Svetlana

    2012-01-01

    Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population. PMID

  11. Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum

    PubMed Central

    2010-01-01

    Background The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome. Results Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings. Conclusions The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology. PMID:20074347

  12. Estrogen therapy increases BDNF expression and improves post-stroke depression in ovariectomy-treated rats

    PubMed Central

    Su, Qiaoer; Cheng, Yifan; Jin, Kunlin; Cheng, Jianhua; Lin, Yuanshao; Lin, Zhenzhen; Wang, Liuqing; Shao, Bei

    2016-01-01

    The present study investigated the effect of exogenous estrogen on post-stroke depression. Rats were exposed to chronic mild stress following middle cerebral artery occlusion. The occurrence of post-stroke depression was evaluated according to the changes in preference for sucrose and performance in a forced swimming test. Estrogen therapy significantly improved these neurological symptoms, indicating that estrogen is effective in treating post-stroke depression. Increased brain-derived neurotrophic factor (BDNF) expression was reported in the hippocampus of rats that had been treated with estrogen for two weeks, suggesting that BDNF expression may be an important contributor to the improvement of post-stroke depression that is observed following estrogen therapy. PMID:27602095

  13. Mouse skin passage of Streptococcus pyogenes results in increased streptokinase expression and activity.

    PubMed

    Rezcallah, Myrna S; Boyle, Michael D P; Sledjeski, Darren D

    2004-02-01

    The plasminogen activator streptokinase has been proposed to be a key component of a complex mechanism that promotes skin invasion by Streptococcus pyogenes. This study was designed to compare ska gene message and protein levels in wild-type M1 serotype isolate 1881 and a more invasive variant recovered from the spleen of a lethally infected mouse. M1 isolates selected for invasiveness demonstrated enhanced levels of active plasminogen activator activity in culture. This effect was due to a combination of increased expression of the ska gene and decreased expression of the speB gene. The speB gene product, SpeB, was found to efficiently degrade streptokinase in vitro. PMID:14766914

  14. Expression of MMP-2 correlates with increased angiogenesis in CNS metastasis of lung carcinoma

    PubMed Central

    Rojiani, Mumtaz V; Alidina, Janeen; Esposito, Nicole; Rojiani, Amyn M

    2010-01-01

    Matrix metalloproteinases (MMP) have been implicated in increased invasive and metastatic potential of tumors, possibly via interactions with the extracellular matrix and angiogenesis. This study investigates the relationship between MMP-2 immunoexpression and angiogenesis in a series of lung carcinomas metastatic to the central nervous system (CNS). Twenty eight metastatic carcinoma cases with adequate brain-tumor interface were identified from the archives at the Moffitt Cancer Center. MMP-2 expression was determined by immunohistochemistry using an antibody directed against pro and active forms (NeoMarkers). Similarly, microvessels were identified on parallel sections with anti-CD34 antibody (Biogenix). Angiogenesis profiles within the tumor and at the CNS/tumor interface were morphometrically assessed by the Image Pro Plus image analysis system. Briefly, CD34 positive vessels were quantitated and correlated with presence or absence of MMP-2 expression in the tumor. Mean microvessel area (MMVA) and mean microvessel number (MMVN) were assessed within areas of brain-tumor interface and within the tumor and expressed as a ratio relative to the tumor. Sixteen (57.14%) metastatic tumors were strongly immunoreac-tive for MMP-2, while 12 (42.86%) were negative. MMP-2 positive tumors had a higher MMVA and MMVN ratio at the CNS/tumor interface in comparison to MMP-2 negative neoplasms. MMP-2 expression thus appears to confer enhanced vascular proliferation particularly at the brain-tumor interface which would support the contention of enhanced capability of growth and invasion within the CNS, possibly modulated by MMP2. The relationship between MMP-2 expression and angiogenesis has been previously reported and its biological and therapeutic implications remain the focus of investigations. PMID:21151391

  15. Expression of MMP-2 correlates with increased angiogenesis in CNS metastasis of lung carcinoma.

    PubMed

    Rojiani, Mumtaz V; Alidina, Janeen; Esposito, Nicole; Rojiani, Amyn M

    2010-01-01

    Matrix metalloproteinases (MMP) have been implicated in increased invasive and metastatic potential of tumors, possibly via interactions with the extracellular matrix and angiogenesis. This study investigates the relationship between MMP-2 immunoexpression and angiogenesis in a series of lung carcinomas metastatic to the central nervous system (CNS). Twenty eight metastatic carcinoma cases with adequate brain-tumor interface were identified from the archives at the Moffitt Cancer Center. MMP-2 expression was determined by immunohistochemistry using an antibody directed against pro and active forms (NeoMarkers). Similarly, microvessels were identified on parallel sections with anti-CD34 antibody (Biogenix). Angiogenesis profiles within the tumor and at the CNS/tumor interface were morphometrically assessed by the Image Pro Plus image analysis system. Briefly, CD34 positive vessels were quantitated and correlated with presence or absence of MMP-2 expression in the tumor. Mean microvessel area (MMVA) and mean microvessel number (MMVN) were assessed within areas of brain-tumor interface and within the tumor and expressed as a ratio relative to the tumor. Sixteen (57.14%) metastatic tumors were strongly immunoreac-tive for MMP-2, while 12 (42.86%) were negative. MMP-2 positive tumors had a higher MMVA and MMVN ratio at the CNS/tumor interface in comparison to MMP-2 negative neoplasms. MMP-2 expression thus appears to confer enhanced vascular proliferation particularly at the brain-tumor interface which would support the contention of enhanced capability of growth and invasion within the CNS, possibly modulated by MMP2. The relationship between MMP-2 expression and angiogenesis has been previously reported and its biological and therapeutic implications remain the focus of investigations. PMID:21151391

  16. The loss of NDRG2 expression improves depressive behavior through increased phosphorylation of GSK3β.

    PubMed

    Ichikawa, Tomonaga; Nakahata, Shingo; Tamura, Tomohiro; Manachai, Nawin; Morishita, Kazuhiro

    2015-10-01

    N-myc downstream-regulated gene 2 (NDRG2) is one of the important stress-inducible genes and plays a critical role in negatively regulating PI3K/AKT signaling during hypoxia and inflammation. Through recruitment of PP2A phosphatase, NDRG2 maintains the dephosphorylated status of PTEN to suppress excessive PI3K/AKT signaling, and loss of NDRG2 expression is frequently seen in various types of cancer with enhanced activation of PI3K/AKT signaling. Because NDRG2 is highly expressed in the nervous system, we investigated whether NDRG2 plays a functional role in the nervous system using Ndrg2-deficient mice. Ndrg2-deficient mice do not display any gross abnormalities in the nervous system, but they have a diminished behavioral response associated with anxiety. Ndrg2-deficient mice exhibited decreased immobility and increased head-dipping and rearing behavior in two behavioral models, indicating an improvement of emotional anxiety-like behavior. Moreover, treatment of wild-type mice with the antidepressant drug imipramine reduced the expression of Ndrg2 in the frontal cortex, which was due to the degradation of HIF-1α through reduced expression of HSP90 protein. Furthermore, we found that the down-regulation of Ndrg2 in Ndrg2-deficient mice and imipramine treatment improved mood behavior with enhanced phosphorylation of GSK3β through activation of PI3K/AKT signaling, suggesting that the expression level of NDRG2 has a causal influence on mood-related phenotypes. Collectively, these results suggest that NDRG2 may be a potential target for mood disorders such as depression and anxiety. PMID:26208882

  17. Progestin increases the expression of gonadotropins in pituitaries of male zebrafish.

    PubMed

    Wang, Cuili; Liu, Dongteng; Chen, Weiting; Ge, Wei; Hong, Wanshu; Zhu, Yong; Chen, Shi X

    2016-07-01

    Our previous study showed that the in vivo positive effects of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), the major progestin in zebrafish, on early spermatogenesis was much stronger than the ex vivo ones, which may suggest an effect of DHP on the expression of gonadotropins. In our present study, we first observed that fshb and lhb mRNA levels in the pituitary of male adult zebrafish were greatly inhibited by 3 weeks exposure to 10nM estradiol (E2). However, an additional 24h 100nM DHP exposure not only reversed the E2-induced inhibition, but also significantly increased the expression of fshb and lhb mRNA. These stimulatory effects were also observed in male adult fish without E2 pretreatment, and a time course experiment showed that it took 24h for fshb and 12h for lhb to respond significantly. Because these stimulatory activities were partially antagonized by a nuclear progesterone receptor (Pgr) antagonist mifepristone, we generated a Pgr-knockout (pgr(-/-)) model using the TALEN technique. With and without DHP in vivo treatment, fshb and lhb mRNA levels of pgr(-/-) were significantly lower than those of pgr(+/+) Furthermore, ex vivo treatment of pituitary fragments of pgr(-/-) with DHP stimulated lhb, but not fshb mRNA expression. Results from double-colored fluorescent in situ hybridization showed that pgr mRNA was expressed only in fshb-expressing cells. Taken together, our results indicated that DHP participated in the regulation of neuroendocrine control of reproduction in male zebrafish, and exerted a Pgr-mediated direct stimulatory effect on fshb mRNA at pituitary level. PMID:27113852

  18. Increased midkine expression correlates with desmoid tumour recurrence: a potential biomarker and therapeutic target.

    PubMed

    Colombo, Chiara; Creighton, Chad J; Ghadimi, Markus P; Bolshakov, Svetlana; Warneke, Carla L; Zhang, Yiqun; Lusby, Kristelle; Zhu, Shirley; Lazar, Alexander J; West, Robert B; van de Rijn, Matt; Lev, Dina

    2011-12-01

    Desmoid tumours (DTs) are soft tissue monoclonal neoplasms exhibiting a unique phenotype, consisting of aggressive local invasiveness without metastatic capacity. While DTs can infrequently occur as part of familial adenomatosis polyposis, most cases arise sporadically. Sporadic DTs harbour a high prevalence of CTNNB1 mutations and hence increased β-catenin signalling. However, β-catenin downstream transcriptional targets and other molecular deregulations operative in DT inception and progression are currently not well defined, contributing to the lack of sensitive molecular prognosticators and efficacious targeted therapeutic strategies. We compared the gene expression profiles of 14 sporadic DTs to those of five corresponding normal tissues and six solitary fibrous tumour specimens. A DT expression signature consisting of 636 up- and 119 down-regulated genes highly enriched for extracellular matrix, cell adhesion and wound healing-related proteins was generated. Furthermore, 98 (15%) of the over-expressed genes were demonstrated to contain a TCF/LEF consensus binding site in their promoters, possibly heralding direct β-catenin downstream targets relevant to DT. The protein products of three of the up-regulated DT genes: ADAM12, MMP2 and midkine, were found to be commonly expressed in a large cohort of human DT samples assembled on a tissue microarray. Interestingly, enhanced midkine expression significantly correlated with a higher propensity and decreased time for primary DT recurrence (log-rank p = 0.0025). Finally, midkine was found to enhance the migration and invasion of primary DT cell cultures. Taken together, these studies provide insights into potential DT molecular aberrations and novel β-catenin transcriptional targets. Further studies to confirm the utility of midkine as a clinical DT molecular prognosticator and a potential therapeutic target are therefore warranted. Raw gene array data can be found at: http://smd.stanford.edu/ PMID:21826666

  19. Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum.

    PubMed

    Dietrich, Christiane; Nato, Aimé; Bost, Bruno; Le Maréchal, Pierre; Guyonvarch, Armel

    2009-04-01

    Corynebacterium glutamicum is a biotin-auxotrophic bacterium and some strains efficiently produce glutamic acid under biotin-limiting conditions. In an effort to understand C. glutamicum metabolism under biotin limitation, growth of the type strain ATCC 13032 was investigated in batch cultures and a time-course analysis was performed. A transient excretion of organic acids was observed and we focused our attention on lactate synthesis. Lactate synthesis was due to the ldh-encoded l-lactate dehydrogenase (Ldh). Features of Ldh activity and ldh transcription were analysed. The ldh gene was shown to be regulated at the transcriptional level by SugR, a pleiotropic transcriptional repressor also acting on most phosphotransferase system (PTS) genes. Electrophoretic mobility shift assays (EMSAs) and site-directed mutagenesis allowed the identification of the SugR-binding site. Effector studies using EMSAs and analysis of ldh expression in a ptsF mutant revealed fructose 1-phosphate as a highly efficient negative effector of SugR. Fructose 1,6-bisphosphate also affected SugR binding. PMID:19332837

  20. Increased microRNA-155 expression in the serum and peripheral monocytes in chronic HCV infection

    PubMed Central

    2012-01-01

    Background Hepatitis C Virus (HCV), a single stranded RNA virus, affects millions of people worldwide and leads to chronic infection characterized by chronic inflammation in the liver and in peripheral immune cells. Chronic liver inflammation leads to progressive liver damage. MicroRNAs (miRNA) regulate inflammation (miR-155, -146a and -125b) as well as hepatocyte function (miR-122). Methods Here we hypothesized that microRNAs are dysregulated in chronic HCV infection. We examined miRNAs in the circulation and in peripheral monocytes of patients with chronic HCV infection to evaluate if specific miRNA expression correlated with HCV infection. Results We found that monocytes from chronic HCV infected treatment-naïve (cHCV) but not treatment responder patients showed increased expression of miR-155, a positive regulator of TNFα, and had increased TNFα production compared to monocytes of normal controls. After LPS stimulation, miR-155 levels were higher in monocytes from cHCV patients compared to controls. MiR-125b, which has negative regulatory effects on inflammation, was decreased in cHCV monocytes compared to controls. Stimulation of normal monocytes with TLR4 and TLR8 ligands or HCV core, NS3 and NS5 recombinant proteins induced a robust increase in both miR-155 expression and TNFα production identifying potential mechanisms for in vivo induction of miR-155. Furthermore, we found increased serum miR-155 levels in HCV patients compared to controls. Serum miR-125b and miR-146a levels were also increased in HCV patients. Serum levels of miR-122 were elevated in cHCV patients and correlated with increased ALT and AST levels and serum miR-155 levels. Conclusion In conclusion, our novel data demonstrate that miR-155, a positive regulator of inflammation, is upregulated both in monocytes and in the serum of patients with chronic HCV infection. Our study suggests that HCV core, NS3, and NS5 proteins or TLR4 and TLR8 ligands can mediate increased miR-155 and TNF

  1. Tiaogan Qingxin Granule treatment increases myocardial connexin 43 expression in a rat model of arrhythmia.

    PubMed

    Yao, J H; Qu, C H; Ma, L; Chang, X Z

    2016-01-01

    Myocardial ischemia-induced arrhythmia, especially ventricular arrhythmia, is the main reason for sudden cardiac death. Therefore, ischemic ventricular arrhythmia-targeted treatments are urgently needed. The mechanism of Tiaogan Qingxin Granule in premature ventricular beat (PVB) treatment was explored in arrhythmic rats pretreated with Tiaogan Qingxin Granule. Sprague-Dawley rats (N = 40) were randomly divided into 4 groups: sham-operated, arrhythmia model, Wenxin Granule, and Tiaogan Qingxin Granule. The ischemic arrhythmia model was established by ligating the left anterior descending coronary artery. The Tiaogan Qingxin Granule group was treated intragastrically for 7 days before surgery. Sham-operated rats underwent thoracotomy without coronary artery ligation. Myocardial infarction rate was measured using the triphenyltetrazolium chloride method and Cx43 expression was quantified by western blotting. Compared to the arrhythmia model group, the Tiaogan Qingxin Granule group showed a significant reduction in the myocardial infarct size and myocardial infarction rate (P < 0.01). Cx43 expression in the left ventricular myocardial tissues was significantly lower in the arrhythmia model group than in the sham-operated group (P < 0.01), but significantly higher in the Tiaogan Qingxin Granule group (P < 0.01). Intergroup difference in the relative Cx43 expression between the Tiaogan Qingxin Granule and Wenxin Granule groups was not significant (P > 0.05). Thus, Tiaogan Qingxin Granule reduced the myocardial infarct size, lowered the myocardial infarction rate, and increased Cx43 expression, possibly by increasing blood supply to the cardiac muscles. In conclusion, Tiaogan Qingxin Granule may be useful for treating ischemic PVB. PMID:27323125

  2. Increased dysbindin-1B isoform expression in schizophrenia and its propensity in aggresome formation

    PubMed Central

    Xu, Yiliang; Sun, Yuhui; Ye, Haihong; Zhu, Li; Liu, Jianghong; Wu, Xiaofeng; Wang, Le; He, Tingting; Shen, Yan; Wu, Jane Y; Xu, Qi

    2015-01-01

    Genetic variations in the human dysbindin-1 gene (DTNBP1) have been associated with schizophrenia. As a result of alternative splicing, the human DTNBP1 gene generates at least three distinct protein isoforms, dysbindin-1A, -1B and -1C. Significant effort has focused on dysbindin-1A, an important player in multiple steps of neurodevelopment. However, the other isoforms, dysbindin-1B and dysbindin-1C have not been well characterized. Nor have been associated with human diseases. Here we report an increase in expression of DTNBP1b mRNA in patients with paranoid schizophrenia as compared with healthy controls. A single-nucleotide polymorphism located in intron 9, rs117610176, has been identified and associated with paranoid schizophrenia, and its C allele leads to an increase of DTNBP1b mRNA splicing. Our data show that different dysbindin splicing isoforms exhibit distinct subcellular distribution, suggesting their distinct functional activities. Dysbindin-1B forms aggresomes at the perinuclear region, whereas dysbindin-1A and -1C proteins exhibit diffused patterns in the cytoplasm. Dysbindin-1A interacts with dysbindin-1B, getting recruited to the aggresome structure when co-expressed with dysbindin-1B. Moreover, cortical neurons over-expressing dysbindin-1B show reduction in neurite outgrowth, suggesting that dysbindin-1B may interfere with dysbindin-1A function in a dominant-negative manner. Taken together, our study uncovers a previously unknown association of DTNBP1b expression with schizophrenia in addition to its distinct biochemical and functional properties.

  3. Treatment with estrogen protects against ovariectomy-induced hepatic steatosis by increasing AQP7 expression.

    PubMed

    Fu, Xiaohua; Xing, Lili; Xu, Weihai; Shu, Jing

    2016-07-01

    Recent evidence has suggested that the marked decrease in ovarian secretion of estrogens in postmenopausal women may be associated with the development of non-alcoholic fatty liver disease. The present study aimed to elucidate the mechanisms by which low levels of estrogen induce fatty liver disease using an ovariectomized (OVX) mouse model and an in vitro cell model. A total of 24 female C57/BL6 mice were divided into four groups: Sham operation, sham operation plus subcutaneous implantation of tamoxifen (TAM), bilateral OVX, and OVX plus subcutaneous implantation of 17β-estradiol (E2). Marked hepatic steatosis and increased expression of lipogenic genes (acetyl‑CoA carboxylase, fatty acid synthase and glycerol-3-phosphate acyltransferase) was observed in the estrogen‑depleted mice (TAM and OVX groups), as compared with in the sham operation group. Treatment with E2 significantly improved hepatic steatosis by decreasing the expression of the aforementioned lipogenic genes. Furthermore, hepatic aquaporin 7 (AQP7) expression was decreased in the estrogen‑depleted mice, but was increased in the OVX + E2 treatment group, as compared with in the sham operation group. These results suggested an association between AQP7 and low estrogen‑induced hepatic steatosis. Subsequently, the functions of AQP7 in hepatic steatosis were investigated using an oleic acid‑induced HepG2 cell model of steatosis. Treatment with E2 alleviated lipid accumulation and decreased the expression of lipogenic genes in vitro; however, such effects were attenuated following transfection with AQP7 small interfering RNA. The present study suggested a mechanism by which low levels of estrogen induce fatty liver disease, and may provide useful information regarding the prevention and treatment of fatty liver disease in postmenopausal women. PMID:27176782

  4. TIE2-expressing macrophages limit the therapeutic efficacy of the vascular-disrupting agent combretastatin A4 phosphate in mice

    PubMed Central

    Welford, Abigail F.; Biziato, Daniela; Coffelt, Seth B.; Nucera, Silvia; Fisher, Matthew; Pucci, Ferdinando; Di Serio, Clelia; Naldini, Luigi; De Palma, Michele; Tozer, Gillian M.; Lewis, Claire E.

    2011-01-01

    Vascular-disrupting agents (VDAs) such as combretastatin A4 phosphate (CA4P) selectively disrupt blood vessels in tumors and induce tumor necrosis. However, tumors rapidly repopulate after treatment with such compounds. Here, we show that CA4P-induced vessel narrowing, hypoxia, and hemorrhagic necrosis in murine mammary tumors were accompanied by elevated tumor levels of the chemokine CXCL12 and infiltration by proangiogenic TIE2-expressing macrophages (TEMs). Inhibiting TEM recruitment to CA4P-treated tumors either by interfering pharmacologically with the CXCL12/CXCR4 axis or by genetically depleting TEMs in tumor-bearing mice markedly increased the efficacy of CA4P treatment. These data suggest that TEMs limit VDA-induced tumor injury and represent a potential target for improving the clinical efficacy of VDA-based therapies. PMID:21490397

  5. TIE2-expressing macrophages limit the therapeutic efficacy of the vascular-disrupting agent combretastatin A4 phosphate in mice.

    PubMed

    Welford, Abigail F; Biziato, Daniela; Coffelt, Seth B; Nucera, Silvia; Fisher, Matthew; Pucci, Ferdinando; Di Serio, Clelia; Naldini, Luigi; De Palma, Michele; Tozer, Gillian M; Lewis, Claire E

    2011-05-01

    Vascular-disrupting agents (VDAs) such as combretastatin A4 phosphate (CA4P) selectively disrupt blood vessels in tumors and induce tumor necrosis. However, tumors rapidly repopulate after treatment with such compounds. Here, we show that CA4P-induced vessel narrowing, hypoxia, and hemorrhagic necrosis in murine mammary tumors were accompanied by elevated tumor levels of the chemokine CXCL12 and infiltration by proangiogenic TIE2-expressing macrophages (TEMs). Inhibiting TEM recruitment to CA4P-treated tumors either by interfering pharmacologically with the CXCL12/CXCR4 axis or by genetically depleting TEMs in tumor-bearing mice markedly increased the efficacy of CA4P treatment. These data suggest that TEMs limit VDA-induced tumor injury and represent a potential target for improving the clinical efficacy of VDA-based therapies. PMID:21490397

  6. Glugacon-like peptide-2: broad receptor expression, limited therapeutic effect on intestinal inflammation and novel role in liver regeneration.

    PubMed

    El-Jamal, Noura; Erdual, Edmone; Neunlist, Michel; Koriche, Dine; Dubuquoy, Caroline; Maggiotto, Francois; Chevalier, Julien; Berrebi, Dominique; Dubuquoy, Laurent; Boulanger, Eric; Cortot, Antoine; Desreumaux, Pierre

    2014-08-01

    The glucagon-like peptide 2 (GLP-2) is an intestinotrophic hormone with growth promoting and anti-inflammatory actions. However, the full biological functions of GLP-2 and the localization of its receptor (GLP-2R) remain controversial. Among cell lines tested, the expression of GLP-2R transcript was detected in human colonic myofibroblasts (CCD-18Co) and in primary culture of rat enteric nervous system but not in intestinal epithelial cell lines, lymphocytes, monocytes, or endothelial cells. Surprisingly, GLP-2R was expressed in murine (GLUTag), but not human (NCI-H716) enteroendocrine cells. The screening of GLP-2R mRNA in mice organs revealed an increasing gradient of GLP-2R toward the distal gut. An unexpected expression was detected in the mesenteric fat, mesenteric lymph nodes, bladder, spleen, and liver, particularly in hepatocytes. In two mice models of trinitrobenzene sulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced colitis, the colonic expression of GLP-2R mRNA was decreased by 60% compared with control mice. Also, GLP-2R mRNA was significantly downregulated in intestinal tissues of inflammatory bowel disease patients. Therapeutically, GLP-2 showed a weak restorative effect on intestinal inflammation during TNBS-induced colitis as assessed by macroscopic score and inflammatory markers. Finally, GLP-2 treatment accelerated mouse liver regeneration following partial hepatectomy as assessed by histological and molecular analyses. In conclusion, the limited therapeutic effect of GLP-2 on colonic inflammation dampens its utility in the management of severe inflammatory intestinal disorders. However, the role of GLP-2 in liver regeneration is a novelty that might introduce GLP-2 into the management of liver diseases and emphasizes on the importance of elucidating other extraintestinal functions of GLP-2. PMID:24875097

  7. The Potential for Increasing Net Incomes on Limited-Resource Farms in Eastern Kentucky. Research Report 24.

    ERIC Educational Resources Information Center

    Stewart, Fred J.; And Others

    The study identified possibilities for improving farm incomes on limited-resource farms in eastern Kentucky. Objectives were to describe farm operations of full-time Appalachian farmers who had gross sales of less than $5,000 in 1972, estimate potential increases in net farm incomes from given resources, and identify nonresource constraints on…

  8. 26 CFR 1.960-6 - Overpayments resulting from increase in limitation for taxable year of exclusion.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 10 2011-04-01 2011-04-01 false Overpayments resulting from increase in limitation for taxable year of exclusion. 1.960-6 Section 1.960-6 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Controlled Foreign Corporations § 1.960-6...

  9. 26 CFR 1.960-6 - Overpayments resulting from increase in limitation for taxable year of exclusion.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 10 2010-04-01 2010-04-01 false Overpayments resulting from increase in limitation for taxable year of exclusion. 1.960-6 Section 1.960-6 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES Controlled Foreign Corporations § 1.960-6 Overpayments resulting...

  10. Increased expression of endocytosis-Related proteins in rat hippocampus following 10-day electroconvulsive seizure treatment.

    PubMed

    Enomoto, Shingo; Shimizu, Kunio; Nibuya, Masashi; Toda, Hiroyuki; Yoshino, Aihide; Suzuki, Eiji; Kondo, Takashi; Fukuda, Hiroshi

    2016-06-15

    Although electroconvulsive therapy (ECT) is clinically used for severe depression and drug-resistant Parkinson's disease, its exact biological background and mechanism have not yet been fully elucidated. Two potential explanations have been presented so far to explain the increased neuroplastic and resilient profiles of multiple ECT administrations. One is the alteration of central neurotransmitter receptor densities and the other is the expressional upregulation of brain derived neurotrophic factor in various brain regions with enhanced hippocampal neurogenesis and mossy fiber sprouting. In the present report, western blot analyses revealed significantly upregulated expression of various endocytosis-related proteins following 10-day electroconvulsive seizure (ECS) treatment in rat hippocampal homogenates and hippocampal lipid raft fractions extracted using an ultracentrifugation procedure. Upregulated proteins included endocytosis-related scaffolding proteins (caveolin-1, flotillin-1, and heavy and light chains of clathrin) and small GTPases (Rab5, Rab7, Rab11, and Rab4) specifically expressed on various types of endosomes. Two scaffolding proteins, caveolin-1 and flotillin-1, were also increased in the lipid raft fraction. Together with our previous finding of increased autophagy-related proteins in the hippocampal region, the present results suggest membrane trafficking machinery is enhanced following 10-day ECS treatment. We consider that the membrane trafficking machinery that transports functional proteins in the neuronal cells and from or into the synaptic membranes is one of the new candidates supporting the cellular and behavioral neuroplastic profiles of ECS treatments in animal experiments and ECT administrations in clinical settings. PMID:27177725

  11. Adult male mice conceived by in vitro fertilization exhibit increased glucocorticoid receptor expression in fat tissue.

    PubMed

    Simbulan, R K; Liu, X; Feuer, S K; Maltepe, E; Donjacour, A; Rinaudo, P

    2016-02-01

    Prenatal development is highly plastic and readily influenced by the environment. Adverse conditions have been shown to alter organ development and predispose offspring to chronic diseases, including diabetes and hypertension. Notably, it appears that the changes in glucocorticoid hormones or glucocorticoid receptor (GR) levels in peripheral tissues could play a role in the development of chronic diseases. We have previously demonstrated that in vitro fertilization (IVF) and preimplantation embryo culture is associated with growth alterations and glucose intolerance in mice. However, it is unknown if GR signaling is affected in adult IVF offspring. Here we show that GR expression is increased in inbred (C57Bl6/J) and outbred (CF-1× B6D2F1/J) blastocysts following in vitro culture and elevated levels are also present in the adipose tissue of adult male mice. Importantly, genes involved in lipolysis and triglyceride synthesis and responsive to GR were also increased in adipose tissue, indicating that increased GR activates downstream gene pathways. The promoter region of GR, previously reported to be epigenetically modified by perinatal manipulation, showed no changes in DNA methylation status. Our findings demonstrate that IVF results in a long-term change in GR gene expression in a sex- and tissue-specific manner. These changes in adipose tissues may well contribute to the metabolic phenotype in mice conceived by IVF. PMID:26511158

  12. Increased expression of sialic acid in cervical biopsies with squamous intraepithelial lesions

    PubMed Central

    2010-01-01

    Background Altered sialylation has been observed during oncogenic transformation. Sialylated oligosaccharides of glycoproteins and glycolipids have been implicated in tumor progression and metastases. In the cervical cancer high levels of sialic acid have been reported in the patients serum, and an increased of total sialic acid concentration has been reported for the cervical neoplasia and cervical cancer. This study investigates the changes in expression and distribution of α2,3-linked sialic acid and α2,6- linked sialic acid in low and high squamous intraepithelial lesions and in normal tissue. Methods Lectin histochemistry was used to examine the expression and distribution of sialic acid in different grades of cervical neoplasia. We applied Maackia amurensis lectin, which interacts with α2,3-linked sialic acid and Sambucus nigra lectin specific for α2,6-linked sialic acid. Results The histochemical analysis showed that α2,3-linked sialic acid and α2,6- linked sialic acid increased in intensity and distribution in concordance with the grade of squamous intraepithelial lesion (SIL). These results are in concordance with a previous study that reports increased RNAm levels of three sialyltransferases. Conclusions These results show that the change in sialylation occurs before cancer development and may play an important role in cellular transformation. These findings provide the basis for more detailed studies of the possible role of cell surface glycoconjugates bearing sialic acid in the cellular cervix transformation. PMID:21092209

  13. Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression

    PubMed Central

    Suchanski, Jaroslaw; Olbromski, Mateusz; Gomulkiewicz, Agnieszka; Owczarek, Tomasz; Kruczak, Anna; Ambicka, Aleksandra; Rys, Janusz; Ugorski, Maciej; Podhorska-Okolow, Marzena; Dziegiel, Piotr

    2015-01-01

    It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients’ shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases. PMID:25933064

  14. Increased expression of factor XIII-A in patients with chronic rhinosinusitis with nasal polyps

    PubMed Central

    Takabayashi, Tetsuji; Kato, Atsushi; Peters, Anju T.; Hulse, Kathryn E.; Suh, Lydia A.; Carter, Roderick; Norton, James; Grammer, Leslie C.; Tan, Bruce K.; Chandra, Rakesh K.; Conley, David B.; Kern, Robert C.; Fujieda, Shigeharu; Schleimer, Robert P.

    2013-01-01

    Background Profound edema or formation of a pseudocyst containing plasma proteins is a prominent characteristic of Nasal polyps (NP). However, the mechanisms underlying NP retention of plasma proteins in the submucosa remain unclear. Recently, we reported that impairment of fibrinolysis causes excessive fibrin deposition in NP and this might be involved in retention of plasma proteins. Although the coagulation cascade plays a critical role in fibrin clot formation at extravascular sites, the expression and role of coagulation factors in NP remain unclear. Objective The objective of this study was to investigate the expression of coagulation factors in patients with chronic rhinosinusitis (CRS). Methods Sinonasal tissues were collected from patients with CRS and control subjects. We assayed mRNA for factor XIII-A (FXIII-A) by using real-time PCR and measured FXIII-A protein by means of ELISA, immunohistochemistry and immunofluorescence. Results FXIII-A mRNA levels were significantly increased in NP from patients with CRS with nasal polyps (CRSwNP; P < .001) compared with uncinate tissue from patients with CRS or control subjects. Similarly, FXIII-A protein levels were increased in NP. Immunofluorescence analysis revealed FXIII-A expression in inflammatory cells, and FXIII-A+ cell numbers were significantly increased in NP. Most FXIII-A staining was observed within CD68+/CD163+ M2 macrophages in NP. Levels of FXIII-A correlated with markers of M2 macrophages, suggesting that M2 macrophages are major FXIIIA producing cells in NP. Conclusion Overproduction of FXIII-A by M2 macrophages might contribute to the excessive fibrin deposition in the submucosa of NP, which might contribute to the tissue remodeling and pathogenesis of CRSwNP. PMID:23541322

  15. Increased expression of Trpv1 in peripheral terminals mediates thermal nociception in Fabry disease mouse model

    PubMed Central

    Lakomá, Jarmila; Rimondini, Roberto; Ferrer Montiel, Antonio; Donadio, Vincenzo; Liguori, Rocco

    2016-01-01

    Fabry disease is a X-linked lysosomal storage disorder caused by deficient function of the alpha-galactosidase A (α-GalA) enzyme. α-GalA deficiency leads to multisystemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide (Gb3) in the endothelium and vascular smooth muscles. A hallmark symptom of Fabry disease patients is neuropathic pain that appears in the early stage of the disease as a result of peripheral small fiber damage. The α-GalA gene null mouse model (α-GalA(−/0)) has provided molecular evidence for the molecular alterations in small type-C nociceptors in Fabry disease that may underlie their hyperexcitability, although the specific mechanism remains elusive. Here, we have addressed this question and report that small type-C nociceptors from α-GalA(−/0) mice exhibit a significant increase in the expression and function of the TRPV1 channel, a thermoTRP channel implicated in painful heat sensation. Notably, male α-GalA(−/0) mice displayed a ≈2-fold higher heat sensitivity than wild-type animals, consistent with the augmented expression levels and activity of TRPV1 in α-GalA(−/0) nociceptors. Intriguingly, blockade of neuronal exocytosis with peptide DD04107, a process that inhibits among others the algesic membrane recruitment of TRPV1 channels in peptidergic nociceptors, virtually eliminated the enhanced heat nociception of α-GalA(−/0) mice. Together, these findings suggest that the augmented expression of TRPV1 in α-GalA(−/0) nociceptors may underly at least in part their increased heat sensitivity, and imply that blockade of peripheral neuronal exocytosis may be a valuable pharmacological strategy to reduce pain in Fabry disease patients, increasing their quality of life. PMID:27531673

  16. Repetitive acute intermittent hypoxia increases growth/neurotrophic factor expression in non-respiratory motor neurons.

    PubMed

    Satriotomo, I; Nichols, N L; Dale, E A; Emery, A T; Dahlberg, J M; Mitchell, G S

    2016-05-13

    Repetitive acute intermittent hypoxia (rAIH) increases growth/trophic factor expression in respiratory motor neurons, thereby eliciting spinal respiratory motor plasticity and/or neuroprotection. Here we demonstrate that rAIH effects are not unique to respiratory motor neurons, but are also expressed in non-respiratory, spinal alpha motor neurons and upper motor neurons of the motor cortex. In specific, we used immunohistochemistry and immunofluorescence to assess growth/trophic factor protein expression in spinal sections from rats exposed to AIH three times per week for 10weeks (3×wAIH). 3×wAIH increased brain-derived neurotrophic factor (BDNF), its high-affinity receptor, tropomyosin receptor kinase B (TrkB), and phosphorylated TrkB (pTrkB) immunoreactivity in putative alpha motor neurons of spinal cervical 7 (C7) and lumbar 3 (L3) segments, as well as in upper motor neurons of the primary motor cortex (M1). 3×wAIH also increased immunoreactivity of vascular endothelial growth factor A (VEGFA), the high-affinity VEGFA receptor (VEGFR-2) and an important VEGF gene regulator, hypoxia-inducible factor-1α (HIF-1α). Thus, rAIH effects on growth/trophic factors are characteristic of non-respiratory as well as respiratory motor neurons. rAIH may be a useful tool in the treatment of disorders causing paralysis, such as spinal injury and motor neuron disease, as a pretreatment to enhance motor neuron survival during disease, or as preconditioning for cell-transplant therapies. PMID:26944605

  17. Increased expression of nuclear factor kappa-B p65 subunit in adenomyosis

    PubMed Central

    Park, Hana; Cho, Yoo Mi; Ihm, Hyo Jin; Oh, Young Sang; Hong, Seung Hwa; Chae, Hee Dong; Kim, Chung-Hoon; Kang, Byung Moon

    2016-01-01

    Objective Nuclear factor kappa-B (NF-κB) is a critical proinflammatory regulator that has been suggested to play a pivotal role in the pathogenesis and pathophysiology of endometriosis. In the present study, we aimed to evaluate whether the expression of NF-κB p65 subunit is increased in the eutopic endometrium and/or in the adenomyosis nodule of women with adenomyosis. Methods Thirty-three women with histologically confirmed adenomyosis after laparoscopic or transabdominal hysterectomy were recruited. Women with carcinoma in situ of uterine cervix without evidence of adenomyosis or endometriosis (n=32) served as controls. Formalin-fixed, paraffin-embedded archival tissues were sectioned and immunostained utilizing a monoclonal anti-human NF-κB p65 subunit antibody, and the immunoreactivity of NF-κB p65 subunit was compared between women with and without adenomyosis. Results The immunoreactivities of both the nuclear and the cytoplasmic NF-κB p65 subunit were significantly increased in the stromal cells in the eutopic endometrium as well as in the adenomyosis nodule of women with adenomyosis compared with controls, respectively. The nuclear expression of NF-κB p65 subunit was significantly higher in the glandular cells in the eutopic endometrium as well as the adenomyosis nodule of women with adenomyosis compared with controls, respectively. Conclusion The expression of NF-κB p65 is increased in the eutopic endometrium and adenomyosis nodule of women with adenomyosis, which strongly suggest that NF-κB plays a critical role in the pathogenesis and/or pathophysiology of adenomyosis. PMID:27004203

  18. Increased expression of Trpv1 in peripheral terminals mediates thermal nociception in Fabry disease mouse model.

    PubMed

    Lakomá, Jarmila; Rimondini, Roberto; Ferrer Montiel, Antonio; Donadio, Vincenzo; Liguori, Rocco; Caprini, Marco

    2016-01-01

    Fabry disease is a X-linked lysosomal storage disorder caused by deficient function of the alpha-galactosidase A (α-GalA) enzyme. α-GalA deficiency leads to multisystemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide (Gb3) in the endothelium and vascular smooth muscles. A hallmark symptom of Fabry disease patients is neuropathic pain that appears in the early stage of the disease as a result of peripheral small fiber damage. The α-GalA gene null mouse model (α-GalA(-/0)) has provided molecular evidence for the molecular alterations in small type-C nociceptors in Fabry disease that may underlie their hyperexcitability, although the specific mechanism remains elusive. Here, we have addressed this question and report that small type-C nociceptors from α-GalA(-/0) mice exhibit a significant increase in the expression and function of the TRPV1 channel, a thermoTRP channel implicated in painful heat sensation. Notably, male α-GalA(-/0) mice displayed a ≈2-fold higher heat sensitivity than wild-type animals, consistent with the augmented expression levels and activity of TRPV1 in α-GalA(-/0) nociceptors. Intriguingly, blockade of neuronal exocytosis with peptide DD04107, a process that inhibits among others the algesic membrane recruitment of TRPV1 channels in peptidergic nociceptors, virtually eliminated the enhanced heat nociception of α-GalA(-/0) mice. Together, these findings suggest that the augmented expression of TRPV1 in α-GalA(-/0) nociceptors may underly at least in part their increased heat sensitivity, and imply that blockade of peripheral neuronal exocytosis may be a valuable pharmacological strategy to reduce pain in Fabry disease patients, increasing their quality of life. PMID:27531673

  19. Over-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations.

    PubMed

    Fuentes, S I; Allen, D J; Ortiz-Lopez, A; Hernández, G

    2001-05-01

    Nitrogen, which is a major limiting nutrient for plant growth, is assimilated as ammonium by the concerted action of glutamine synthetase (GS) and glutamate synthase (GOGAT). GS catalyses the critical incorporation of inorganic ammonium into the amino acid glutamine. Two types of GS isozymes, located in the cytosol (GS1) and in the chloroplast (GS2) have been identified in plants. Tobacco (Nicotiana tabacum) transformants, over-expressing GS1 driven by the constitutive CaMV 35S promoter were analysed. GS in leaves of GS-5 and GS-8 plants was up-regulated, at the level of RNA and proteins. These transgenic plants had six times higher leaf GS activity than controls. Under optimum nitrogen fertilization conditions there was no effect of GS over-expression on photosynthesis or growth. However, under nitrogen starvation the GS transgenics had c. 70% higher shoot and c. 100% greater root dry weight as well as 50% more leaf area than low nitrogen controls. This was achieved by the maintenance of photosynthesis at rates indistinguishable from plants under high nitrogen, while photosynthesis in control plants was inhibited by 40-50% by nitrogen deprivation. It was demonstrated that manipulation of GS activity has the potential to maintain crop photosynthetic productivity while reducing nitrogen fertilization and the concomitant pollution. PMID:11432923

  20. Directed evolution increases desaturation of a cyanobacterial fatty acid desaturase in eukaryotic expression systems.

    PubMed

    Bai, Shuangyi; Wallis, James G; Denolf, Peter; Browse, John

    2016-07-01

    Directed evolution of a cyanobacterial Δ9 fatty acid desaturase (DSG) from Synechococcus elongatus, PCC6301 created new, more productive desaturases and revealed the importance of certain amino acid residues to increased desaturation. A codon-optimized DSG open reading frame with an endoplasmic-reticulum retention/retrieval signal appended was used as template for random mutagenesis. Increased desaturation was detected using a novel screen based on complementation of the unsaturated fatty acid auxotrophy of Saccharomyces cerevisiae mutant ole1Δ. Amino acid residues whose importance was discovered by the random processes were further examined by saturation mutation to determine the best amino acid at each identified location in the peptide chain and by combinatorial analysis. One frequently-detected single amino acid change, Q240R, yielded a nearly 25-fold increase in total desaturation in S. cerevisiae. Several other variants of the protein sequence with multiple amino acid changes increased total desaturation more than 60-fold. Many changes leading to increased desaturation were in the vicinity of the canonical histidine-rich regions known to be critical for electron transfer mediated by these di-iron proteins. Expression of these evolved proteins in the seed of Arabidopsis thaliana altered the fatty acid composition, increasing monounsaturated fatty acids and decreasing the level of saturated fatty acid, suggesting a potential application of these desaturases in oilseed crops. Biotechnol. Bioeng. 2016;113: 1522-1530. © 2016 Wiley Periodicals, Inc. PMID:26724425

  1. Dietary resveratrol administration increases MnSOD expression and activity in mouse brain

    SciTech Connect

    Robb, Ellen L.; Winkelmolen, Lieke; Visanji, Naomi; Brotchie, Jonathan; Stuart, Jeffrey A.

    2008-07-18

    trans-Resveratrol (3,4',5-trihydroxystilbene; RES) is of interest for its reported protective effects in a variety of pathologies, including neurodegeneration. Many of these protective properties have been attributed to the ability of RES to reduce oxidative stress. In vitro studies have shown an increase in antioxidant enzyme activities following exposure to RES, including upregulation of mitochondrial superoxide dismutase, an enzyme that is capable of reducing both oxidative stress and cell death. We sought to determine if a similar increase in endogenous antioxidant enzymes is observed with RES treatment in vivo. Three separate modes of RES delivery were utilized; in a standard diet, a high fat diet and through a subcutaneous osmotic minipump. RES given in a high fat diet proved to be effective in elevating antioxidant capacity in brain resulting in an increase in both MnSOD protein level (140%) and activity (75%). The increase in MnSOD was not due to a substantial proliferation of mitochondria, as RES treatment induced a 10% increase in mitochondrial abundance (Citrate Synthase activity). The potential neuroprotective properties of MnSOD have been well established, and we demonstrate that a dietary delivery of RES is able to increase the expression and activity of this enzyme in vivo.

  2. Imiquimod Increases Cutaneous VEGF Expression in Imiquimod-induced Psoriatic Mouse Model.

    PubMed

    Wu, Hui-Hui; Xie, Wen-Lin; Zhao, Yu-Kun; Liu, Juan-Hua; Luo, Di-Qing

    2016-01-01

    Psoriasis is a chronic skin disease of unknown aetiology but increasing evidence suggests that cutaneous angiogenesis plays an important role. Vascular endothelial growth factor (VEGF) is one of the pro-angiogenic cytokines which is related to the pathogenesis of psoriasis. Our study evaluated the influence of imiquimod (IMQ) on VEGF in IMQ-induced mouse model. Balb/c female mice (n=16) 8-12 weeks of age were randomly divided into an experimental group (5% IMQ cream) and the control group (Vaseline cream). Serum levels of circulating VEGF-A were quantified by enzyme-linked immunosorbent assay. VEGF protein expression in tested skin was measured by western blotting and immunohistochemical staining. The tested skin in the experimental group expressed higher levels of VEGF protein than in the control group (p=0.012); immunohistochemical staining revealed that the cells over-expressing VEGF localized predominantly in the epidermis and vascular endothelium. Circulating VEGF-A levels showed no significant difference between the experimental and control groups (p=0.445). The IMQ-induced mouse psoriatic model showed an upregulation of VEGF in the skin lesions mimicking human psoriasis but the circulating VEGF-A levels showed no difference. This model may be useful to investigate the role of angiogenesis in psoriasis. PMID:26733387

  3. Expression of Mouse MGAT in Arabidopsis Results in Increased Lipid Accumulation in Seeds

    PubMed Central

    El Tahchy, Anna; Petrie, James R.; Shrestha, Pushkar; Vanhercke, Thomas; Singh, Surinder P.

    2015-01-01

    Worldwide demand for vegetable oil is projected to double within the next 30 years due to increasing food, fuel, and industrial requirements. There is therefore great interest in metabolic engineering strategies that boost oil accumulation in plant tissues, however, efforts to date have only achieved levels of storage lipid accumulation in plant tissues far below the benchmark to meet demand. Monoacylglycerol acyltransferase (MGAT) is predominantly associated with lipid absorption and resynthesis in the animal intestine where it catalyzes monoacylglycerol (MAG) to form diacylglycerol (DAG), and then triacylglycerol (TAG). In contrast plant lipid biosynthesis routes do not include MGAT. Rather, DAG and TAG are either synthesized from glycerol-3-phosphate by a series of three subsequent acylation reactions, or originated from phospholipids via an acyl editing pathway. Mouse MGATs 1 and 2 have been shown to increase oil content transiently in Nicotiana benthamiana leaf tissue by 2.6 fold. Here we explore the feasibility of this approach to increase TAG in Arabidopsis thaliana seed. The stable MGAT2 expression resulted in a significant increase in seed oil content by 1.32 fold. We also report evidence of the MGAT2 activity based on in vitro assays. Up to 3.9 fold increase of radiolabeled DAG were produced in seed lysate which suggest that the transgenic MGAT activity can result in DAG re-synthesis by salvaging the MAG product of lipid breakdown. The expression of MGAT2 therefore creates an independent and complementary TAG biosynthesis route to the endogenous Kennedy pathway and other glycerolipid synthesis routes. PMID:26834753

  4. Swertianlarin, isolated from Swertia mussotii Franch, increases detoxification enzymes and efflux transporters expression in rats.

    PubMed

    Feng, Xin-Chan; Du, Xiaohuang; Chen, Sheng; Yue, Dongmei; Cheng, Ying; Zhang, Liangjun; Gao, Yu; Li, Shaoxue; Chen, Lei; Peng, Zhihong; Yang, Yong; Luo, Weizao; Wang, Rongquan; Chen, Wensheng; Chai, Jin

    2015-01-01

    Swertianlarin, isolated from Swertia mussotii Franch and Enicostemma axillare, has hepatoprotective effects against cholestasis in rat models of hepatotoxicity. However, the underlying molecular mechanism is not clear. We then treated rats with swertianlarin for 7 d and then measured serum liver injury markers, lipids, and bile salts, as well as the expression of bile acid synthesis and detoxification enzymes (e.g. Cyp7a1 and Cyp3a), membrane influx and efflux transporters (e.g. Ntcp and Mrp3), nuclear receptors (e.g. Pxr and Fxr/Shp) and transcriptional factors (e.g. Nrf2 and Hnf3β) in the liver. We found a significant induction of the expression of the basolateral efflux transporters Mrp3 and Mrp4 and canalicular transporter Mdr1 in rats treated with swertianlarin, compared with the controls (1.9-fold and 2.2-fold, P < 0.005, and 3.4-fold, P < 0.05, respectively). The expression of detoxification enzymes Cyp3a, Ugt2b, Sult2a1 and Gsta1 in rats treated with swertianlarin was significantly higher than that in controls (3.7-fold, 2.8-fold, 2.1-fold, and 1.7-fold, respectively, all P < 0.05). Expression of the synthetic enzyme, Cyp8b1, was higher in rats treated with swertianlarin than that in controls (1.8-fold at mRNA level and 3.4-flod at protein level, P < 0.05). Elevated serum levels of the conjugated bile acids, taurocholic acid and taurodeoxycholic acid, and a reduction in levels of serum ALP, unconjugated bile acid αMCA, and TG were observed (all P < 0.05). In conclusion, swertianlarin significantly up-regulates hepatic bile acid detoxification enzymes and efflux transporters in rats, which can increase the water solubility of hydrophobic bile acids and elimination of conjugated bile acids. PMID:25755705

  5. Swertianlarin, isolated from Swertia mussotii Franch, increases detoxification enzymes and efflux transporters expression in rats

    PubMed Central

    Feng, Xin-Chan; Du, Xiaohuang; Chen, Sheng; Yue, Dongmei; Cheng, Ying; Zhang, Liangjun; Gao, Yu; Li, Shaoxue; Chen, Lei; Peng, Zhihong; Yang, Yong; Luo, Weizao; Wang, Rongquan; Chen, Wensheng; Chai, Jin

    2015-01-01

    Swertianlarin, isolated from Swertia mussotii Franch and Enicostemma axillare, has hepatoprotective effects against cholestasis in rat models of hepatotoxicity. However, the underlying molecular mechanism is not clear. We then treated rats with swertianlarin for 7 d and then measured serum liver injury markers, lipids, and bile salts, as well as the expression of bile acid synthesis and detoxification enzymes (e.g. Cyp7a1 and Cyp3a), membrane influx and efflux transporters (e.g. Ntcp and Mrp3), nuclear receptors (e.g. Pxr and Fxr/Shp) and transcriptional factors (e.g. Nrf2 and Hnf3β) in the liver. We found a significant induction of the expression of the basolateral efflux transporters Mrp3 and Mrp4 and canalicular transporter Mdr1 in rats treated with swertianlarin, compared with the controls (1.9-fold and 2.2-fold, P < 0.005, and 3.4-fold, P < 0.05, respectively). The expression of detoxification enzymes Cyp3a, Ugt2b, Sult2a1 and Gsta1 in rats treated with swertianlarin was significantly higher than that in controls (3.7-fold, 2.8-fold, 2.1-fold, and 1.7-fold, respectively, all P < 0.05). Expression of the synthetic enzyme, Cyp8b1, was higher in rats treated with swertianlarin than that in controls (1.8-fold at mRNA level and 3.4-flod at protein level, P < 0.05). Elevated serum levels of the conjugated bile acids, taurocholic acid and taurodeoxycholic acid, and a reduction in levels of serum ALP, unconjugated bile acid αMCA, and TG were observed (all P < 0.05). In conclusion, swertianlarin significantly up-regulates hepatic bile acid detoxification enzymes and efflux transporters in rats, which can increase the water solubility of hydrophobic bile acids and elimination of conjugated bile acids. PMID:25755705

  6. Increased Expression of Pattern Recognition Receptors and Nitric Oxide Synthase in Patients with Endometriosis

    PubMed Central

    Yeo, Seung Geun; Won, Yong Sung; Lee, Ho Yun; Kim, Young Il; Lee, Jin-Woo; Park, Dong Choon

    2013-01-01

    Objective: Endometriosis is characterized by repeated inflammatory changes and serious adhesions, inducing innate and adaptive immune responses within the abdominal cavity. To assess these immune responses, we evaluated the levels of expression of Toll-like receptors (TLR)-1, -2, -4, -5, and -9; nucleotide-binding oligomerization domains (NOD)-1 and -2; interleukins-1β, -6, -8, -10, and -12; interferon-γ; tumor necrosis factor-α; inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS); and immunoglobulins (Igs) in patients with endometriosis. Methods: The levels of TLRs, NODs, cytokines, and NOS mRNAs in peritoneal effusions were assessed by real time reverse transcription-polymerase chain reaction; and IgG, IgA and IgM concentrations were measured by enzyme-linked immunosorbent assays (ELISA) in 40 patients with and 40 without endometriosis. Findings from the two groups were compared. Results: We observed expression of all pattern recognition receptors (PRRs), cytokines, and NOS mRNAs and Igs in the effusion fluid of patients with and without endometriosis. The levels of TLR-2 and -9; NOD-1 and -2; iNOS and eNOS mRNAs and CA 125 were significantly higher in the endometriosis than in the non-endometriosis group (p<0.05 each). Moreover, PRR, cytokine, and NOS expression showed significant correlations (p<0.05). Conclusions: PRRs, cytokines, and NOS, which act cooperatively in the innate immune response, are closely associated with endometriosis. Increased expression of TLR-2, TLR -9, NOD-1, NOD-2, and NOS mRNA in peritoneal fluid may be associated with endometriosis. PMID:23935397

  7. The anorexigenic effects of metformin involve increases in hypothalamic leptin receptor expression.

    PubMed

    Aubert, Grégory; Mansuy, Virginie; Voirol, Marie-Jeanne; Pellerin, Luc; Pralong, François P

    2011-03-01

    Metformin demonstrates anorectic effects in vivo and inhibits neuropeptide Y expression in cultured hypothalamic neurons. Here we investigated the mechanisms implicated in the modulation of feeding by metformin in animals rendered obese by long-term high-fat diet (diet-induced obesity [DIO]) and in animals resistant to obesity (diet resistant [DR]). Male Long-Evans rats were kept on normal chow feeding (controls) or on high-fat diet (DIO, DR) for 6 months. Afterward, rats were treated 14 days with metformin (75 mg/kg) or isotonic sodium chloride solution and killed. Energy efficiency, metabolic parameters, and gene expression were analyzed at the end of the high-fat diet period and after 14 days of metformin treatment. At the end of the high-fat diet period, despite higher leptin levels, DIO rats had higher levels of hypothalamic neuropeptide Y expression than DR or control rats, suggesting a central leptin resistance. In DIO but also in DR rats, metformin treatment induced significant reductions of food intake accompanied by decreases in body weight. Interestingly, the weight loss achieved by metformin was correlated with pretreatment plasma leptin levels. This effect was paralleled by a stimulation of the expression of the leptin receptor gene (ObRb) in the arcuate nucleus. These data identify the hypothalamic ObRb as a gene modulated after metformin treatment and suggest that the anorectic effects of the drug are potentially mediated via an increase in the central sensitivity to leptin. Thus, they provide a rationale for novel therapeutic approaches associating leptin and metformin in the treatment of obesity. PMID:20303124

  8. Sulindac sulfide selectively increases sensitivity of ABCC1 expressing tumor cells to doxorubicin and glutathione depletion

    PubMed Central

    Whitt, Jason D.; Keeton, Adam B.; Gary, Bernard D.; Sklar, Larry A.; Sodani, Kamlesh; Chen, Zhe-Sheng; Piazza, Gary A.

    2016-01-01

    Abstract ATP-binding cassette (ABC) transpo rters ABCC1 (MRP1), ABCB1 (P-gp), and ABCG2 (BCRP) contribute to chemotherapy failure. The primary goals of this study were to characterize the efficacy and mechanism of the non­steroidal anti-inflammatory drug (NSAID), sulindac sulfide, to reverse ABCC1 mediated resistance to chemother­apeutic drugs and to determine if sulindac sulfide can influence sensitivity to chemotherapeutic drugs independently of drug efflux. Cytotoxicity assays were performed to measure resistance of ABC-expressing cell lines to doxoru­bicin and other chemotherapeutic drugs. NSAIDs were tested for the ability to restore sensitivity to resistance selected tumor cell lines, as well as a large panel of standard tumor cell lines. Other experiments characterized the mechanism by which sulindac sulfide inhibits ABCC1 substrate and co-substrate (GSH) transport in isolated membrane vesicles and intact cells. Selective reversal of multi-drug resistance (MDR), decreased efflux of doxor­ubicin, and fluorescent substrates were demonstrated by sulindac sulfide and a related NSAID, indomethacin, in resistance selected and engineered cell lines expressing ABCC1, but not ABCB1 or ABCG2. Sulindac sulfide also inhibited transport of leukotriene C4 into membrane vesicles. Sulindac sulfide enhanced the sensitivity to doxoru­bicin in 24 of 47 tumor cell lines, including all melanoma lines tested (7-7). Sulindac sulfide also decreased intra­cellular GSH in ABCC1 expressing cells, while the glutathione synthesis inhibitor, BSO, selectively increased sensitivity to sulindac sulfide induced cytotoxicity. Sulindac sulfide potently and selectively reverses ABCC1-mediated MDR at clinically achievable concentrations. ABCC1 expressing tumors may be highly sensitive to the direct cytotoxicity of sulindac sulfide, and in combination with chemotherapeutic drugs that induce oxidative stress.

  9. Increased expression of S100A4, a metastasis-associated gene, in human colorectal adenocarcinomas.

    PubMed

    Takenaga, K; Nakanishi, H; Wada, K; Suzuki, M; Matsuzaki, O; Matsuura, A; Endo, H

    1997-12-01

    The S100A4 gene (also known as pEL98/mts1/p9Ka/18A2/42A/calvasculin /FSP1/CAPL) encoding an S100-related calcium-binding protein is implied to be involved in the invasion and metastasis of murine tumor cells. In the present study, the expression of S100A4 in human colorectal adenocarcinoma cell lines (SW837, LoVo, DLD-1, HT-29, SW480, SW620, WiDr, and Colo201) and surgically resected neoplastic tissues was examined to investigate whether S100A4 plays a role in the invasion and metastasis of human tumor cells. Northern blot analysis using total RNA isolated from the adenocarcinoma cell lines revealed that five of the eight cell lines expressed substantial amounts of S100A4 mRNA. Normal colon fibroblasts (CCD-18Co) expressed little of the RNA. Using surgically resected specimens, it seemed that the amount of S100A4 mRNA in adenomas was nearly equal to that in normal colonic mucosa, whereas adenocarcinomas expressed a significantly higher amount of the RNA than did the adjacent normal colonic mucosa. Immunohistochemical analysis using formalin-fixed paraffin-embedded surgical specimens and monoclonal anti-S100A4 antibody demonstrated that none of 12 adenoma specimens were immunopositive, whereas 8 of 18 (44%) focal carcinomas in carcinoma in adenoma specimens and 50 of 53 (94%) adenocarcinoma specimens were immunopositive. Interestingly, the incidence of immunopositive cells increased according to the depth of invasion, and nearly all of the carcinoma cells in 14 metastases in the liver were positive. These results suggest that S100A4 may be involved in the progression and the metastatic process of human colorectal neoplastic cells. PMID:9815629

  10. Atorvastatin increases miR-124a expression: a mechanism of Gamt modulation in liver cells.

    PubMed

    Phulukdaree, Alisa; Moodley, Devapregasan; Khan, Sajidah; Chuturgoon, Anil A

    2015-11-01

    Atorvastatin is used to control cholesterol and lipid levels in hyperlipidaemic and hypercholesterolaemic patients. Myopathy and hepatotoxicity, however, have been reported as side effects in a small percentage of statin users. This study aimed to investigate the cytotoxicity and the effect of atorvastatin on microRNA expression in HepG2 cells. The methylthiazol tetrazolium assay was used to assess hepatocyte viability and at 20 μM atorvastatin (24 h) treatment were 82 ± 1.5% viable (P = 0.0002). Levels of intracellular ATP in cells treated with 20 μM atorvastatin were reduced by 1.25-fold, P = 0.002. Cytotoxicity, measured by the release of intracellular lactate dehydrogenase, was increased from 0.95 ± 0.29 units in control cells to 1.12 ± 0.02 units (P = 0.002) in atorvastatin treated cells. A panel of 84-miRNA species was used to evaluate the effect of atorvastatin on miRNA expression. MiR-124a was significantly up-regulated by atorvastatin (12.94-fold). A significant decrease in GAMT expression (3.54-fold) was observed in atorvastatin treated cells following quantitative PCR analysis. In addition, western blotting data showed GAMT protein levels were significantly lower than the controls (3.02-fold) and analysis of creatine levels in treated cells showed a significant decrease in the atorvastatin treated culture supernatant compared to control culture supernatant (32.33 ± 3.51 μM/l vs. 59.67 ± 1.52μM/l, P = 0.0056). This is the first study to show that atorvastatin up-regulates miR-124a levels and consequently modulates GAMT expression in hepatocytes. PMID:25926069

  11. Heme oxygenase-1 gene expression modulates angiotensin II-induced increase in blood pressure.

    PubMed

    Yang, Liming; Quan, Shuo; Nasjletti, Alberto; Laniado-Schwartzman, Michal; Abraham, Nader G

    2004-06-01

    The heme-heme oxygenase (HO) system has been implicated in the regulation of vascular reactivity and blood pressure. This study examines the notion that overexpression of HO decreases pressor responsiveness to angiotensin II (Ang II). Five-day-old Sprague-Dawley rats received an intraleft ventricular injection of approximately 5x10(9) cfu/mL of retroviruses containing human HO-1 sense (LSN-HHO-1), rat HO-1 antisense (LSN-RHO-1-AS), or control retrovirus (LXSN). Three months later, rats were instrumented with femoral arterial and venous catheters for mean arterial pressure (MAP) determination and Ang II administration, respectively. Rats injected with LSN-HHO-1, but not with LXSN, expressed human HO-1 mRNA and protein in several tissues. BP increased with administration of Ang II in rats expressing and not expressing human HO-1. However, the Ang II-induced pressor response (mm Hg) in LSN-HHO-1 rats (16+/-3, 27+/-3, and 38+/-3 at 0.5, 2, and 10 ng) was surpassed (P<0.05) in LXSN rats (23+/-1, 37+/-2, and 52+/-2 at 0.5, 2, and 10 ng). Importantly, treating LSN-HHO-1 rats with the HO inhibitor tin mesoporphyrin (SnMP) enhanced (P<0.05) the Ang II-induced pressor response to a level not different from that observed in LXSN rats. Rats injected with LSN-RHO-1-AS showed a decrease in renal HO-1 protein expression and HO activity relative to control LXSN rats. Administration of Ang II (0.1 to 2 ng) caused small (4 to 5 mm Hg) but significant increases in MAP in rats injected with LSN-RHO-1-AS (P<0.05) compared with rats injected with LXSN. These data demonstrate that overexpression of HO-1 brings about a reduction in pressor responsiveness to Ang II, which is most likely due to increased generation of an HO-1 product, presumably CO, with the ability to inhibit vascular reactivity to constrictor stimuli. PMID:15166181

  12. Increased apomixis expression concurrent with genetic and epigenetic variation in a newly synthesized Eragrostis curvula polyploid

    NASA Astrophysics Data System (ADS)

    Zappacosta, Diego C.; Ochogavía, Ana C.; Rodrigo, Juan M.; Romero, José R.; Meier, Mauro S.; Garbus, Ingrid; Pessino, Silvina C.; Echenique, Viviana C.

    2014-04-01

    Eragrostis curvula includes biotypes reproducing through obligate and facultative apomixis or, rarely, full sexuality. We previously generated a ``tetraploid-dihaploid-tetraploid'' series of plants consisting of a tetraploid apomictic plant (T), a sexual dihaploid plant (D) and a tetraploid artificial colchiploid (C). Initially, plant C was nearly 100% sexual. However, its capacity to form non-reduced embryo sacs dramatically increased over a four year period (2003-2007) to reach levels of 85-90%. Here, we confirmed high rates of apomixis in plant C, and used AFLPs and MSAPs to characterize the genetic and epigenetic variation observed in this plant in 2007 as compared to 2003. Of the polymorphic sequences, some had no coding potential whereas others were homologous to retrotransposons and/or protein-coding-like sequences. Our results suggest that in this particular plant system increased apomixis expression is concurrent with genetic and epigenetic modifications, possibly involving transposable elements.

  13. Increased apomixis expression concurrent with genetic and epigenetic variation in a newly synthesized Eragrostis curvula polyploid.

    PubMed

    Zappacosta, Diego C; Ochogavía, Ana C; Rodrigo, Juan M; Romero, José R; Meier, Mauro S; Garbus, Ingrid; Pessino, Silvina C; Echenique, Viviana C

    2014-01-01

    Eragrostis curvula includes biotypes reproducing through obligate and facultative apomixis or, rarely, full sexuality. We previously generated a "tetraploid-dihaploid-tetraploid" series of plants consisting of a tetraploid apomictic plant (T), a sexual dihaploid plant (D) and a tetraploid artificial colchiploid (C). Initially, plant C was nearly 100% sexual. However, its capacity to form non-reduced embryo sacs dramatically increased over a four year period (2003-2007) to reach levels of 85-90%. Here, we confirmed high rates of apomixis in plant C, and used AFLPs and MSAPs to characterize the genetic and epigenetic variation observed in this plant in 2007 as compared to 2003. Of the polymorphic sequences, some had no coding potential whereas others were homologous to retrotransposons and/or protein-coding-like sequences. Our results suggest that in this particular plant system increased apomixis expression is concurrent with genetic and epigenetic modifications, possibly involving transposable elements. PMID:24710346

  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. A pragmatic approach to increase expressive language skills in young autistic children.

    PubMed

    Beisler, J M; Tsai, L Y

    1983-09-01

    This paper is a description of a pilot communication program for autistic children designed to increase communication skills in the context of establishing reciprocal communication exchanges. The methods involved intensive modeling of verbal responses within joint activity routines and using a reinforcement system based on fulfilling the intent of the child's communication. Five male subjects ranging in age from 36 to 68 months participated in the communication program for 6 weeks during an inpatient stay at the Iowa Autism Program, Child Psychiatry Service. Communication exchanges involving requests for actions and objects, descriptions of actions and objects, comments on existence and nonexistence, and requests for recurrence were targeted. Individual gains for each subject are outlined. Posttreatment results indicated a significant increase in the mean length of response, in the mean receptive language level, and in the mean number of semantic-grammatical rules expressed. Suggestions regarding future research are discussed. PMID:6643374

  16. Lupus Risk Variant Increases pSTAT1 Binding and Decreases ETS1 Expression

    PubMed Central

    Lu, Xiaoming; Zoller, Erin E.; Weirauch, Matthew T.; Wu, Zhiguo; Namjou, Bahram; Williams, Adrienne H.; Ziegler, Julie T.; Comeau, Mary E.; Marion, Miranda C.; Glenn, Stuart B.; Adler, Adam; Shen, Nan; Nath, Swapan K.; Stevens, Anne M.; Freedman, Barry I.; Tsao, Betty P.; Jacob, Chaim O.; Kamen, Diane L.; Brown, Elizabeth E.; Gilkeson, Gary S.; Alarcón, Graciela S.; Reveille, John D.; Anaya, Juan-Manuel; James, Judith A.; Sivils, Kathy L.; Criswell, Lindsey A.; Vilá, Luis M.; Alarcón-Riquelme, Marta E.; Petri, Michelle; Scofield, R. Hal; Kimberly, Robert P.; Ramsey-Goldman, Rosalind; Joo, Young Bin; Choi, Jeongim; Bae, Sang-Cheol; Boackle, Susan A.; Graham, Deborah Cunninghame; Vyse, Timothy J.; Guthridge, Joel M.; Gaffney, Patrick M.; Langefeld, Carl D.; Kelly, Jennifer A.; Greis, Kenneth D.; Kaufman, Kenneth M.; Harley, John B.; Kottyan, Leah C.

    2015-01-01

    Genetic variants at chromosomal region 11q23.3, near the gene ETS1, have been associated with systemic lupus erythematosus (SLE), or lupus, in independent cohorts of Asian ancestry. Several recent studies have implicated ETS1 as a critical driver of immune cell function and differentiation, and mice deficient in ETS1 develop an SLE-like autoimmunity. We performed a fine-mapping study of 14,551 subjects from multi-ancestral cohorts by starting with genotyped variants and imputing to all common variants spanning ETS1. By constructing genetic models via frequentist and Bayesian association methods, we identified 16 variants that are statistically likely to be causal. We functionally assessed each of these variants on the basis of their likelihood of affecting transcription factor binding, miRNA binding, or chromatin state. Of the four variants that we experimentally examined, only rs6590330 differentially binds lysate from B cells. Using mass spectrometry, we found more binding of the transcription factor signal transducer and activator of transcription 1 (STAT1) to DNA near the risk allele of rs6590330 than near the non-risk allele. Immunoblot analysis and chromatin immunoprecipitation of pSTAT1 in B cells heterozygous for rs6590330 confirmed that the risk allele increased binding to the active form of STAT1. Analysis with expression quantitative trait loci indicated that the risk allele of rs6590330 is associated with decreased ETS1 expression in Han Chinese, but not other ancestral cohorts. We propose a model in which the risk allele of rs6590330 is associated with decreased ETS1 expression and increases SLE risk by enhancing the binding of pSTAT1. PMID:25865496

  17. Single Nucleotide Polymorphisms that Increase Expression of the GTPase RAC1 are Associated with Ulcerative Colitis

    PubMed Central

    Muise, Aleixo M; Walters, Thomas; Xu, Wei; Shen-Tu, Grace; Guo, Cong-Hui; Fattouh, Ramzi; Lam, Grace Y; Wolters, Victorien M; Bennitz, Joshua; Van Limbergen, Johan; Renbaum, Paul; Kasirer, Yair; Ngan, Bo-Yee; Turner, Dan; Denson, Lee A; Sherman, Philip M; Duerr, Richard H; Cho, Judy; Lees, Charlie W; Satsangi, Jack; Wilson, David C; Paterson, Andrew D; Griffiths, Anne M; Glogauer, Michael; Silverberg, Mark S; Brumell, John H

    2011-01-01

    Background & Aims RAC1 is a GTPase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases (IBD) are associated with dysregulation of immune defenses. We studied the role of RAC1 in IBD using human genetic and functional studies and animal models of colitis. Methods We used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms (SNPs) in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 mRNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulphate sodium (DSS). Results We observed a genetic association between RAC1 with ulcerative colitis (UC) in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the SNPs rs10951982 (Pcombined UC = 3.3 × 10–8, odds ratio [OR]=1.43 [1.26–1.63]) and rs4720672 (Pcombined UC=4.7 × 10–6, OR=1.36 [1.19–1.58]). Patients with IBD who had the rs10951982 risk allele had increased expression of RAC1, compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected them against DSS-induced colitis. Conclusion Studies of human tissue samples and knockout mice demonstrated a role for the GTPase RAC1 in the development of UC; increased expression of RAC1 was associated with susceptibility to colitis. PMID:21684284

  18. Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.

    PubMed

    Aberg, Karin M; Radek, Katherine A; Choi, Eung-Ho; Kim, Dong-Kun; Demerjian, Marianne; Hupe, Melanie; Kerbleski, Joseph; Gallo, Richard L; Ganz, Tomas; Mauro, Theodora; Feingold, Kenneth R; Elias, Peter M

    2007-11-01

    The skin is the first line of defense against microbial infection, and psychological stress (PS) has been shown to have adverse effects on cutaneous barrier function. Here we show that PS increased the severity of group A Streptococcus pyogenes (GAS) cutaneous skin infection in mice; this was accompanied by increased production of endogenous glucocorticoids (GCs), which inhibited epidermal lipid synthesis and decreased lamellar body (LB) secretion. LBs encapsulate antimicrobial peptides (AMPs), and PS or systemic or topical GC administration downregulated epidermal expression of murine AMPs cathelin-related AMP and beta-defensin 3. Pharmacological blockade of the stress hormone corticotrophin-releasing factor or of peripheral GC action, as well as topical administration of physiologic lipids, normalized epidermal AMP levels and delivery to LBs and decreased the severity of GAS infection during PS. Our results show that PS decreases the levels of 2 key AMPs in the epidermis and their delivery into LBs and that this is attributable to increased endogenous GC production. These data suggest that GC blockade and/or topical lipid administration could normalize cutaneous antimicrobial defense during PS or GC increase. We believe this to be the first mechanistic link between PS and increased susceptibility to infection by microbial pathogens. PMID:17975669

  19. Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice

    PubMed Central

    Aberg, Karin M.; Radek, Katherine A.; Choi, Eung-Ho; Kim, Dong-Kun; Demerjian, Marianne; Hupe, Melanie; Kerbleski, Joseph; Gallo, Richard L.; Ganz, Tomas; Mauro, Theodora; Feingold, Kenneth R.; Elias, Peter M.

    2007-01-01

    The skin is the first line of defense against microbial infection, and psychological stress (PS) has been shown to have adverse effects on cutaneous barrier function. Here we show that PS increased the severity of group A Streptococcus pyogenes (GAS) cutaneous skin infection in mice; this was accompanied by increased production of endogenous glucocorticoids (GCs), which inhibited epidermal lipid synthesis and decreased lamellar body (LB) secretion. LBs encapsulate antimicrobial peptides (AMPs), and PS or systemic or topical GC administration downregulated epidermal expression of murine AMPs cathelin-related AMP and β-defensin 3. Pharmacological blockade of the stress hormone corticotrophin-releasing factor or of peripheral GC action, as well as topical administration of physiologic lipids, normalized epidermal AMP levels and delivery to LBs and decreased the severity of GAS infection during PS. Our results show that PS decreases the levels of 2 key AMPs in the epidermis and their delivery into LBs and that this is attributable to increased endogenous GC production. These data suggest that GC blockade and/or topical lipid administration could normalize cutaneous antimicrobial defense during PS or GC increase. We believe this to be the first mechanistic link between PS and increased susceptibility to infection by microbial pathogens. PMID:17975669

  20. Increased antioxidant capacity in tomato by ectopic expression of the strawberry D-galacturonate reductase gene.

    PubMed

    Amaya, Iraida; Osorio, Sonia; Martinez-Ferri, Elsa; Lima-Silva, Viviana; Doblas, Veronica G; Fernández-Muñoz, Rafael; Fernie, Alisdair R; Botella, Miguel A; Valpuesta, Victoriano

    2015-03-01

    Increasing L-ascorbic acid (AsA, vitamin C) content in fruits is a common goal in current breeding programs due to its beneficial effect on human health. Attempts to increase AsA content by genetic engineering have resulted in variable success likely due to AsA's complex regulation. Here, we report the effect of ectopically expressing in tomato the D-galacturonate reductase (FaGalUR) gene from strawberry, involved in AsA biosynthesis, either under the control of the constitutive 35S or the tomato fruit-specific polygalucturonase (PG) promoters. Although transgenic lines showed a moderate increase on AsA content, complex changes in metabolites were found in transgenic fruits. Metabolomic analyses of ripe fruits identified a decrease in citrate, glutamate, asparagine, glucose, and fructose, accompanied by an increase of sucrose, galactinol, and chlorogenic acid. Significant metabolic changes also occurred in leaves of 35S-FaGalUR lines, which showed higher non-photochemical fluorescence quenching (NPQ), indicative of a higher constitutive photo-protective capacity. Overall, overexpression of FaGalUR increased total antioxidant capacity in fruits and the results suggest a tight control of AsA content, probably linked to a complex regulation of cellular redox state and metabolic adjustment. PMID:25143316

  1. A Single Oral Administration of Theaflavins Increases Energy Expenditure and the Expression of Metabolic Genes

    PubMed Central

    Kudo, Naoto; Arai, Yasunori; Suhara, Yoshitomo; Ishii, Takeshi; Nakayama, Tsutomu; Osakabe, Naomi

    2015-01-01

    Theaflavins are polyphenols found in black tea, whose physiological activities are not well understood. This study on mice evaluated the influence of a single oral administration of theaflavins on energy metabolism by monitoring the initial metabolic changess in skeletal muscle and brown adipose tissue (BAT). Oxygen consumption (VO2) and energy expenditure (EE) were increased significantly in mice treated with theaflavin rich fraction (TF) compared with the group administered vehicle alone. There was no difference in locomotor activity. Fasting mice were euthanized under anesthesia before and 2 and 5, 20-hr after treatment with TF or vehicle. The mRNA levels of uncoupling protein-1 (UCP-1) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in BAT were increased significantly 2-hr after administration ofTF. The levels of UCP-3 and PGC-1α in the gastrocnemius muscle were increased significantly 2 and 5-hr after administration of TF. The concentration of phosphorylated AMP-activated protein kinase (AMPK) 1α was also increased significantly in the gastrocnemius 2 and 5-hr after treatment with TF. These results indicate that TF significantly enhances systemic energy expenditure, as evidenced by an increase in expression of metabolic genes. PMID:26375960

  2. A Single Oral Administration of Theaflavins Increases Energy Expenditure and the Expression of Metabolic Genes.

    PubMed

    Kudo, Naoto; Arai, Yasunori; Suhara, Yoshitomo; Ishii, Takeshi; Nakayama, Tsutomu; Osakabe, Naomi

    2015-01-01

    Theaflavins are polyphenols found in black tea, whose physiological activities are not well understood. This study on mice evaluated the influence of a single oral administration of theaflavins on energy metabolism by monitoring the initial metabolic changess in skeletal muscle and brown adipose tissue (BAT). Oxygen consumption (VO2) and energy expenditure (EE) were increased significantly in mice treated with theaflavin rich fraction (TF) compared with the group administered vehicle alone. There was no difference in locomotor activity. Fasting mice were euthanized under anesthesia before and 2 and 5, 20-hr after treatment with TF or vehicle. The mRNA levels of uncoupling protein-1 (UCP-1) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in BAT were increased significantly 2-hr after administration ofTF. The levels of UCP-3 and PGC-1α in the gastrocnemius muscle were increased significantly 2 and 5-hr after administration of TF. The concentration of phosphorylated AMP-activated protein kinase (AMPK) 1α was also increased significantly in the gastrocnemius 2 and 5-hr after treatment with TF. These results indicate that TF significantly enhances systemic energy expenditure, as evidenced by an increase in expression of metabolic genes. PMID:26375960

  3. Both harmful and (some) helpful behaviours from others are associated with increased expression of schizotypal traits.

    PubMed

    Badcock, Johanna C; Panton, Kirsten; Cohen, Alex; Badcock, David R

    2016-05-30

    Negative treatment from others is related to elevated levels of trait schizotypy, signifying increased risk for psychosis, but associations with helpful behaviour have been much less studied. Using the Stereotype Content Model we tested the hypothesis that passive and active forms of help would be associated with increased and decreased expression of schizotypy, respectively. Schizotypal traits were assessed in students (N=631) using positive (Perceptual Aberration) and negative (Social Anhedonia) subscales of the Wisconsin Schizotypy Scales-Brief. Experiences of active (intentional) and passive (less deliberative) harm and help were assessed with the Behaviour from Intergroup Affect and Stereotypes Treatment Scale. As predicted, the results showed that experiences of passive help from others were associated with a 2-3 fold increase in scores on schizotypy scales, whilst reports of active help tended to be associated with a decrease in scores on these scales. Results also showed that increased reports of active and passive harm were associated with elevated scores on negative and positive schizotypy subscales, consistent with prior research. These findings, bridging research on social stereotyping and schizotypal personality, challenge the assumption that helpful behaviour from others is always beneficial for individuals with schizotypal traits who are at increased risk for psychosis. PMID:27058156

  4. miR-17 inhibitor suppressed osteosarcoma tumor growth and metastasis via increasing PTEN expression

    SciTech Connect

    Gao, Yong; Luo, Ling-hui; Li, Shuai; Yang, Cao

    2014-02-07

    Highlights: • miR-17 was increased in OS tissues and cell lines. • Inhibition of miR-17 suppressed OS cell proliferation. • Inhibition of miR-17 suppressed OS cell migration and invasion. • PTEN was a target of miR-17. • miR-17 was negatively correlated with PTEN in OS tissues. - Abstract: MicroRNAs (miRNAs) play essential roles in cancer development and progression. Here, we investigated the role of miR-17 in the progression and metastasis of osteosarcoma (OS). miR-17 was frequently increased in OS tissues and cell lines. Inhibition of miR-17 in OS cell lines substantially suppressed cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a target of miR-17, and ectopic expression of miR-17 inhibited PTEN by direct binding to its 3′-untranslated region (3′-UTR). Expression of miR-17 was negatively correlated with PTEN in OS tissues. Together, these findings indicate that miR-17 acts as an oncogenic miRNA and may contribute to the progression and metastasis of OS, suggesting miR-17 as a potential novel diagnostic and therapeutic target of OS.

  5. Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus.

    PubMed

    Chakraborty, S; Chakraborty, N; Datta, A

    2000-03-28

    Improvement of nutritive value of crop plants, in particular the amino acid composition, has been a major long-term goal of plant breeding programs. Toward this end, we reported earlier the cloning of the seed albumin gene AmA1 from Amaranthus hypochondriacus. The AmA1 protein is nonallergenic in nature and is rich in all essential amino acids, and the composition corresponds well with the World Health Organization standards for optimal human nutrition. In an attempt to improve the nutritional value of potato, the AmA1 coding sequence was successfully introduced and expressed in tuber-specific and constitutive manner. There was a striking increase in the growth and production of tubers in transgenic populations and also of the total protein content with an increase in most essential amino acids. The expressed protein was localized in the cytoplasm as well as in the vacuole of transgenic tubers. Thus we have been able to use a seed albumin gene with a well-balanced amino acid composition as a donor protein to develop a transgenic crop plant. The results document, in addition to successful nutritional improvement of potato tubers, the feasibility of genetically modifying other crop plants with novel seed protein composition. PMID:10716698

  6. Cerium oxide nanoparticles inhibit the migration and proliferation of gastric cancer by increasing DHX15 expression

    PubMed Central

    Xiao, Yu-Feng; Li, Jian-Mei; Wang, Su-Min; Yong, Xin; Tang, Bo; Jie, Meng-Meng; Dong, Hui; Yang, Xiao-Chao; Yang, Shi-Ming

    2016-01-01

    Gastric cancer is one of the leading causes of tumor-related deaths in the world. Current treatment options do not satisfy doctors and patients, and new therapies are therefore needed. Cerium oxide nanoparticles (CNPs) have been studied as a potential therapeutic approach for treating many diseases. However, their effects on human gastric cancer are currently unknown. Therefore, in this study, we aimed to characterize the effects of CNPs on human gastric cancer cell lines (MKN28 and BGC823). Gastric cancer cells were cocultured with different concentrations of CNPs, and proliferation and migration were measured both in vitro and in vivo. We found that CNPs inhibited the migration of gastric cancer cells when applied at different concentrations, but only a relatively high concentration (10 µg/mL) of CNPs suppressed proliferation. Furthermore, we found that CNPs increased the expression of DHX15 and its downstream signaling pathways. We therefore provide evidence showing that CNPs may be a promising approach to suppress malignant activity of gastric cancer by increasing the expression of DHX15. PMID:27486320

  7. Arterial calcifications and increased expression of vitamin D receptor targets in mice lacking TIF1α

    PubMed Central

    Ignat, Mihaela; Teletin, Marius; Tisserand, Johan; Khetchoumian, Konstantin; Dennefeld, Christine; Chambon, Pierre; Losson, Régine; Mark, Manuel

    2008-01-01

    Calcification of arteries is a major risk factor for cardiovascular mortality in humans. Using genetic approaches, we demonstrate here that the transcriptional intermediary factor 1α (TIF1α), recently shown to function as a tumor suppressor in murine hepatocytes, also participates in a molecular cascade that prevents calcifications in arterioles and medium-sized arteries. We further provide genetic evidence that this function of TIF1α is not exerted in hepatocytes. The sites of ectopic calcifications in mutant mice lacking TIF1α resemble those seen in mice carrying an activating mutation of the calcium sensor receptor (Casr) gene and, in TIF1α-deficient kidneys, Casr expression is increased together with that of many other vitamin D receptor (VDR) direct target genes, namely Car2, Cyp24a1, Trpv5, Trpv6, Calb1, S100g, Pthlh, and Spp1. Thus, our data indicate that TIF1α represses the VDR pathway in kidney and suggest that an up-regulation of Casr expression in this organ could account for ectopic calcifications generated upon TIF1α deficiency. Interestingly, the calcifying arteriopathy of TIF1α-null mutant mice shares features with the human age-related Mönckeberg's disease and, overall, the TIF1α-null mutant pathological phenotype supports the hypothesis that aging is promoted by increased activity of the vitamin D signaling pathway. PMID:18287084

  8. Magnesium ions increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis.

    PubMed

    Zou, Chun; Duan, Xuguo; Wu, Jing

    2016-08-01

    Addition of MgCl2 to the culture medium has been found to dramatically increase the activity of Bacillus deramificans pullulanase expressed by Brevibacillus choshinensis. The specific activity of the pullulanase obtained from medium supplemented with MgCl2 was also higher than that obtained in culture medium without added magnesium ions. In this work, the mechanism of this increase was studied. When cultured in medium without added magnesium ions, B. choshinensis mainly produced a thermolabile, inactive form of pullulanase. The addition of magnesium ions led to the production of a thermostable, active form of pullulanase. Circular dichroism assays revealed considerable differences in secondary structure between the active and inactive pullulanase forms. Transmission electron microscopy suggested that magnesium ion addition inhibits the shedding of cell wall protein (HWP) layers from the cell surface. Quantitative real-time PCR showed that magnesium ion addition represses transcription of HWP. Because the pullulanase gene and HWP have identical promoters, pullulanase gene transcription was also inhibited. These results suggest that when pullulanase is expressed slowly, it tends to fold into an active form. PMID:27026175

  9. Huqi San-Evoked Rat Colonic Anion Secretion through Increasing CFTR Expression

    PubMed Central

    Xue, Xiaowei; Shi, Zhengming; Wang, Wen; Yu, Xiaotong; Feng, Ping; Zhang, Min; Wang, Xuejiang; Xu, Jingdong

    2015-01-01

    Huqi San (HQS) is a Chinese herbal preparation of eight medicinal herbs that promote diuresis, detoxification, blood circulation, and cholestasis. Defects in transporter expression and function can cause cholestasis and jaundice. However, the mechanism of the cholestasis underlying HQS effects, especially on the gastrointestinal tract ion secretion, has not been elucidated. Real-time RT-PCR and Western blotting were used to study the expression and localization of cystic fibrosis transmembrane conductance regulator (CFTR) and α-ENaC in rat alimentary tract, and then the effect of HQS on the ion transport in rat distal colon mucosa was investigated using the short-circuit current (ISC) technique. The results showed that pretreatment with HQS significantly enhanced mRNA transcripts and protein content of CFTR in liver and distal colon but not α-ENaC in alimentary organs. HQS increases ISC and decreases the transepithelial resistance. Pretreatment with epithelial Na+ channel blocker did not affect the ISC responses elicited by HQS, but removal of extracellular Cl− or pretreatment with Cl− channel or Na+-K+-2Cl− cotransporter blocker inhibited HQS-elicited ISC responses. These findings demonstrated that HQS, RA, and RP can stimulate Cl− secretion in the distal colon by increasing the mRNA transcripts and protein content of CFTR in liver and distal colon. PMID:26290673

  10. Perivascular Cells Increase Expression of Ciliary Neurotrophic Factor Following Partial Denervation of the Rat Neurohypophysis

    PubMed Central

    Lo, David; SunRhodes, Neil; Watt, John A.

    2008-01-01

    The expression of ciliary neurotrophic factor (CNTF) was investigated immunocytochemically during the axonal degeneration and collateral axonal sprouting response that follows partial denervation of the rat neurohypophysis. A significant increase in the number of CNTF-immunoreactive (CNTF-ir) cells was observed in the neurohypophysis of partially denervated animals compared to age-matched sham-operated controls by 5 days post-denervation, remaining elevated throughout the 30 day post denervation period. Stereometric assessment of the numbers of CNTF-ir cells within the partially denervated neurohypophysis demonstrated a 36% increase by 3 days following denervation reaching 130% of control values by 10 days post-lesion. The cell numbers remained elevated throughout the 30 days post-lesion period suggesting that CNTF may play a role in the neurosecretory axonal sprouting process known to occur between 10 and 30 days post-denervation. Subsequent preparations pairing anti-CNTF with antibodies against ED1, CR3, p75 low affinity neurotrophin receptor (p75LNGFR), and S100β, demonstrated that CNTF was exclusively localized in a phenotypically-distinct population of perivascular cells. The association of perivascular cells with phagocytic activity was confirmed by dual label fluorescence microscopy showing the colocalization of P75LNGFR-ir and OX-42-ir in cells expressing the ED-1 antigen. No increase in CNTF-ir was observed in non-injured animals in which heightened levels of neurosecretory activity were induced physiologically. These results suggest that increased CNTF-ir occurs in response to conditions which induce high levels of phagocytic activity by perivascular cells in the axotomized neurohypophysis which is sustained throughout a period in which axonal sprouting is known to occur in the partially denervated neurohypophysis. PMID:18805412

  11. Local overexpression of the myostatin propeptide increases glucose transporter expression and enhances skeletal muscle glucose disposal

    PubMed Central

    Jarmin, S.; Eilers, W.; Elashry, M.; Andersen, D. K.; Dickson, G.; Foster, K.

    2014-01-01

    Insulin resistance (IR) in skeletal muscle is a prerequisite for type 2 diabetes and is often associated with obesity. IR also develops alongside muscle atrophy in older individuals in sarcopenic obesity. The molecular defects that underpin this syndrome are not well characterized, and there is no licensed treatment. Deletion of the transforming growth factor-β family member myostatin, or sequestration of the active peptide by overexpression of the myostatin propeptide/latency-associated peptide (ProMyo) results in both muscle hypertrophy and reduced obesity and IR. We aimed to establish whether local myostatin inhibition would have a paracrine/autocrine effect to enhance glucose disposal beyond that simply generated by increased muscle mass, and the mechanisms involved. We directly injected adeno-associated virus expressing ProMyo in right tibialis cranialis/extensor digitorum longus muscles of rats and saline in left muscles and compared the effects after 17 days. Both test muscles were increased in size (by 7 and 11%) and showed increased radiolabeled 2-deoxyglucose uptake (26 and 47%) and glycogen storage (28 and 41%) per unit mass during an intraperitoneal glucose tolerance test. This was likely mediated through increased membrane protein levels of GLUT1 (19% higher) and GLUT4 (63% higher). Interestingly, phosphorylation of phosphoinositol 3-kinase signaling intermediates and AMP-activated kinase was slightly decreased, possibly because of reduced expression of insulin-like growth factor-I in these muscles. Thus, myostatin inhibition has direct effects to enhance glucose disposal in muscle beyond that expected of hypertrophy alone, and this approach may offer potential for the therapy of IR syndromes. PMID:24473441

  12. Increased forest carbon storage with increased atmospheric CO2 despite nitrogen limitation: a game-theoretic allocation model for trees in competition for nitrogen and light.

    PubMed

    Dybzinski, Ray; Farrior, Caroline E; Pacala, Stephen W

    2015-03-01

    Changes in resource availability often cause competitively driven changes in tree allocation to foliage, wood, and fine roots, either via plastic changes within individuals or through turnover of individuals with differing strategies. Here, we investigate how optimally competitive tree allocation should change in response to elevated atmospheric CO2 along a gradient of nitrogen and light availability, together with how those changes should affect carbon storage in living biomass. We present a physiologically-based forest model that includes the primary functions of wood and nitrogen. From a tree's perspective, wood is an offensive and defensive weapon used against neighbors in competition for light. From a biogeochemical perspective, wood is the primary living reservoir of stored carbon. Nitrogen constitutes a tree's photosynthetic machinery and the support systems for that machinery, and its limited availability thus reduces a tree's ability to fix carbon. This model has been previously successful in predicting allocation to foliage, wood, and fine roots along natural productivity gradients. Using game theory, we solve the model for competitively optimal foliage, wood, and fine root allocation strategies for trees in competition for nitrogen and light as a function of CO2 and nitrogen mineralization rate. Instead of down-regulating under nitrogen limitation, carbon storage under elevated CO2 relative to carbon storage at ambient CO2 is approximately independent of the nitrogen mineralization rate. This surprising prediction is a consequence of both increased competition for nitrogen driving increased fine root biomass and increased competition for light driving increased allocation to wood under elevated CO2 . PMID:25392967

  13. Increasing the Upper Temperature Oxidation Limit of Alumina Forming Austenitic Stainless Steels in Air with Water Vapor

    SciTech Connect

    Brady, Michael P; Unocic, Kinga A; Lance, Michael J; Santella, Michael L; Yamamoto, Yukinori; Walker, Larry R

    2011-01-01

    A family of alumina-forming austenitic (AFA) stainless steels is under development for use in aggressive oxidizing conditions from {approx}600-900 C. These alloys exhibit promising mechanical properties but oxidation resistance in air with water vapor environments is currently limited to {approx}800 C due to a transition from external protective alumina scale formation to internal oxidation of aluminum with increasing temperature. The oxidation behavior of a series of AFA alloys was systematically studied as a function of Cr, Si, Al, C, and B additions in an effort to provide a basis to increase the upper-temperature oxidation limit. Oxidation exposures were conducted in air with 10% water vapor environments from 800-1000 C, with post oxidation characterization of the 900 C exposed samples by electron probe microanalysis (EPMA), scanning and transmission electron microscopy, and photo-stimulated luminescence spectroscopy (PSLS). Increased levels of Al, C, and B additions were found to increase the upper-temperature oxidation limit in air with water vapor to between 950 and 1000 C. These findings are discussed in terms of alloy microstructure and possible gettering of hydrogen from water vapor at second phase carbide and boride precipitates.

  14. Low-dose oral cadmium increases airway reactivity and lung neuronal gene expression in mice.

    PubMed

    Chandler, Joshua D; Wongtrakool, Cherry; Banton, Sophia A; Li, Shuzhao; Orr, Michael L; Barr, Dana Boyd; Neujahr, David C; Sutliff, Roy L; Go, Young-Mi; Jones, Dean P

    2016-07-01

    Inhalation of cadmium (Cd) is associated with lung diseases, but less is known concerning pulmonary effects of Cd found in the diet. Cd has a decades-long half-life in humans and significant bioaccumulation occurs with chronic dietary intake. We exposed mice to low-dose CdCl2 (10 mg/L in drinking water) for 20 weeks, which increased lung Cd to a level similar to that of nonoccupationally exposed adult humans. Cd-treated mice had increased airway hyperresponsiveness to methacholine challenge, and gene expression array showed that Cd altered the abundance of 443 mRNA transcripts in mouse lung. In contrast to higher doses, low-dose Cd did not elicit increased metallothionein transcripts in lung. To identify pathways most affected by Cd, gene set enrichment of transcripts was analyzed. Results showed that major inducible targets of low-dose Cd were neuronal receptors represented by enriched olfactory, glutamatergic, cholinergic, and serotonergic gene sets. Olfactory receptors regulate chemosensory function and airway hypersensitivity, and these gene sets were the most enriched. Targeted metabolomics analysis showed that Cd treatment also increased metabolites in pathways of glutamatergic (glutamate), serotonergic (tryptophan), cholinergic (choline), and catecholaminergic (tyrosine) receptors in the lung tissue. Protein abundance measurements showed that the glutamate receptor GRIN2A was increased in mouse lung tissue. Together, these results show that in mice, oral low-dose Cd increased lung Cd to levels comparable to humans, increased airway hyperresponsiveness and disrupted neuronal pathways regulating bronchial tone. Therefore, dietary Cd may promote or worsen airway hyperresponsiveness in multiple lung diseases including asthma. PMID:27401458

  15. Increased miR-132-3p expression is associated with chronic neuropathic pain.

    PubMed

    Leinders, M; Üçeyler, N; Pritchard, R A; Sommer, C; Sorkin, L S

    2016-09-01

    Alterations in the neuro-immune balance play a major role in the pathophysiology of chronic neuropathic pain. MicroRNAs (miRNA) can regulate both immune and neuronal processes and may function as master switches in chronic pain development and maintenance. We set out to analyze the role of miR-132-3p, first in patients with peripheral neuropathies and second in an animal model of neuropathic pain. We initially determined miR-132-3p expression by measuring its levels in white blood cells (WBC) of 30 patients and 30 healthy controls and next in sural nerve biopsies of 81 patients with painful or painless inflammatory or non-inflammatory neuropathies based on clinical diagnosis. We found a 2.6 fold increase in miR-132-3p expression in WBC of neuropathy patients compared to healthy controls (p<0.001). MiR-132-3p expression was also slightly up-regulated in sural nerve biopsies from neuropathy patients suffering from neuropathic pain compared to those without pain (1.2 fold; p<0.001). These promising findings were investigated further in an animal model of neuropathic pain, the spared nerve injury model (SNI). For this purpose miR-132-3p expression levels were measured in dorsal root ganglia and spinal cord of rats. Subsequently, miR-132-3p expression was pharmacologically modulated with miRNA antagonists or mimetics, and evoked pain and pain aversion were assessed. Spinal miR-132-3p levels were highest 10days after SNI, a time when persistent allodynia was established (p<0.05). Spinal administration of miR-132-3p antagonists via intrathecal (i.t.) catheters dose dependently reversed mechanical allodyina (p<0.001) and eliminated pain behavior in the place escape avoidance paradigm (p<0.001). Intrathecal administration of miR-132-3p mimetic dose-dependently induced pain behavior in naïve rats (p<0.001). Taken together these results indicate a pro-nociceptive effect of miR-132-3p in chronic neuropathic pain. PMID:27349406

  16. Increased programmed death ligand-1 expression predicts poor prognosis in hepatocellular carcinoma patients

    PubMed Central

    Gu, Xiaobin; Gao, Xian-Shu; Xiong, Wei; Guo, Wei; Han, Linjun; Bai, Yun; Peng, Chuan; Cui, Ming; Xie, Mu

    2016-01-01

    Purpose Accumulating studies have investigated the prognostic and clinical significance of programmed death ligand-1 (PD-L1) expression in patients with hepatocellular carcinoma (HCC); however, the results were conflicting and inconclusive. We conducted a meta-analysis to combine controversial data to precisely evaluate this issue. Methods Relevant studies were thoroughly searched on PubMed, Web of Science, and Embase until April 2016. Eligible studies were evaluated by selection criteria. Hazard ratio (HR) with 95% confidence interval (CI) was used to estimate the prognostic role of PD-L1 for overall survival (OS) and disease-free survival (DFS)/recurrence-free survival (RFS). Odds ratio (OR) with 95% CI were selected to assess the relationship between PD-L1 and clinicopathological features of HCC patients. Publication bias was tested using Begg’s funnel plot. Results A total of seven studies published from 2009 to 2016 were included for meta-analysis. The data showed that high PD-L1 expression was correlated to shorter OS (HR =2.09, 95% CI: 1.66–2.64, P<0.001) as well as poor DFS/RFS (HR =2.3, 95% CI: 1.46–3.62, P<0.001). In addition, increased PD-L1 expression was also associated with tumor differentiation (HR =1.51, 95% CI: 1–2.29, P=0.05), vascular invasion (HR =2.16, 95% CI: 1.43–3.27, P<0.001), and α-fetoprotein (AFP; HR =1.46, 95% CI: 1–2.14, P=0.05), but had no association with tumor stage, tumor size, hepatitis history, sex, age, or tumor multiplicity. No publication bias was found for all analyses. Conclusion This meta-analysis revealed that overexpression of PD-L1 was predictive for shortened OS and DFS/RFS in HCC. Furthermore, increased PD-L1 expression was associated with less differentiation, vascular invasion, and AFP elevation. PMID:27536144

  17. Probing the Limits to MicroRNA-Mediated Control of Gene Expression.

    PubMed

    Martirosyan, Araks; Figliuzzi, Matteo; Marinari, Enzo; De Martino, Andrea

    2016-01-01

    According to the 'ceRNA hypothesis', microRNAs (miRNAs) may act as mediators of an effective positive interaction between long coding or non-coding RNA molecules, carrying significant potential implications for a variety of biological processes. Here, inspired by recent work providing a quantitative description of small regulatory elements as information-conveying channels, we characterize the effectiveness of miRNA-mediated regulation in terms of the optimal information flow achievable between modulator (transcription factors) and target nodes (long RNAs). Our findings show that, while a sufficiently large degree of target derepression is needed to activate miRNA-mediated transmission, (a) in case of differential mechanisms of complex processing and/or transcriptional capabilities, regulation by a post-transcriptional miRNA-channel can outperform that achieved through direct transcriptional control; moreover, (b) in the presence of large populations of weakly interacting miRNA molecules the extra noise coming from titration disappears, allowing the miRNA-channel to process information as effectively as the direct channel. These observations establish the limits of miRNA-mediated post-transcriptional cross-talk and suggest that, besides providing a degree of noise buffering, this type of control may be effectively employed in cells both as a failsafe mechanism and as a preferential fine tuner of gene expression, pointing to the specific situations in which each of these functionalities is maximized. PMID:26812364

  18. Probing the Limits to MicroRNA-Mediated Control of Gene Expression

    PubMed Central

    Martirosyan, Araks; Figliuzzi, Matteo

    2016-01-01

    According to the ‘ceRNA hypothesis’, microRNAs (miRNAs) may act as mediators of an effective positive interaction between long coding or non-coding RNA molecules, carrying significant potential implications for a variety of biological processes. Here, inspired by recent work providing a quantitative description of small regulatory elements as information-conveying channels, we characterize the effectiveness of miRNA-mediated regulation in terms of the optimal information flow achievable between modulator (transcription factors) and target nodes (long RNAs). Our findings show that, while a sufficiently large degree of target derepression is needed to activate miRNA-mediated transmission, (a) in case of differential mechanisms of complex processing and/or transcriptional capabilities, regulation by a post-transcriptional miRNA-channel can outperform that achieved through direct transcriptional control; moreover, (b) in the presence of large populations of weakly interacting miRNA molecules the extra noise coming from titration disappears, allowing the miRNA-channel to process information as effectively as the direct channel. These observations establish the limits of miRNA-mediated post-transcriptional cross-talk and suggest that, besides providing a degree of noise buffering, this type of control may be effectively employed in cells both as a failsafe mechanism and as a preferential fine tuner of gene expression, pointing to the specific situations in which each of these functionalities is maximized. PMID:26812364

  19. Expression of Umbelopsis ramanniana DGAT2A in Seed Increases Oil in Soybean1[OA

    PubMed Central

    Lardizabal, Kathryn; Effertz, Roger; Levering, Charlene; Mai, Jennifer; Pedroso, M.C.; Jury, Tom; Aasen, Eric; Gruys, Ken; Bennett, Kristen

    2008-01-01

    Oilseeds are the main source of lipids used in both food and biofuels. The growing demand for vegetable oil has focused research toward increasing the amount of this valuable component in oilseed crops. Globally, soybean (Glycine max) is one of the most important oilseed crops grown, contributing about 30% of the vegetable oil used for food, feed, and industrial applications. Breeding efforts in soy have shown that multiple loci contribute to the final content of oil and protein stored in seeds. Genetically, the levels of these two storage products appear to be inversely correlated with an increase in oil coming at the expense of protein and vice versa. One way to overcome the linkage between oil and protein is to introduce a transgene that can specifically modulate one pathway without disrupting the other. We describe the first, to our knowledge, transgenic soy crop with increased oil that shows no major impact on protein content or yield. This was achieved by expressing a codon-optimized version of a diacylglycerol acyltransferase 2A from the soil fungus Umbelopsis (formerly Mortierella) ramanniana in soybean seed during development, resulting in an absolute increase in oil of 1.5% (by weight) in the mature seed. PMID:18633120

  20. Expression of lactoferrin receptors is increased in the mesencephalon of patients with Parkinson disease.

    PubMed Central

    Faucheux, B A; Nillesse, N; Damier, P; Spik, G; Mouatt-Prigent, A; Pierce, A; Leveugle, B; Kubis, N; Hauw, J J; Agid, Y

    1995-01-01

    The degeneration of nigral dopaminergic neurons in Parkinson disease is believed to be associated with oxidative stress. Since iron levels are increased in the substantia nigra of parkinsonian patients and this metal catalyzes the formation of free radicals, it may be involved in the mechanisms of nerve cell death. The cause of nigral iron increase is not understood. Iron acquisition by neurons may occur from iron-transferrin complexes with a direct interaction with specific membrane receptors, but recent results have shown a low density of transferrin receptors in the substantia nigra. To investigate whether neuronal death in Parkinson disease may be associated with changes in a pathway supplementary to that of transferrin, lactoferrin (lactotransferrin) receptor expression was studied in the mesencephalon. In this report we present evidence from immunohistochemical staining of postmortem human brain tissue that lactoferrin receptors are localized on neurons (perikarya, dendrites, axons), cerebral microvasculature, and, in some cases, glial cells. In parkinsonian patients, lactoferrin receptor immunoreactivity on neurons and microvessels was increased and more pronounced in those regions of the mesencephalon where the loss of dopaminergic neurons is severe. Moreover, in the substantia nigra, the intensity of immunoreactivity on neurons and microvessels was higher for patients with higher nigral dopaminergic loss. These data suggest that lactoferrin receptors on vulnerable neurons may increase intraneuronal iron levels and contribute to the degeneration of nigral dopaminergic neurons in Parkinson disease. Images Fig. 1 Fig. 2 PMID:7568181

  1. Water deficit in field-grown Gossypium hirsutum primarily limits net photosynthesis by decreasing stomatal conductance, increasing photorespiration, and increasing the ratio of dark respiration to gross photosynthesis.

    PubMed

    Chastain, Daryl R; Snider, John L; Collins, Guy D; Perry, Calvin D; Whitaker, Jared; Byrd, Seth A

    2014-11-01

    Much effort has been expended to improve irrigation efficiency and drought tolerance of agronomic crops; however, a clear understanding of the physiological mechanisms that interact to decrease source strength and drive yield loss has not been attained. To elucidate the underlying mechanisms contributing to inhibition of net carbon assimilation under drought stress, three cultivars of Gossypium hirsutum were grown in the field under contrasting irrigation regimes during the 2012 and 2013 growing season near Camilla, Georgia, USA. Physiological measurements were conducted on three sample dates during each growing season (providing a broad range of plant water status) and included, predawn and midday leaf water potential (ΨPD and ΨMD), gross and net photosynthesis, dark respiration, photorespiration, and chlorophyll a fluorescence. End-of-season lint yield was also determined. ΨPD ranged from -0.31 to -0.95MPa, and ΨMD ranged from -1.02 to -2.67MPa, depending upon irrigation regime and sample date. G. hirsutum responded to water deficit by decreasing stomatal conductance, increasing photorespiration, and increasing the ratio of dark respiration to gross photosynthesis, thereby limiting PN and decreasing lint yield (lint yield declines observed during the 2012 growing season only). Conversely, even extreme water deficit, causing a 54% decline in PN, did not negatively affect actual quantum yield, maximum quantum yield, or photosynthetic electron transport. It is concluded that PN is primarily limited in drought-stressed G. hirsutum by decreased stomatal conductance, along with increases in respiratory and photorespiratory carbon losses, not inhibition or down-regulation of electron transport through photosystem II. It is further concluded that ΨPD is a reliable indicator of drought stress and the need for irrigation in field-grown cotton. PMID:25151126

  2. Increased influence of nitrogen limitation on CO2 emissions from future land use and land-use change

    NASA Astrophysics Data System (ADS)

    Jain, A. K.; Meiyappan, P.; House, J.

    2015-12-01

    In the latest projections of future greenhouse gas emissions for the Intergovernmental Panel on Climate Change (IPCC), few Earth System Models included the effect of nitrogen limitation, a key process limiting forest regrowth. We estimate the impacts of nitrogen limitation on the CO2 emissions from land use and land-use change (LULUC), including wood harvest, for the period 1900-2100. We use a land-surface model that includes a fully coupled carbon and nitrogen cycle, and accounts for forest regrowth processes following agricultural abandonment and wood harvest. Future projections are based on the four Representation Concentration Pathways used in the IPCC Fifth Assessment Report, and we account for uncertainty in future climate for each scenario based on ensembles of climate model outputs. Results show that excluding nitrogen limitation will underestimate global LULUC emissions by 34-52 PgC (20-30%) during the 20th century (range across three different historical LULUC reconstructions) and by 128-187 PgC (90-150%) during the 21st century (range across the four IPCC scenarios). The full range for estimated LULUC emissions during the 21st century including climate model uncertainty is 91 to 227 PgC (with nitrogen limitation included). The underestimation increases with time because: (1) Projected annual wood harvest rates from forests summed over the 21st century are 380-1080% higher compared to those of the 20th century, resulting in more regrowing secondary forests, (2) Nitrogen limitation reduces the CO2 fertilization effect on net primary production of regrowing secondary forests following wood harvest and agricultural abandonment, and (3) Nitrogen limitation effect is aggravated by the gradual loss of soil nitrogen from LULUC disturbance. Our study implies that: (1) Nitrogen limitation of CO2 uptake is substantial and sensitive to nitrogen inputs, (2) If LULUC emissions are larger than previously estimated in studies without nitrogen limitation, then meeting

  3. Increased influence of nitrogen limitation on CO2 emissions from future land use and land use change

    NASA Astrophysics Data System (ADS)

    Meiyappan, Prasanth; Jain, Atul K.; House, Joanna I.

    2015-09-01

    In the latest projections of future greenhouse gas emissions for the Intergovernmental Panel on Climate Change (IPCC), few Earth System Models included the effect of nitrogen limitation, a key process limiting forest regrowth. Few included forest management (wood harvest). We estimate the impacts of nitrogen limitation on the CO2 emissions from land use and land use change (LULUC), including wood harvest, for the period 1900-2100. We use a land surface model that includes a fully coupled carbon and nitrogen cycle and accounts for forest regrowth processes following agricultural abandonment and wood harvest. Future projections are based on the four Representation Concentration Pathways used in the IPCC Fifth Assessment Report, and we account for uncertainty in future climate for each scenario based on ensembles of climate model outputs. Results show that excluding nitrogen limitation will underestimate global LULUC emissions by 34-52 PgC (20-30%) during the 20th century (range across three different historical LULUC reconstructions) and by 128-187 PgC (90-150%) during the 21st century (range across the four IPCC scenarios). The full range for estimated LULUC emissions during the 21st century including climate model uncertainty is 91 to 227 PgC (with nitrogen limitation included). The underestimation increases with time because (1) projected annual wood harvest rates from forests summed over the 21st century are 380-1080% higher compared to those of the 20th century, resulting in more regrowing secondary forests; (2) nitrogen limitation reduces the CO2 fertilization effect on net primary production of regrowing secondary forests following wood harvest and agricultural abandonment; and (3) nitrogen limitation effect is aggravated by the gradual loss of soil nitrogen from LULUC disturbance. Our study implies that (1) nitrogen limitation of CO2 uptake is substantial and sensitive to nitrogen inputs; (2) if LULUC emissions are larger than previously estimated in studies

  4. Constitutive homologous expression of phosphoglucomutase and transaldolase increases the metabolic flux of Fusarium oxysporum

    PubMed Central

    2014-01-01

    Background Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism. Results Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher biomass to xylose yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth on glucose revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source. Conclusions Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics. PMID:24649884

  5. Inflammatory mediator bradykinin increases population of sensory neurons expressing functional T-type Ca(2+) channels.

    PubMed

    Huang, Dongyang; Liang, Ce; Zhang, Fan; Men, Hongchao; Du, Xiaona; Gamper, Nikita; Zhang, Hailin

    2016-04-29

    T-type Ca(2+) channels are important regulators of peripheral sensory neuron excitability. Accordingly, T-type Ca(2+) currents are often increased in various pathological pain conditions, such as inflammation or nerve injury. Here we investigated effects of inflammation on functional expression of T-type Ca(2+) channels in small-diameter cultured dorsal root ganglion (DRG) neurons. We found that overnight treatment of DRG cultures with a cocktail of inflammatory mediators bradykinin (BK), adenosine triphosphate (ATP), norepinephrine (NE) and prostaglandin E2 (PGE2) strongly increased the population size of the small-diameter neurons displaying low-voltage activated (LVA, T-type) Ca(2+) currents while having no effect on the peak LVA current amplitude. When applied individually, BK and ATP also increased the population size of LVA-positive neurons while NE and PGE2 had no effect. The PLC inhibitor U-73122 and B2 receptor antagonist, Hoe-140, both abolished the increase of the population of LVA-positive DRG neurons. Inflammatory treatment did not affect CaV3.2 mRNA or protein levels in DRG cultures. Furthermore, an ubiquitination inhibitor, MG132, did not increase the population of LVA-positive neurons. Our data suggest that inflammatory mediators BK and ATP increase the abundance of LVA-positive DRG neurons in total neuronal population by stimulating the recruitment of a 'reserve pool' of CaV3.2 channels, particularly in neurons that do not display measurable LVA currents under control conditions. PMID:26944020

  6. Inflammatory mediator bradykinin increases population of sensory neurons expressing functional T-type Ca2+ channels

    PubMed Central

    Huang, Dongyang; Liang, Ce; Zhang, Fan; Men, Hongchao; Du, Xiaona; Gamper, Nikita; Zhang, Hailin

    2016-01-01

    T-type Ca2+ channels are important regulators of peripheral sensory neuron excitability. Accordingly, T-type Ca2+ currents are often increased in various pathological pain conditions, such as inflammation or nerve injury. Here we investigated effects of inflammation on functional expression of T-type Ca2+ channels in small-diameter cultured dorsal root ganglion (DRG) neurons. We found that overnight treatment of DRG cultures with a cocktail of inflammatory mediators bradykinin (BK), adenosine triphosphate (ATP), norepinephrine (NE) and prostaglandin E2 (PGE2) strongly increased the population size of the small-diameter neurons displaying low-voltage activated (LVA, T-type) Ca2+ currents while having no effect on the peak LVA current amplitude. When applied individually, BK and ATP also increased the population size of LVA-positive neurons while NE and PGE2 had no effect. The PLC inhibitor U-73122 and B2 receptor antagonist, Hoe-140, both abolished the increase of the population of LVA-positive DRG neurons. Inflammatory treatment did not affect CaV3.2 mRNA or protein levels in DRG cultures. Furthermore, an ubiquitination inhibitor, MG132, did not increase the population of LVA-positive neurons. Our data suggest that inflammatory mediators BK and ATP increase the abundance of LVA-positive DRG neurons in total neuronal population by stimulating the recruitment of a ‘reserve pool’ of CaV3.2 channels, particularly in neurons that do not display measurable LVA currents under control conditions. PMID:26944020

  7. Estimation of molecular upper remission limit for monitoring minimal residual disease in peripheral blood of acute myeloid leukemia patients by WT1 expression

    PubMed Central

    POLÁK, JAROSLAV; HÁJKOVÁ, HANA; MAALAUFOVÁ-SOUKUPOVÁ, JACQUELINE; MARKOVÁ, JANA; ŠÁLEK, CYRIL; SCHWARZ, JIŘÍ; HAŠKOVEC, CEDRIK

    2012-01-01

    To date, approximately one half of acute myeloid leukaemia (AML) patients do not have a suitable specific molecular marker for monitoring minimal residual disease (MRD). The Wilm’s tumour gene (WT1) has been suggested as a possible molecular marker of MRD in AML. The expression of WT1 in peripheral blood (PB) was measured using quantitative real-time reverse transcription-polymerase chain reaction in peripheral leukocytes from 151 patients with AML at diagnosis. WT1 expression was significantly elevated, i.e. up to 3 orders of magnitude in the majority (80%) of AML patients at diagnosis compared to the PB of healthy donors. Sequence samples of the long-term followed-up AML patients treated with chemotherapy and/or allogeneic bone marrow transplantation were analysed for WT1 expression. The results revealed that the hematological relapses were preceded (median, 1.8 months) by an increase in WT1 gene expression. For the practical utility of this gene as a molecular marker of relapse, it was necessary to determine an upper remission limit, crossing which would signal hematological relapse. The upper remission limit was determined in our set of patients to be 0.02 WT1/ABL. The AML patients who consequently relapsed crossed this upper remission limit; however, those in permanent remission did not. Therefore, this upper remission limit could be taken as the border of molecular relapse of AML patients. Moreover, insufficient decline of WT1 expression under the upper remission limit following induction and/or consolidation therapy was associated with markedly high risk of relapse. The results show that our upper remission limit can be taken as the border of molecular relapse of AML patients and WT1 levels following initial therapy as a beneficial prognostic marker. PMID:22969857

  8. Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

    PubMed Central

    Creelman, Robert A.; Griffith, Cara; Ahrens, Jeffrey E.; Taylor, J. Philip; Murphy, Lesley R.; Manjunath, Siva; Thompson, Rebecca L.; Lingard, Matthew J.; Back, Stephanie L.; Larue, Huachun; Brayton, Bonnie R.; Burek, Amanda J.; Tiwari, Shiv; Adam, Luc; Morrell, James A.; Caldo, Rico A.; Huai, Qing; Kouadio, Jean-Louis K.; Kuehn, Rosemarie; Sant, Anagha M.; Wingbermuehle, William J.; Sala, Rodrigo; Foster, Matt; Kinser, Josh D.; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E.; Huang, Mingya G.; Kuriakose, Saritha V.; Skottke, Kyle; Repetti, Peter P.; Reuber, T. Lynne; Ruff, Thomas G.; Petracek, Marie E.; Loida, Paul J.

    2014-01-01

    ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658

  9. Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency.

    PubMed

    Maddox, J F; Aherne, K M; Reddy, C C; Sordillo, L M

    1999-05-01

    Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation. Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome. Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH-Px) and plays an important role in the maintenance of the redox state of a cell. Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland. However, the mechanisms by which Se modulates neutrophil migration have not been elucidated. Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC. The effect of Se on functional activities was examined by measuring neutrophil adherence to Se-deficient and Se-supplemented EC. Se-deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor alpha (TNF-alpha) for 4 or 24 h, interleukin-1 for 12 h, or H2O2 for 20 min (P < 0.05). To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM-1, E-selectin, and P-selectin were examined at the molecular level by a competitive reverse transcription-polymerase chain reaction. Results revealed higher mRNA expression for E-selectin and ICAM-1 in Se-deficient EC stimulated with TNF-alpha for 3 and 6 h, and greater expression of P-selectin mRNA in Se-supplemented EC with 3-h TNF-alpha stimulation. These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium. PMID:10331495

  10. Epileptiform stimulus increases Homer 1a expression to modulate synapse number and activity in hippocampal cultures

    PubMed Central

    Li, Yan; Popko, Jonathan; Krogh, Kelly A.

    2013-01-01

    Neurons adapt to seizure activity structurally and functionally to attenuate hyperactive neural circuits. Homer proteins provide a scaffold in the postsynaptic density (PSD) by binding to ligands through an EVH1 domain and to other Homer proteins by a coiled-coil domain. The short Homer isoform 1a (H1a) has a ligand-binding domain but lacks a coiled-coil domain and thus acts in a dominant-negative manner to uncouple Homer scaffolds. Here, we show that treating rat hippocampal cultures with bicuculline and 4-aminopyridine (Bic+4-AP) evoked epileptiform activity and synchronized Ca2+ spiking, measured with whole cell current-clamp and fura-2-based digital imaging; Bic+4-AP increased H1a mRNA through the activation of metabotropic glutamate receptor 5 (mGluR5). Treatment with Bic+4-AP for 4 h attenuated burst firing and induced synapse loss. Synaptic changes were measured using a confocal imaging-based assay that quantified clusters of PSD-95 fused to green fluorescent protein. Treatment with an mGluR5 antagonist blocked H1a expression, synapse loss, and burst attenuation. Overexpression of H1a inhibited burst firing similar to Bic+4-AP treatment. Furthermore, knockdown of H1a using a short hairpin RNA (shRNA) strategy reduced synapse loss and burst attenuation induced by Bic+4-AP treatment. Thus an epileptiform stimulus applied to hippocampal neurons in culture induced burst firing and H1a expression through the activation of mGluR5; a 4-h exposure to this stimulus resulted in synapse loss and burst attenuation. These results suggest that H1a expression functions in a negative-feedback manner to reduce network excitability by regulating the number of synapses. PMID:23274309

  11. Increasing TRPV4 expression restores flow-induced dilation impaired in mesenteric arteries with aging

    PubMed Central

    Du, Juan; Wang, Xia; Li, Jie; Guo, Jizheng; Liu, Limei; Yan, Dejun; Yang, Yunyun; Li, Zhongwen; Zhu, Jinhang; Shen, Bing

    2016-01-01

    The flow-stimulated intracellular Ca2+ concentration ([Ca2+]i) rise in endothelial cells is an important early event leading to flow-induced blood vessel dilation. Transient receptor potential vanilloid subtype 4 (TRPV4), a Ca2+-permeable cation channel, facilitates the flow-stimulated [Ca2+]i rise. To determine whether TRPV4 is involved in age-related flow-induced blood vessel dilation impairment, we measured blood vessel diameter and nitric oxide (NO) levels and performed Ca2+ imaging, immunoblotting, and immunostaining assays in rats. We found that the flow-induced and TRPV4 activator 4α-PDD-induced dilation of mesenteric arteries from aged rats were significantly decreased compared with those from young rats. The flow- or 4α-PDD-induced [Ca2+]i rise was also markedly reduced in primary cultured mesenteric artery endothelial cells (MAECs) from aged rats. Immunoblotting and immunostaining results showed an age-related decrease of TRPV4 expression levels in MAECs. Additionally, the 4α-PDD-induced NO production was significantly reduced in aged MAECs. Compared with lentiviral GFP-treated aged rats, lentiviral vector delivery of TRPV4 increased TRPV4 expression level in aged MAECs and restored the flow- and 4α-PDD-induced vessel dilation in aged mesenteric arteries. We concluded that impaired TRPV4-mediated Ca2+ signaling causes endothelial dysfunction and that TRPV4 is a potential target for clinical treatment of age-related vascular system diseases. PMID:26947561

  12. Suppressive expression of CD274 increases tumorigenesis and cancer stem cell phenotypes in cholangiocarcinoma

    PubMed Central

    Tamai, Keiichi; Nakamura, Mao; Mizuma, Masamichi; Mochizuki, Mai; Yokoyama, Misa; Endo, Hiroyuki; Yamaguchi, Kazunori; Nakagawa, Takayuki; Shiina, Masaaki; Unno, Michiaki; Muramoto, Koji; Sato, Ikuro; Satoh, Kennichi; Sugamura, Kazuo; Tanaka, Nobuyuki

    2014-01-01

    Cholangiocarcinoma is an aggressive malignant tumor originating from intrahepatic or extrahepatic bile ducts. Its malignant phenotypes may be assumed by cancer stem cells (CSC). Here, we demonstrate that CD274 (PD-L1), known as an immunomodulatory ligand, has suppressive effects on CSC-related phenotypes of cholangiocarcinoma. Using two human cholangiocarcinoma cell lines, RBE and HuCCT1, we attempted to isolate the CD274low and CD274high cells from each cell line, and xenografted them into immunodeficient NOD/scid/γcnull (NOG) mice. We found that the CD274low cells isolated from both RBE and HuCCT1 are highly tumorigenic in NOG mice compared with CD274high cells. Furthermore, the CD274low cells possess several CSC-related characteristics, such as high aldehyde dehydrogenase (ALDH) activity, reduced reactive oxygen species production and a dormant state in the cell cycle. Furthermore, depletion of CD274 expression by shRNA in RBE cells enhances their tumorigenicity and increases ALDH activity. These findings are compatible with our observation that clinical cholangiocarcinoma specimens are classified into low and high groups for CD274 expression, and the CD274 low group shows poorer prognosis when compared with the CD274 high group. These results strongly suggest that CD274 has a novel function in the negative regulation of CSC-related phenotypes in human cholangiocarcinoma, which is distinct from its immunomodulatory actions. PMID:24673799

  13. [Expression of cecropin P1 gene increases resistance of Camelina sativa (L.) plants to microbial phytopathogenes].

    PubMed

    Zakharchenko, N S; Kaliaeva, M A; Bur'ianov, Ia I

    2013-05-01

    Transgenic plants of camelina (Camelina sativa (L.) Crantz) with the synthetic gene of antimicrobial peptide cecropin P1 (cecP1) were obtained. Agrobacterium-mediated transformation is performed using the binary vector pGA482::cecP1 by vacuum infiltration of flower buds. The presence of the cecP1 gene in the genome of plants was confirmed by PCR. cecP1 gene expression in transgenic plants was shown by Western blot analysis and by antimicrobial activity of plant extracts against the bacterial phytopathogene Erwinia carotovora. The plants of F0 and F1 generations had the normal phenotype and retained the ability to form viable seeds in self-pollination. cecP1 plants exhibit enhanced resistance to bacterial and fungal phytopathogens: Erwinia carotovora and Fusarium sporotrichioides. The increased sustainability of cecropin P1-expressing plants against salt stress is shown. The possibility of the integration of the cecP1 gene into the overall protective system of plants against biotic and abiotic stresses is discussed. PMID:24159802

  14. Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety.

    PubMed

    Panda, R; Ariyarathna, H; Amnuaycheewa, P; Tetteh, A; Pramod, S N; Taylor, S L; Ballmer-Weber, B K; Goodman, R E

    2013-02-01

    Premarket, genetically modified (GM) plants are assessed for potential risks of food allergy. The major risk would be transfer of a gene encoding an allergen or protein nearly identical to an allergen into a different food source, which can be assessed by specific serum testing. The potential that a newly expressed protein might become an allergen is evaluated based on resistance to digestion in pepsin and abundance in food fractions. If the modified plant is a common allergenic source (e.g. soybean), regulatory guidelines suggest testing for increases in the expression of endogenous allergens. Some regulators request evaluating endogenous allergens for rarely allergenic plants (e.g. maize and rice). Since allergic individuals must avoid foods containing their allergen (e.g. peanut, soybean, maize, or rice), the relevance of the tests is unclear. Furthermore, no acceptance criteria are established and little is known about the natural variation in allergen concentrations in these crops. Our results demonstrate a 15-fold difference in the major maize allergen, lipid transfer protein between nine varieties, and complex variation in IgE binding to various soybean varieties. We question the value of evaluating endogenous allergens in GM plants unless the intent of the modification was production of a hypoallergenic crop. PMID:23205714

  15. Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression

    PubMed Central

    Leventhal, Jeremy S.; Ni, Jie; Osmond, Morgan; Lee, Kyung; Gusella, G. Luca; Salem, Fadi; Ross, Michael J.

    2016-01-01

    Sepsis related acute kidney injury (AKI) is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC) from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS), a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO). Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3) and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling. PMID:26990086

  16. Ectopic expression of RNF168 and 53BP1 increases mutagenic but not physiological non-homologous end joining

    PubMed Central

    Zong, Dali; Callén, Elsa; Pegoraro, Gianluca; Lukas, Claudia; Lukas, Jiri; Nussenzweig, André

    2015-01-01

    DNA double strand breaks (DSBs) formed during S phase are preferentially repaired by homologous recombination (HR), whereas G1 DSBs, such as those occurring during immunoglobulin class switch recombination (CSR), are repaired by non-homologous end joining (NHEJ). The DNA damage response proteins 53BP1 and BRCA1 regulate the balance between NHEJ and HR. 53BP1 promotes CSR in part by mediating synapsis of distal DNA ends, and in addition, inhibits 5’ end resection. BRCA1 antagonizes 53BP1 dependent DNA end-blocking activity during S phase, which would otherwise promote mutagenic NHEJ and genome instability. Recently, it was shown that supra-physiological levels of the E3 ubiquitin ligase RNF168 results in the hyper-accumulation of 53BP1/BRCA1 which accelerates DSB repair. Here, we ask whether increased expression of RNF168 or 53BP1 impacts physiological versus mutagenic NHEJ. We find that the anti-resection activities of 53BP1 are rate-limiting for mutagenic NHEJ but not for physiological CSR. As heterogeneity in the expression of RNF168 and 53BP1 is found in human tumors, our results suggest that deregulation of the RNF168/53BP1 pathway could alter the chemosensitivity of BRCA1 deficient tumors. PMID:25916843

  17. Increased expression of genes involved in uptake and degradation of murein tripeptide under nitrogen starvation in Escherichia coli.

    PubMed

    Choi, Umji; Park, Young-Ha; Kim, Yeon-Ran; Seok, Yeong-Jae; Lee, Chang-Ro

    2016-07-01

    Peptidoglycan (also known as murein) is an important envelope component of bacteria, and its turnover usually takes place at considerable levels during normal growth. Amino sugars and murein tripeptide resulting from murein degradation are used for resynthesis of peptidoglycan or as self-generated nutrients or energy sources for cell growth. PgrR (regulator of peptide glycan recycling; formerly YcjZ) was recently identified as a repressor of several genes participating in uptake and degradation of murein tripeptide. In this study, we identified the ycjG gene involved in murein tripeptide degradation as a new direct target of PgrR. The expression of PgrR-regulated genes including ycjY, mppA, mpaA and ycjG was repressed in the presence of a good nitrogen source, but their expression increased under poor nitrogen conditions. Under nitrogen starvation, the pgrR mutant cells exhibited faster growth than wild-type cells, implying that derepression of genes under the control of PgrR may help cells overcome nitrogen limitation. Therefore, these results suggest that nitrogen starvation induces derepression of PgrR-controlled genes involved in uptake and degradation of murein tripeptide, and this may stimulate the utilization of murein tripeptide as a nitrogen source. PMID:27231238

  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. Activation of D2 dopamine receptor-expressing neurons in the nucleus accumbens increases motivation.

    PubMed

    Soares-Cunha, Carina; Coimbra, Barbara; David-Pereira, Ana; Borges, Sonia; Pinto, Luisa; Costa, Patricio; Sousa, Nuno; Rodrigues, Ana J

    2016-01-01

    Striatal dopamine receptor D1-expressing neurons have been classically associated with positive reinforcement and reward, whereas D2 neurons are associated with negative reinforcement and aversion. Here we demonstrate that the pattern of activation of D1 and D2 neurons in the nucleus accumbens (NAc) predicts motivational drive, and that optogenetic activation of either neuronal population enhances motivation in mice. Using a different approach in rats, we further show that activating NAc D2 neurons increases cue-induced motivational drive in control animals and in a model that presents anhedonia and motivational deficits; conversely, optogenetic inhibition of D2 neurons decreases motivation. Our results suggest that the classic view of D1-D2 functional antagonism does not hold true for all dimensions of reward-related behaviours, and that D2 neurons may play a more prominent pro-motivation role than originally anticipated. PMID:27337658

  20. Activation of D2 dopamine receptor-expressing neurons in the nucleus accumbens increases motivation

    PubMed Central

    Soares-Cunha, Carina; Coimbra, Barbara; David-Pereira, Ana; Borges, Sonia; Pinto, Luisa; Costa, Patricio; Sousa, Nuno; Rodrigues, Ana J.

    2016-01-01

    Striatal dopamine receptor D1-expressing neurons have been classically associated with positive reinforcement and reward, whereas D2 neurons are associated with negative reinforcement and aversion. Here we demonstrate that the pattern of activation of D1 and D2 neurons in the nucleus accumbens (NAc) predicts motivational drive, and that optogenetic activation of either neuronal population enhances motivation in mice. Using a different approach in rats, we further show that activating NAc D2 neurons increases cue-induced motivational drive in control animals and in a model that presents anhedonia and motivational deficits; conversely, optogenetic inhibition of D2 neurons decreases motivation. Our results suggest that the classic view of D1–D2 functional antagonism does not hold true for all dimensions of reward-related behaviours, and that D2 neurons may play a more prominent pro-motivation role than originally anticipated. PMID:27337658

  1. BDNF over-expression increases olfactory bulb granule cell dendritic spine density in vivo.

    PubMed

    McDole, B; Isgor, C; Pare, C; Guthrie, K

    2015-09-24

    Olfactory bulb granule cells (GCs) are axon-less, inhibitory interneurons that regulate the activity of the excitatory output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses located on GC spines. These contacts are established in the distal apical dendritic compartment, while GC basal dendrites and more proximal apical segments bear spines that receive glutamatergic inputs from the olfactory cortices. This synaptic connectivity is vital to olfactory circuit function and is remodeled during development, and in response to changes in sensory activity and lifelong GC neurogenesis. Manipulations that alter levels of the neurotrophin brain-derived neurotrophic factor (BDNF) in vivo have significant effects on dendritic spine morphology, maintenance and activity-dependent plasticity for a variety of CNS neurons, yet little is known regarding BDNF effects on bulb GC spine maturation or maintenance. Here we show that, in vivo, sustained bulbar over-expression of BDNF in transgenic mice produces a marked increase in GC spine density that includes an increase in mature spines on their apical dendrites. Morphometric analysis demonstrated that changes in spine density were most notable in the distal and proximal apical domains, indicating that multiple excitatory inputs are potentially modified by BDNF. Our results indicate that increased levels of endogenous BDNF can promote the maturation and/or maintenance of dendritic spines on GCs, suggesting a role for this factor in modulating GC functional connectivity within adult olfactory circuitry. PMID:26211445

  2. Conditional Expression of the Androgen Receptor Increases Susceptibility of Bladder Cancer in Mice

    PubMed Central

    Luong, Richard; Yu, Eun-Jeong; He, Yongfeng; Gonzalgo, Mark L.; Sun, Zijie

    2016-01-01

    Bladder cancer represents a significant human tumor burden, accounting for about 7.7% and 2.4% of all cancer cases in males and females, respectively. While men have a higher risk of developing bladder cancer, women tend to present at a later stage of disease and with more aggressive tumors. Previous studies have suggested a promotional role of androgen signaling in enhancing bladder cancer development. To directly assess the role of androgens in bladder tumorigenesis, we have developed a novel transgenic mouse strain, R26hARLoxP/+:Upk3aGCE/+, in which the human AR transgene is conditionally expressed in bladder urothelium. Intriguingly, both male and female R26hARLoxP/+:Upk3aGCE/+ mice display a higher incidence of urothelial cell carcinoma (UCC) than the age and sex matched control littermates in response to the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). We detect expression of the human AR transgene in CK5-positive and p63-positive basal cells in bladder urothelium. Further analyses of UCC tissues from R26hARLoxP/+:Upk3aGCE/+ mice showed that the majority of tumor cells are of urothelial basal cell origin. Positive immunostaining of transgenic AR protein was observed in the majority of tumor cells of the transgenic mice, providing a link between transgenic AR expression and oncogenic transformation. We observed an increase in Ki67 positive cells within the UCC lesions of transgenic AR mice. Manipulating endogenous androgen levels by castration and androgen supplementation directly affected bladder tumor development in male and female R26hARLoxP/+:Upk3aGCE/+ mice, respectively. Taken together, our data demonstrate for the first time that conditional activation of transgenic AR expression in bladder urothelium enhances carciongen-induced bladder tumor formation in mice. This new AR transgenic mouse line mimics certain features of human bladder cancer and can be used to study bladder tumorigenesis and for drug development. PMID:26862755

  3. Conditional Expression of the Androgen Receptor Increases Susceptibility of Bladder Cancer in Mice.

    PubMed

    Johnson, Daniel T; Hooker, Erika; Luong, Richard; Yu, Eun-Jeong; He, Yongfeng; Gonzalgo, Mark L; Sun, Zijie

    2016-01-01

    Bladder cancer represents a significant human tumor burden, accounting for about 7.7% and 2.4% of all cancer cases in males and females, respectively. While men have a higher risk of developing bladder cancer, women tend to present at a later stage of disease and with more aggressive tumors. Previous studies have suggested a promotional role of androgen signaling in enhancing bladder cancer development. To directly assess the role of androgens in bladder tumorigenesis, we have developed a novel transgenic mouse strain, R26hARLoxP/+:Upk3aGCE/+, in which the human AR transgene is conditionally expressed in bladder urothelium. Intriguingly, both male and female R26hARLoxP/+:Upk3aGCE/+ mice display a higher incidence of urothelial cell carcinoma (UCC) than the age and sex matched control littermates in response to the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). We detect expression of the human AR transgene in CK5-positive and p63-positive basal cells in bladder urothelium. Further analyses of UCC tissues from R26hARLoxP/+:Upk3aGCE/+ mice showed that the majority of tumor cells are of urothelial basal cell origin. Positive immunostaining of transgenic AR protein was observed in the majority of tumor cells of the transgenic mice, providing a link between transgenic AR expression and oncogenic transformation. We observed an increase in Ki67 positive cells within the UCC lesions of transgenic AR mice. Manipulating endogenous androgen levels by castration and androgen supplementation directly affected bladder tumor development in male and female R26hARLoxP/+:Upk3aGCE/+ mice, respectively. Taken together, our data demonstrate for the first time that conditional activation of transgenic AR expression in bladder urothelium enhances carciongen-induced bladder tumor formation in mice. This new AR transgenic mouse line mimics certain features of human bladder cancer and can be used to study bladder tumorigenesis and for drug development. PMID:26862755

  4. The fibroblast growth factor receptor mediates the increased FGF23 expression in acute and chronic uremia.

    PubMed

    Hassan, Alia; Durlacher, Karina; Silver, Justin; Naveh-Many, Tally; Levi, Ronen

    2016-02-01

    Serum FGF23 is markedly elevated in chronic kidney disease and has been associated with poor long-term outcomes. FGF23 expression is increased by activation of the FGF receptor 1 (FGFR1) in rats with normal renal function and in vitro in bone-derived osteoblast-like cells. We studied the regulation of FGF23 by FGFR1 in vivo in acute and chronic uremia in mice and rats. Folic acid-induced acute kidney injury increased calvaria FGF23 mRNA and serum FGF23 and parathyroid hormone (PTH) levels at 6 h. The FGFR1 receptor inhibitor PD173074 prevented the folic acid-induced increase in both FGF23 mRNA and serum levels but had no effect on serum PTH levels. A more prolonged uremia due to an adenine high-phosphorus diet for 14 days resulted in high levels of FGF23 mRNA and serum FGF23 and PTH. PD173074 decreased serum FGF23 and mRNA levels with no effect on PTH in the adenine high phosphorus-induced uremic rats. Therefore, a derangement in FGF23 regulation starts early in the course of acute kidney injury, is in part independent of the increase in serum PTH, and involves activation of FGFR1. It is possible that FGFR1 in the osteocyte is activated by locally produced canonical FGFs, which are increased in uremia. This is the first demonstration that activation of FGFR1 is essential for the high levels of FGF23 in acute and chronic experimental uremia. PMID:26311115

  5. Zataria multiflora increases insulin sensitivity and PPARγ gene expression in high fructose fed insulin resistant rats

    PubMed Central

    Mohammadi, Abbas; Gholamhoseinian, Ahmad; Fallah, Hossein

    2014-01-01

    Objective(s): In insulin resistance, the insulin action in liver, muscles and adipocytes decreases and result in hyperglycemia, dyslipidemia and hyperinsulinemia. In this study we evaluate the effect of Zataria multiflora extract on insulin sensitivity in high fructose fed insulin resistant rats, since this extract was shown antihyperglycemic effect in streptozotocin induced diabetes in rats. Materials and Methods: Experimental rats were fed with high fructose diet for 6 weeks and then were treated with Z. multiflora extract or a pioglitazone solution for 2 weeks. Blood and tissue samples were collected for analysis at the end of two weeks. Blood glucose, serum level of triglyceride and cholesterol were measured by auto analyzer. Insulin and adiponectin levels were assayed by enzyme-linked immunosorbent assay (ELISA) method. Plasma free fatty acids profile was studied by gas chromatography. Peroxisome proliferator activated receptor (PPAR.γ) and Glucose transporter type 4 (GLUT.4) gene expressions were assessed by real time polymerase chain reaction (PCR) and western blotting. Results: Animals were treated by Z. multiflora extract showed insulin (43±11pmol/l), adiponectin (5.3±0.5 μg/ml), glucose (144±9.8 mg/dl), and triglyceride (120±10 mg/dl) levels significantly improved as compare with the control group [insulin (137±34 pmol/l), adiponectin (3.9±0.15 μg/ml), glucose (187±15mg/dl), and triglycerides (217±18 mg/dl)]. PPARγ protein level, also significantly increased in Zataria multiflora treated group. Conclusion: This study demonstrates the beneficial effects of Zataria multiflora extract on insulin resistance in rats fed with a high-fructose diet through at least three mechanisms including direct insulin like effect, increasing in adiponectin and of PPARγ protein expression. PMID:24904719

  6. Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice.

    PubMed

    Holland, Ruby A; Leonard, John J; Kensey, Nicholas A; Hannikainen, Paavali A; De Jonghe, Bart C

    2014-09-01

    Although rats and mice do not vomit, these species are widely studied as models of energy balance and sickness behavior. Previous work has shown that rats exhibit similar neuroanatomical activation of brain and visceral afferent pathways following cisplatin chemotherapy compared to vomiting species. However, the neural response to cisplatin in mice is understudied. Here, food intake, body weight, and central c-Fos immunofluorescence were analyzed in the hindbrains of male C57BL/6 mice following IP saline or cisplatin (5mg/kg, and 20mg/kg doses). As glutamate receptor signaling is classically linked to inhibitory feeding pathways in the rodent, gene expression of selected α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptor subunits were assessed in the dorsal vagal complex (DVC), parabrachial nucleus (PBN), amygdala, and bed nucleus of the stria terminalis (BNST). Our results show dose-dependent reductions in food intake and body weight following cisplatin treatment, as well as increases in cisplatin-induced c-Fos in the PBN and throughout the DVC. Quantitative PCR analysis shows cisplatin-induced increases in NMDA receptor subunit expression, particularly NR2B, in the DVC, PBN, BNST, and amygdala. In addition, upregulation of AMPA receptor subunits (GluA1 and/or GluA2) were observed in all regions examined except the amygdala. Taken together, these results suggest similar neural pathways mediating cisplatin effects in mice compared to other well-studied species, which are likely mediated by central upregulation of AMPA and NMDA receptors. PMID:24582677

  7. Milk-soluble formula increases food intake and reduces Il6 expression in elderly rat hypothalami.

    PubMed

    Ould Hamouda, Hassina; Delplanque, Bernadette; Benomar, Yacir; Crépin, Delphine; Riffault, Laure; LeRuyet, Pascale; Bonhomme, Cécile; Taouis, Mohammed

    2015-07-01

    Malnutrition in the elderly is accompanied by several metabolic dysfunctions, especially alterations in energy homeostasis regulation and a loss of insulin responsiveness. Nutritional recommendations aim to enrich food with high protein and energy supplements, and protein composition and lipid quality have been widely studied. Despite the numerous studies that have examined attempts to overcome malnutrition in the elderly through such nutritional supplementation, it is still necessary to study the effects of a combination of protein, lipids, and vitamin D (VitD). This can be done in animal models of elderly malnutrition. In the present study, we investigated the effects of several diet formulae on insulin responsiveness, inflammation, and the hypothalamic expression of key genes that are involved in energy homeostasis control. To mimic elderly malnutrition in humans, elderly Wistar rats were food restricted (R, -50%) for 12 weeks and then refed for 4 weeks with one of four different isocaloric diets: a control diet; a diet where milk soluble protein (MSP) replaced casein; a blend of milk fat, rapeseed, and DHA (MRD); or a full formula (FF) diet that combined MSP and a blend of MRD (FF). All of the refeeding diets contained VitD. We concluded that: (i) food restriction led to the upregulation of insulin receptor in liver and adipose tissue accompanied by increased Tnfα in the hypothalamus; (ii) in all of the refed groups, refeeding led to similar body weight gain during the refeeding period; and (iii) refeeding with MSP and MRD diets induced higher food intake on the fourth week of refeeding, and this increase was associated with reduced hypothalamic interleukin 6 expression. PMID:25994005

  8. Over-expression of a novel JAZ family gene from Glycine soja, increases salt and alkali stress tolerance

    SciTech Connect

    Zhu, Dan; Cai, Hua; Luo, Xiao; Bai, Xi; Deyholos, Michael K.; Chen, Qin; Chen, Chao; Ji, Wei; Zhu, Yanming

    2012-09-21

    Highlights: Black-Right-Pointing-Pointer We isolated and characterized a novel JAZ family gene, GsJAZ2, from Glycine soja. Black-Right-Pointing-Pointer Overexpression of GsJAZ2 enhanced plant tolerance to salt and alkali stress. Black-Right-Pointing-Pointer The transcriptions of stress marker genes were higher in GsJAZ2 overexpression lines. Black-Right-Pointing-Pointer GsJAZ2 was localized to nucleus. -- Abstract: Salt and alkali stress are two of the main environmental factors limiting crop production. Recent discoveries show that the JAZ family encodes plant-specific genes involved in jasmonate signaling. However, there is only limited information about this gene family in abiotic stress response, and in wild soybean (Glycine soja), which is a species noted for its tolerance to alkali and salinity. Here, we isolated and characterized a novel JAZ family gene, GsJAZ2, from G. soja. Transcript abundance of GsJAZ2 increased following exposure to salt, alkali, cold and drought. Over-expression of GsJAZ2 in Arabidopsis resulted in enhanced plant tolerance to salt and alkali stress. The expression levels of some alkali stress response and stress-inducible marker genes were significantly higher in the GsJAZ2 overexpression lines as compared to wild-type plants. Subcellular localization studies using a GFP fusion protein showed that GsJAZ2 was localized to the nucleus. These results suggest that the newly isolated wild soybean GsJAZ2 is a positive regulator of plant salt and alkali stress tolerance.

  9. A method for increasing expressivity of Gene Ontology annotations using a compositional approach

    PubMed Central

    2014-01-01

    Background The Gene Ontology project integrates data about the function of gene products across a diverse range of organisms, allowing the transfer of knowledge from model organisms to humans, and enabling computational analyses for interpretation of high-throughput experimental and clinical data. The core data structure is the annotation, an association between a gene product and a term from one of the three ontologies comprising the GO. Historically, it has not been possible to provide additional information about the context of a GO term, such as the target gene or the location of a molecular function. This has limited the specificity of knowledge that can be expressed by GO annotations. Results The GO Consortium has introduced annotation extensions that enable manually curated GO annotations to capture additional contextual details. Extensions represent effector–target relationships such as localization dependencies, substrates of protein modifiers and regulation targets of signaling pathways and transcription factors as well as spatial and temporal aspects of processes such as cell or tissue type or developmental stage. We describe the content and structure of annotation extensions, provide examples, and summarize the current usage of annotation extensions. Conclusions The additional contextual information captured by annotation extensions improves the utility of functional annotation by representing dependencies between annotations to terms in the different ontologies of GO, external ontologies, or an organism’s gene products. These enhanced annotations can also support sophisticated queries and reasoning, and will provide curated, directional links between many gene products to support pathway and network reconstruction. PMID:24885854

  10. A low-salt diet increases the expression of renal sirtuin 1 through activation of the ghrelin receptor in rats.

    PubMed

    Yang, Shao-Yu; Lin, Shuei-Liong; Chen, Yung-Ming; Wu, Vin-Cent; Yang, Wei-Shiung; Wu, Kwan-Dun

    2016-01-01

    Previous studies have shown that sirtuin 1 (Sirt1) is renoprotective; however, details regarding its distribution and functions in the kidney remain unknown. Here, we demonstrated that Sirt1 was mainly expressed in the tubulointerstitial cells of normal rat kidneys and was co-localized with aquaporin 2, indicating it may be involved in water/salt regulation. Renal Sirt1 expression increased in the non-glomerular cytoplasmic portion of the kidney after a 24-h fast, but no significant changes in Sirt1 expression occurred after water loading (50 mL/kg) or 24-h water deprivation. After consuming a low-salt (0.075%) or 60% calorie restriction diet for 7 days, Sirt1 expression in the rat kidney was significantly increased, whereas a high-salt (8%) diet did not change the level of Sirt1 expression. The low-salt diet also increased Sirt1 expression in the heart, muscle, brain, and fat tissues. The increased Sirt1 that was observed in rats on a low-salt diet was associated with increased ghrelin expression in the distal nephron, with both molecules exhibiting similar distribution patterns. An in vitro experiment suggested that ghrelin increases Sirt1 expression in cortical collecting duct cells by activating ghrelin receptors. Our study indicates that this 'ghrelin-Sirt1 system' may participate in regulating sodium reabsorption in the distal nephron. PMID:27600292

  11. A low-salt diet increases the expression of renal sirtuin 1 through activation of the ghrelin receptor in rats

    PubMed Central

    Yang, Shao-Yu; Lin, Shuei-Liong; Chen, Yung-Ming; Wu, Vin-Cent; Yang, Wei-Shiung; Wu, Kwan-Dun

    2016-01-01

    Previous studies have shown that sirtuin 1 (Sirt1) is renoprotective; however, details regarding its distribution and functions in the kidney remain unknown. Here, we demonstrated that Sirt1 was mainly expressed in the tubulointerstitial cells of normal rat kidneys and was co-localized with aquaporin 2, indicating it may be involved in water/salt regulation. Renal Sirt1 expression increased in the non-glomerular cytoplasmic portion of the kidney after a 24-h fast, but no significant changes in Sirt1 expression occurred after water loading (50 mL/kg) or 24-h water deprivation. After consuming a low-salt (0.075%) or 60% calorie restriction diet for 7 days, Sirt1 expression in the rat kidney was significantly increased, whereas a high-salt (8%) diet did not change the level of Sirt1 expression. The low-salt diet also increased Sirt1 expression in the heart, muscle, brain, and fat tissues. The increased Sirt1 that was observed in rats on a low-salt diet was associated with increased ghrelin expression in the distal nephron, with both molecules exhibiting similar distribution patterns. An in vitro experiment suggested that ghrelin increases Sirt1 expression in cortical collecting duct cells by activating ghrelin receptors. Our study indicates that this ‘ghrelin-Sirt1 system’ may participate in regulating sodium reabsorption in the distal nephron. PMID:27600292

  12. Type III secretion system expression in oxygen-limited Pseudomonas aeruginosa cultures is stimulated by isocitrate lyase activity

    PubMed Central

    Chung, Jade C. S.; Rzhepishevska, Olena; Ramstedt, Madeleine; Welch, Martin

    2013-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen and a common cause of chronic infections in individuals with cystic fibrosis (CF). Oxygen limitation was recently reported to regulate the expression of a major virulence determinant in P. aeruginosa, the type III secretion system (T3SS). Here, we show that expression of the T3SS in oxygen-limited growth conditions is strongly dependent on the glyoxylate shunt enzyme, isocitrate lyase (ICL; encoded by aceA), which was previously shown to be highly expressed in CF isolates. ICL-dependent regulation of the T3SS did not alter the expression level of the master transcriptional regulator, ExsA, but did affect expression of the T3 structural proteins, effectors and regulators (ExsC, ExsD and ExsE). An aceA mutant displayed enhanced biofilm formation during anaerobic growth, which suggested that AceA-dependent modulation of type III secretion might impinge upon the RetS/LadS signalling pathways. Indeed, our data suggest that RetS is able to mediate some of its effects through AceA, as expression of aceA in trans partially restored T3SS expression in a retS mutant. Our findings indicate that AceA is a key player in the metabolic regulation of T3SS expression during oxygen-limited growth of P. aeruginosa. To the best of our knowledge, this is the first demonstration that the T3SS can be regulated by factors that do not affect ExsA expression levels. PMID:23363478

  13. Cathinone increases body temperature, enhances locomotor activity, and induces striatal c-fos expression in the Siberian hamster.

    PubMed

    Jones, S; Fileccia, E L; Murphy, M; Fowler, M J; King, M V; Shortall, S E; Wigmore, P M; Green, A R; Fone, K C F; Ebling, F J P

    2014-01-24

    Cathinone is a β-keto alkaloid that is the major active constituent of khat, the leaf of the Catha edulis plant that is chewed recreationally in East Africa and the Middle East. Related compounds, such as methcathinone and mephedrone have been increasing in popularity as recreational drugs, resulting in the recent proposal to classify khat as a Class C drug in the UK. There is still limited knowledge of the pharmacological effects of cathinone. This study examined the acute effects of cathinone on core body temperature, locomotor and other behaviors, and neuronal activity in Siberian hamsters. Adult male hamsters, previously implanted with radio telemetry devices, were treated with cathinone (2 or 5mg/kg i.p.), the behavioral profile scored and core body temperature and locomotor activity recorded by radio telemetry. At the end of the study, hamsters received vehicle or cathinone (5mg/kg) and neuronal activation in the brain was determined using immunohistochemical evaluation of c-fos expression. Cathinone dose-dependently induced significant (p<0.0001) increases in both temperature and locomotor activity lasting 60-90min. Cathinone (2mg/kg) increased rearing (p<0.02), and 5mg/kg increased both rearing (p<0.001) and lateral head twitches (p<0.02). Both cathinone doses decreased the time spent at rest (p<0.001). The number of c-fos immunopositive cells were significantly increased in the striatum (p<0.0001) and suprachiasmatic nucleus (p<0.05) following cathinone, indicating increased neuronal activity. There was no effect of cathinone on food intake or body weight. It is concluded that systemic administration of cathinone induces significant behavioral changes and CNS activation in the hamster. PMID:24287379

  14. Claudin-4 expression in gastric cancer cells enhances the invasion and is associated with the increased level of matrix metalloproteinase-2 and -9 expression

    PubMed Central

    HWANG, TSANN-LONG; CHANGCHIEN, TZU-TSUNG; WANG, CHEE-CHAN; WU, CHI-MING

    2014-01-01

    Claudin-4 is a member of a large family of transmembrane proteins known as claudins, which are essential for the formation and maintenance of tight junctions. Our previous studies have revealed that claudin-4 proteins are overexpressed in metastatic gastric cancer. To clarify the roles of claudin-4 in gastric cancer metastasis, human gastric adenocarcinoma (AGS) cells constitutively expressing wild-type claudin-4 were generated. Expression of claudin-4 in AGS cells was found to increase cell invasion and migration, as measured by Boyden invasion chamber assays. Moreover, the claudin-4-expressing AGS cells were found to have increased matrix metalloproteinase (MMP)-2 and -9 expression, indicating that claudin-mediated increased invasion may be mediated through the activation of the MMP protein. Overall, the results suggest that claudin-4 overexpression may promote gastric cancer metastasis through the increased invasion of gastric cancer cells. PMID:25120725

  15. Changes in granulosa cells' gene expression associated with increased oocyte competence in bovine.

    PubMed

    Nivet, Anne-Laure; Vigneault, Christian; Blondin, Patrick; Sirard, Marc-André

    2013-06-01

    One of the challenges in mammalian reproduction is to understand the basic physiology of oocyte quality. It is believed that the follicle status is linked to developmental competence of the enclosed oocyte. To explore the link between follicles and competence in cows, previous research at our laboratory has developed an ovarian stimulation protocol that increases and then decreases oocyte quality according to the timing of oocyte recovery post-FSH withdrawal (coasting). Using this protocol, we have obtained the granulosa cells associated with oocytes of different qualities at selected times of coasting. Transcriptome analysis was done with Embryogene microarray slides and validation was performed by real-time PCR. Results show that the major changes in gene expression occurred from 20 to 44  h of coasting, when oocyte quality increases. Secondly, among upregulated genes (20-44  h), 25% were extracellular molecules, highlighting potential granulosa signaling cascades. Principal component analysis identified two patterns: one resembling the competence profile and another associated with follicle growth and atresia. Additionally, three major functional changes were identified: (i) the end of follicle growth (BMPR1B, IGF2, and RELN), involving interactions with the extracellular matrix (TFPI2); angiogenesis (NRP1), including early hypoxia, and potentially oxidative stress (GFPT2, TF, and VNN1) and (ii) apoptosis (KCNJ8) followed by iii) inflammation (ANKRD1). This unique window of analysis indicates a progressive hypoxia during coasting mixed with an increase in apoptosis and inflammation. Potential signaling pathways leading to competence have been identified and will require downstream testing. This preliminary analysis supports the potential role of the follicular differentiation in oocyte quality both during competence increase and decrease phases. PMID:23564726

  16. Increased oxidative metabolism and myoglobin expression in zebrafish muscle during chronic hypoxia

    PubMed Central

    Jaspers, Richard T.; Testerink, Janwillem; Della Gaspera, Bruno; Chanoine, Christophe; Bagowski, Christophe P.; van der Laarse, Willem J.

    2014-01-01

    ABSTRACT Fish may be extremely hypoxia resistant. We investigated how muscle fibre size and oxidative capacity in zebrafish (Danio rerio) adapt during severe chronic hypoxia. Zebrafish were kept for either 3 or 6 weeks under chronic constant hypoxia (CCH) (10% air/90%N2 saturated water). We analyzed cross-sectional area (CSA), succinate dehydrogenase (SDH) activity, capillarization, myonuclear density, myoglobin (Mb) concentration and Mb mRNA expression of high and low oxidative muscle fibres. After 3 weeks of CCH, CSA, SDH activity, Mb concentration, capillary and myonuclear density of both muscle fibre types were similar as under normoxia. In contrast, staining intensity for Mb mRNA of hypoxic high oxidative muscle fibres was 94% higher than that of normoxic controls (P<0.001). Between 3 and 6 weeks of CCH, CSA of high and low oxidative muscle fibres increased by 25 and 30%, respectively. This was similar to normoxic controls. Capillary and myonuclear density were not changed by CCH. However, in high oxidative muscle fibres of fish maintained under CCH, SDH activity, Mb concentration as well as Mb mRNA content were higher by 86%, 138% and 90%, respectively, than in muscle fibres of fish kept under normoxia (P<0.001). In low oxidative muscle fibres, SDH activity, Mb and Mb mRNA content were not significantly changed. Under normoxia, the calculated interstitial oxygen tension required to prevent anoxic cores in muscle fibres (PO2crit) of high oxidative muscle fibres was between 1.0 and 1.7 mmHg. These values were similar at 3 and 6 weeks CCH. We conclude that high oxidative skeletal muscle fibres of zebrafish continue to grow and increase oxidative capacity during CCH. Oxygen supply to mitochondria in these fibres may be facilitated by an increased Mb concentration, which is regulated by an increase in Mb mRNA content per myonucleus. PMID:25063194

  17. Adaptation options for wheat in Europe will be limited by increased adverse weather events under climate change.

    PubMed

    Trnka, Miroslav; Hlavinka, Petr; Semenov, Mikhail A

    2015-11-01

    Ways of increasing the production of wheat, the most widely grown cereal crop, will need to be found to meet the increasing demand caused by human population growth in the coming decades. This increase must occur despite the decrease in yield gains now being reported in some regions, increased price volatility and the expected increase in the frequency of adverse weather events that can reduce yields. However, if and how the frequency of adverse weather events will change over Europe, the most important wheat-growing area, has not yet been analysed. Here, we show that the accumulated probability of 11 adverse weather events with the potential to significantly reduce yield will increase markedly across all of Europe. We found that by the end of the century, the exposure of the key European wheat-growing areas, where most wheat production is currently concentrated, may increase more than twofold. However, if we consider the entire arable land area of Europe, a greater than threefold increase in risk was predicted. Therefore, shifting wheat production to new producing regions to reduce the risk might not be possible as the risk of adverse events beyond the key wheat-growing areas increases even more. Furthermore, we found a marked increase in wheat exposure to high temperatures, severe droughts and field inaccessibility compared with other types of adverse events. Our results also showed the limitations of some of the presently debated adaptation options and demonstrated the need for development of region-specific strategies. Other regions of the world could be affected by adverse weather events in the future in a way different from that considered here for Europe. This observation emphasizes the importance of conducting similar analyses for other major wheat regions. PMID:26577595

  18. Adaptation options for wheat in Europe will be limited by increased adverse weather events under climate change

    PubMed Central

    Trnka, Miroslav; Hlavinka, Petr; Semenov, Mikhail A.

    2015-01-01

    Ways of increasing the production of wheat, the most widely grown cereal crop, will need to be found to meet the increasing demand caused by human population growth in the coming decades. This increase must occur despite the decrease in yield gains now being reported in some regions, increased price volatility and the expected increase in the frequency of adverse weather events that can reduce yields. However, if and how the frequency of adverse weather events will change over Europe, the most important wheat-growing area, has not yet been analysed. Here, we show that the accumulated probability of 11 adverse weather events with the potential to significantly reduce yield will increase markedly across all of Europe. We found that by the end of the century, the exposure of the key European wheat-growing areas, where most wheat production is currently concentrated, may increase more than twofold. However, if we consider the entire arable land area of Europe, a greater than threefold increase in risk was predicted. Therefore, shifting wheat production to new producing regions to reduce the risk might not be possible as the risk of adverse events beyond the key wheat-growing areas increases even more. Furthermore, we found a marked increase in wheat exposure to high temperatures, severe droughts and field inaccessibility compared with other types of adverse events. Our results also showed the limitations of some of the presently debated adaptation options and demonstrated the need for development of region-specific strategies. Other regions of the world could be affected by adverse weather events in the future in a way different from that considered here for Europe. This observation emphasizes the importance of conducting similar analyses for other major wheat regions. PMID:26577595

  19. Increasing nitrogen limitation in the Bothnian Sea, potentially caused by inflow of phosphate-rich water from the Baltic Proper.

    PubMed

    Rolff, Carl; Elfwing, Tina

    2015-11-01

    The study showed that the open water of the Bothnian Sea (BS) is likely to have shifted from altering nitrogen and phosphorous limitations of the spring bloom to more nitrogen-limited conditions during the last 20 years. This is affected by the by inflow of phosphate-rich and oxygen-depleted water from depths near the halocline in the northern Baltic Proper, where severe oxygen conditions currently cause extreme phosphate concentrations in the deep water. The change in relation between inorganic nitrogen and phosphorous in the BS occurs first in the deep water and then progresses to the surface water. The change can potentially cause increased production in the BS and more frequent cyanobacterial blooms. There does not appear to be any immediate concern in the short-term perspective for the state of the BS, but a progression of the processes may lead to a more eutrophic state of the BS. PMID:25990584

  20. INCREASED LIVER PATHOLOGY IN HEPATITIS C VIRUS TRANSGENIC MICE EXPRESSING THE HEPATITIS B VIRUS X PROTEIN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was id...

  1. Zinc deficiency increases miR-34a expression in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    miRNAs play a significant role in the regulation of gene expression in a plethora of eukaryotic systems by binding to complementary regions of specific target mRNAs. To explore the possible changes of miRNA expression induced by zinc deficiency, we compared the global miRNA expression in small intes...

  2. Zaprinast impairs spatial memory by increasing PDE5 expression in the rat hippocampus.

    PubMed

    Giorgi, Mauro; Pompili, Assunta; Cardarelli, Silvia; Castelli, Valentina; Biagioni, Stefano; Sancesario, Giuseppe; Gasbarri, Antonella

    2015-02-01

    In this work, we report the effect of post-training intraperitoneal administration of zaprinast on rat memory retention in the Morris water maze task that revealed a significant memory impairment at the intermediate dose of 10mg/kg. Zaprinast is capable of inhibiting both striatal and hippocampal PDE activity but to a different extent which is probably due to the different PDE isoforms expressed in these areas. To assess the possible involvement of cyclic nucleotides in rat memory impairment, we compared the effects obtained 30 min after the zaprinast injection with respect to 24h after injection by measuring both cyclic nucleotide levels and PDE activity. As expected, 30 min after the zaprinast administration, we observed an increase of cyclic nucleotides, which returned to a basal level within 24h, with the exception of the hippocampal cGMP which was significantly decreased at the dose of 10mg/kg of zaprinast. This increase in the hippocampal region is the result of a cGMP-specific PDE5 induction, confirmed by sildenafil inhibition, in agreement with literature data that demonstrate transcriptional regulation of PDE5 by cAMP/cGMP intracellular levels. Our results highlight the possible rebound effect of PDE inhibitors. PMID:25281278

  3. Increased Fos Expression Among Midbrain Dopaminergic Cell Groups during Birdsong Tutoring

    PubMed Central

    Nordeen, E.J.; Holtzman, D.A.; Nordeen, K.W.

    2009-01-01

    During avian vocal learning, birds memorize conspecific song patterns and then use auditory feedback to match their vocal output to this acquired template. Some models of song learning posit that during tutoring, conspecific visual, social, and/or auditory cues activate neuromodulatory systems that encourage acquisition of the tutor’s song and attach incentive value to that specific acoustic pattern. This hypothesis predicts that stimuli experienced during social tutoring activate cell populations capable of signaling reward. Using immunocytochemistry for the protein product of the immediate early gene c-Fos, we found that brief exposure of juvenile male zebra finches to a live familiar male tutor increased the density of Fos+ cells within two brain regions implicated in reward processing; the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). This activation of Fos appears to involve both dopaminergic and non-dopaminergic VTA/SNc neurons. Intriguingly, a familiar tutor was more effective than a novel tutor in stimulating Fos expression within these regions. In the periaqueductal gray (PAG), a dopamine-enriched cell population that has been implicated in emotional processing, Fos labeling also was increased after tutoring, with a familiar tutor once again being more effective than a novel conspecific. Since several neural regions implicated in song acquisition receive strong dopaminergic projections from these midbrain nuclei, their activation in conjunction with hearing the tutor’s song could help establish sensory representations that later guide motor sequence learning. PMID:19686474

  4. Survivin expression increases during aging and enhances the resistance of aged human fibroblasts to genotoxic stress.

    PubMed

    Al-Khalaf, Huda H; Aboussekhra, Abdelilah

    2013-06-01

    Survivin, an important anti-apoptotic protein, is highly expressed in most cancers, which generally arise in cells of older individuals. We have shown here accumulation of survivin and phospho-survivin in aged normal human skin fibroblasts and mice organs. This age-related accumulation of survivin was due to protein stabilization through association with the molecular chaperone Hsp90 protein, which was also up-regulated during aging. Interestingly, Hsp90 binds preferentially to phospho-survivin, which explains its higher stability. In addition, we provide clear evidence that aged cells exhibit apoptosis resistance when challenged with UV light, cisplatin, γ-rays or H2O2 as compared to their younger counterparts. In response to γ-rays and H2O2, the levels of Bcl-2 and both forms of survivin were up-regulated in old cells, but not in their corresponding young ones. This repression of survivin and phospho-survivin in young cells is p53 dependent. Importantly, survivin inhibition/down-regulation with flavopiridol or specific shRNAs increased the apoptotic response of old fibroblasts to various genotoxic agents, and restored the pro-apoptotic Bax/Bcl2 ratio and the increase in the levels of cleaved caspase-3 and PARP in old cells. These results show the role of survivin in the age-dependent resistance of human fibroblasts, and provide new insights into the molecular mechanisms that underlie the complex relationship between aging, apoptosis, and cancer. PMID:22252435

  5. Increased Expression of Alpha-Synuclein Reduces Neurotransmitter Release by Inhibiting Synaptic Vesicle Reclustering After Endocytosis

    PubMed Central

    Nemani, Venu M.; Lu, Wei; Berge, Victoria; Nakamura, Ken; Onoa, Bibiana; Lee, Michael K.; Chaudhry, Farrukh A.; Nicoll, Roger A.; Edwards, Robert H.

    2011-01-01

    Summary The protein α-synuclein accumulates in the brain of patients with sporadic Parkinson’s disease (PD), and increased gene dosage causes a severe, dominantly inherited form of PD, but we know little about the effects of synuclein that precede degeneration. α-Synuclein localizes to the nerve terminal, but the knockout has little if any effect on synaptic transmission. In contrast, we now find that the modest over-expression of α-synuclein, in the range predicted for gene multiplication and in the absence of overt toxicity, markedly inhibits neurotransmitter release. The mechanism, elucidated by direct imaging of the synaptic vesicle cycle, involves a specific reduction in size of the synaptic vesicle recycling pool. Ultrastructural analysis demonstrates reduced synaptic vesicle density at the active zone, and imaging further reveals a defect in the reclustering of synaptic vesicles after endocytosis. Increased levels of α-synuclein thus produce a specific, physiological defect in synaptic vesicle recycling that precedes detectable neuropathology. PMID:20152114

  6. Neurotoxic Methamphetamine Doses Increase LINE-1 Expression in the Neurogenic Zones of the Adult Rat Brain

    PubMed Central

    Moszczynska, Anna; Flack, Amanda; Qiu, Ping; Muotri, Alysson R.; Killinger, Bryan A.

    2015-01-01

    Methamphetamine (METH) is a widely abused psychostimulant with the potential to cause neurotoxicity in the striatum and hippocampus. Several epigenetic changes have been described after administration of METH; however, there are no data regarding the effects of METH on the activity of transposable elements in the adult brain. The present study demonstrates that systemic administration of neurotoxic METH doses increases the activity of Long INterspersed Element (LINE-1) in two neurogenic niches in the adult rat brain in a promoter hypomethylation-independent manner. Our study also demonstrates that neurotoxic METH triggers persistent decreases in LINE-1 expression and increases the LINE-1 levels within genomic DNA in the striatum and dentate gyrus of the hippocampus, and that METH triggers LINE-1 retrotransposition in vitro. We also present indirect evidence for the involvement of glutamate (GLU) in LINE-1 activation. The results suggest that LINE-1 activation might occur in neurogenic areas in human METH users and might contribute to METH abuse-induced hippocampus-dependent memory deficits and impaired performance on several cognitive tasks mediated by the striatum. PMID:26463126

  7. Cecal ligation and puncture sepsis is associated with attenuated expression of adenylyl cyclase 9 and increased miR142-3p.

    PubMed

    Risøe, Petter K; Ryg, Una; Wang, Yun Yong; Rutkovskiy, Arkady; Smedsrød, Bård; Valen, Guro; Dahle, Maria K

    2011-10-01

    The host inflammatory response in sepsis may be resolved by endogenous anti-inflammatory immune cell responses, avoiding fatal pathogenesis, organ injury, and death. The intracellular signaling mediator cyclic 3'5'-adenosine monophosphate is a potent modulator of inflammatory responses and initiates the polarization of immune cells in a direction that suppresses inflammatory activation. Cyclic 3'5'-adenosine monophosphate is enzymatically produced by adenylyl cyclases (ACs). The expression of ACs is previously shown to be reduced in rat organs after in vivo endotoxemia, concurrent with the progressing systemic inflammation. In the present study, tissue AC gene expression and regulation are explored in a rat model of cecal ligation and puncture (CLP) sepsis. Eighteen hours after CLP operation, expression of several AC isoforms in the liver, spleen, and kidney was reduced, significantly so for AC9 in all tissues. AC9 expression is regulated by the microRNA miR142-3p in T cells. When microRNA was extracted and amplified for miR142-3p expression, it was increasingly expressed 18 h after CLP. A correlation between increased miR142-3p and decreased AC9 expression was found in the liver, kidney, and spleen, and when hepatocytes, Kupffer cells (KCs), and liver sinusoidal endothelial cells were isolated after CLP, reduced AC expression and increased miR142-3p expression were found in KCs and liver sinusoidal endothelial cells. Transfecting a miR142-3p inhibitor probe in rat KCs abolished LPS-mediated AC9 inhibition in vitro. These results indicate that CLP leads to miR142-3p-mediated AC9 reduction in liver macrophages, which may further limit cyclic 3'5'-adenosine monophosphate signaling and the ability of macrophages to resolve the proinflammatory response. PMID:21701418

  8. An Individual-Based Diploid Model Predicts Limited Conditions Under Which Stochastic Gene Expression Becomes Advantageous

    PubMed Central

    Matsumoto, Tomotaka; Mineta, Katsuhiko; Osada, Naoki; Araki, Hitoshi

    2015-01-01

    Recent studies suggest the existence of a stochasticity in gene expression (SGE) in many organisms, and its non-negligible effect on their phenotype and fitness. To date, however, how SGE affects the key parameters of population genetics are not well understood. SGE can increase the phenotypic variation and act as a load for individuals, if they are at the adaptive optimum in a stable environment. On the other hand, part of the phenotypic variation caused by SGE might become advantageous if individuals at the adaptive optimum become genetically less-adaptive, for example due to an environmental change. Furthermore, SGE of unimportant genes might have little or no fitness consequences. Thus, SGE can be advantageous, disadvantageous, or selectively neutral depending on its context. In addition, there might be a genetic basis that regulates magnitude of SGE, which is often referred to as “modifier genes,” but little is known about the conditions under which such an SGE-modifier gene evolves. In the present study, we conducted individual-based computer simulations to examine these conditions in a diploid model. In the simulations, we considered a single locus that determines organismal fitness for simplicity, and that SGE on the locus creates fitness variation in a stochastic manner. We also considered another locus that modifies the magnitude of SGE. Our results suggested that SGE was always deleterious in stable environments and increased the fixation probability of deleterious mutations in this model. Even under frequently changing environmental conditions, only very strong natural selection made SGE adaptive. These results suggest that the evolution of SGE-modifier genes requires strict balance among the strength of natural selection, magnitude of SGE, and frequency of environmental changes. However, the degree of dominance affected the condition under which SGE becomes advantageous, indicating a better opportunity for the evolution of SGE in different genetic

  9. Inefficient assembly limits transport and cell surface expression of HLA-Cw4 molecules in C1R.

    PubMed

    Zemmour, J

    1996-12-01

    HLA-C antigens are expressed to the cell surface at roughly 10% the level of HLA-B or -A, and their serological definition remains persistently difficult. To characterize the factors limiting surface expression, the processes of assembly and intracellular transport of HLA-Cw4 molecules were investigated in the C1R cell line. When appropriate peptides were added to cultured cells or in cell lysates significant amounts of conformed HLA-C molecules that associate with beta 2-microglobulin (beta 2 m) are detected, but are indeed not sufficient to restore expression to the level observed for HLA-A or -B molecules. Furthermore, a precursor/product relationship exists between the free class I heavy chain and the mature conformation of HLA-Cw4 molecules. Thus, HLA-C assembly promotes the conversion of HC-10-reactive molecules (weakly-beta 2m-associated non-ligand associated free HC form) into the beta 2m-associated class I molecules recognized by W6/32. To further investigate the factors that regulate cell surface expression, intracellular transport of HLA-Cw4 was studied in pulse chase analysis. In contrast to some HLA-A and B, maturation of HLA-Cw4 heavy chains and their export to the medial and trans-Golgi compartments are quite inefficient. After 4 h of chase period, roughly half of the pulse-labeled HLA-Cw4 molecules have transited to the medial-Golgi and acquired complex oligosaccharides characteristic of mature form. In addition, treatment with gamma-interferon does not appear to improve maturation of HLA-Cw4 heavy chains, suggesting that increased supply of peptides does not influence intracellular transport. Moreover, only a small fraction in the pool of HLA-Cw4 molecules was subsequently transported through the trans-Golgi network, as indicated by their acquisition of sialic acids. Taken together these studies show that HLA-Cw4 molecules are inefficiently transported through the Golgi apparatus and presumably retained in the endoplasmic reticulum or cis

  10. Filamin-A Increases the Stability and Plasma Membrane Expression of Polycystin-2

    PubMed Central

    Wang, Qian; Zheng, Wang; Wang, Zuocheng; Yang, JungWoo; Hussein, Shaimaa; Tang, Jingfeng; Chen, Xing-Zhen

    2015-01-01

    Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in ~15% of autosomal dominant polycystic kidney disease. Filamins are actin-binding proteins implicated in scaffolding and membrane stabilization. Here we studied the effects of filamin on PC2 stability using filamin-deficient human melanoma M2, filamin-A (FLNA)-replete A7, HEK293 and IMCD cells together with FLNA siRNA/shRNA knockdown (KD). We found that the presence of FLNA is associated with higher total and plasma membrane PC2 protein expression. Western blotting analysis in combination with FLNA KD showed that FLNA in A7 cells represses PC2 degradation, prolonging the half-life from 2.3 to 4.4 hours. By co-immunoprecipitation and Far Western blotting we found that the FLNA C-terminus (FLNAC) reduces the FLNA-PC2 binding and PC2 expression, presumably through competing with FLNA for binding PC2. We further found that FLNA mediates PC2 binding with actin through forming complex PC2-FLNA-actin. FLNAC acted as a blocking peptide and disrupted the link of PC2 with actin through disrupting the PC2-FLNA-actin complex. Finally, we demonstrated that the physical interaction of PC2-FLNA is Ca-dependent. Taken together, our current study indicates that FLNA anchors PC2 to the actin cytoskeleton through complex PC2-FLNA-actin to reduce degradation and increase stability, and possibly regulate PC2 function in a Ca-dependent manner. PMID:25861040

  11. Filamin-a increases the stability and plasma membrane expression of polycystin-2.

    PubMed

    Wang, Qian; Zheng, Wang; Wang, Zuocheng; Yang, JungWoo; Hussein, Shaimaa; Tang, Jingfeng; Chen, Xing-Zhen

    2015-01-01

    Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in ~15% of autosomal dominant polycystic kidney disease. Filamins are actin-binding proteins implicated in scaffolding and membrane stabilization. Here we studied the effects of filamin on PC2 stability using filamin-deficient human melanoma M2, filamin-A (FLNA)-replete A7, HEK293 and IMCD cells together with FLNA siRNA/shRNA knockdown (KD). We found that the presence of FLNA is associated with higher total and plasma membrane PC2 protein expression. Western blotting analysis in combination with FLNA KD showed that FLNA in A7 cells represses PC2 degradation, prolonging the half-life from 2.3 to 4.4 hours. By co-immunoprecipitation and Far Western blotting we found that the FLNA C-terminus (FLNAC) reduces the FLNA-PC2 binding and PC2 expression, presumably through competing with FLNA for binding PC2. We further found that FLNA mediates PC2 binding with actin through forming complex PC2-FLNA-actin. FLNAC acted as a blocking peptide and disrupted the link of PC2 with actin through disrupting the PC2-FLNA-actin complex. Finally, we demonstrated that the physical interaction of PC2-FLNA is Ca-dependent. Taken together, our current study indicates that FLNA anchors PC2 to the actin cytoskeleton through complex PC2-FLNA-actin to reduce degradation and increase stability, and possibly regulate PC2 function in a Ca-dependent manner. PMID:25861040

  12. Metformin-mediated increase in DICER1 regulates microRNA expression and cellular senescence.

    PubMed

    Noren Hooten, Nicole; Martin-Montalvo, Alejandro; Dluzen, Douglas F; Zhang, Yongqing; Bernier, Michel; Zonderman, Alan B; Becker, Kevin G; Gorospe, Myriam; de Cabo, Rafael; Evans, Michele K

    2016-06-01

    Metformin, an oral hypoglycemic agent, has been used for decades to treat type 2 diabetes mellitus. Recent studies indicate that mice treated with metformin live longer and have fewer manifestations of age-related chronic disease. However, the molecular mechanisms underlying this phenotype are unknown. Here, we show that metformin treatment increases the levels of the microRNA-processing protein DICER1 in mice and in humans with diabetes mellitus. Our results indicate that metformin upregulates DICER1 through a post-transcriptional mechanism involving the RNA-binding protein AUF1. Treatment with metformin altered the subcellular localization of AUF1, disrupting its interaction with DICER1 mRNA and rendering DICER1 mRNA stable, allowing DICER1 to accumulate. Consistent with the role of DICER1 in the biogenesis of microRNAs, we found differential patterns of microRNA expression in mice treated with metformin or caloric restriction, two proven life-extending interventions. Interestingly, several microRNAs previously associated with senescence and aging, including miR-20a, miR-34a, miR-130a, miR-106b, miR-125, and let-7c, were found elevated. In agreement with these findings, treatment with metformin decreased cellular senescence in several senescence models in a DICER1-dependent manner. Metformin lowered p16 and p21 protein levels and the abundance of inflammatory cytokines and oncogenes that are hallmarks of the senescence-associated secretory phenotype (SASP). These data lead us to hypothesize that changes in DICER1 levels may be important for organismal aging and to propose that interventions that upregulate DICER1 expression (e.g., metformin) may offer new pharmacotherapeutic approaches for age-related disease. PMID:26990999

  13. Hypoxic culture conditions induce increased metabolic rate and collagen gene expression in ACL-derived cells.

    PubMed

    Kowalski, Tomasz J; Leong, Natalie L; Dar, Ayelet; Wu, Ling; Kabir, Nima; Khan, Adam Z; Eliasberg, Claire D; Pedron, Andrew; Karayan, Ashant; Lee, Siyoung; Di Pauli von Treuheim, Theodor; Jiacheng, Jin; Wu, Ben M; Evseenko, Denis; McAllister, David R; Petrigliano, Frank A

    2016-06-01

    There has been substantial effort directed toward the application of bone marrow and adipose-derived mesenchymal stromal cells (MSCs) in the regeneration of musculoskeletal tissue. Recently, resident tissue-specific stem cells have been described in a variety of mesenchymal structures including ligament, tendon, muscle, cartilage, and bone. In the current study, we systematically characterize three novel anterior cruciate ligament (ACL)-derived cell populations with the potential for ligament regeneration: ligament-forming fibroblasts (LFF: CD146(neg) , CD34(neg) CD44(pos) , CD31(neg) , CD45(neg) ), ligament perivascular cells (LPC: CD146(pos) CD34(neg) CD44(pos) , CD31(neg) , CD45(neg) ) and ligament interstitial cells (LIC: CD34(pos) CD146(neg) , CD44(pos) , CD31(neg) , CD45(neg) )-and describe their proliferative and differentiation potential, collagen gene expression and metabolism in both normoxic and hypoxic environments, and their trophic potential in vitro. All three groups of cells (LIC, LPC, and LFF) isolated from adult human ACL exhibited progenitor cell characteristics with regard to proliferation and differentiation potential in vitro. Culture in low oxygen tension enhanced the collagen I and III gene expression in LICs (by 2.8- and 3.3-fold, respectively) and LFFs (by 3- and 3.5-fold, respectively) and increased oxygen consumption rate and extracellular acidification rate in LICs (by 4- and 3.5-fold, respectively), LFFs (by 5.5- and 3-fold, respectively), LPCs (by 10- and 4.5-fold, respectively) as compared to normal oxygen concentration. In summary, this study demonstrates for the first time the presence of three novel progenitor cell populations in the adult ACL that demonstrate robust proliferative and matrix synthetic capacity; these cells may play a role in local ligament regeneration, and consequently represent a potential cell source for ligament engineering applications. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc

  14. Risk of Anterior Cruciate Ligament Fatigue Failure Is Increased by Limited Internal Femoral Rotation During In Vitro Repeated Pivot Landings

    PubMed Central

    Beaulieu, Mélanie L.; Wojtys, Edward M.; Ashton-Miller, James A.

    2015-01-01

    Background A reduced range of hip internal rotation is associated with increased peak anterior cruciate ligament (ACL) strain and risk for injury. It is unknown, however, whether limiting the available range of internal femoral rotation increases the susceptibility of the ACL to fatigue failure. Hypothesis Risk of ACL failure is significantly greater in female knee specimens with a limited range of internal femoral rotation, smaller femoral-ACL attachment angle, and smaller tibial eminence volume during repeated in vitro simulated single-leg pivot landings. Study Design Controlled laboratory study. Methods A custom-built testing apparatus was used to simulate repeated single-leg pivot landings with a 4×-body weight impulsive load that induces knee compression, knee flexion, and internal tibial torque in 32 paired human knee specimens from 8 male and 8 female donors. These test loads were applied to each pair of specimens, in one knee with limited internal femoral rotation and in the contralateral knee with femoral rotation resisted by 2 springs to simulate the active hip rotator muscles’ resistance to stretch. The landings were repeated until ACL failure occurred or until a minimum of 100 trials were executed. The angle at which the ACL originates from the femur and the tibial eminence volume were measured on magnetic resonance images. Results The final Cox regression model (P = .024) revealed that range of internal femoral rotation and sex of donor were significant factors in determining risk of ACL fatigue failure. The specimens with limited range of internal femoral rotation had a failure risk 17.1 times higher than did the specimens with free rotation (P = .016). The female knee specimens had a risk of ACL failure 26.9 times higher than the male specimens (P = .055). Conclusion Limiting the range of internal femoral rotation during repetitive pivot landings increases the risk of an ACL fatigue failure in comparison with free rotation in a cadaveric model

  15. Increased expression and activity of nuclear cathepsin L in cancer cells suggests a novel mechanism of cell transformation.

    PubMed

    Goulet, Brigitte; Sansregret, Laurent; Leduy, Lam; Bogyo, Matthew; Weber, Ekkehard; Chauhan, Shyam S; Nepveu, Alain

    2007-09-01

    It is generally accepted that the role of cathepsin L in cancer involves its activities outside the cells once it has been secreted. However, cathepsin L isoforms that are devoid of a signal peptide were recently shown to be present in the nucleus where they proteolytically process the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor. A role for nuclear cathepsin L in cell proliferation could be inferred from the observation that the CDP/Cux processed isoform can accelerate entry into S phase. Here, we report that in many transformed cells the proteolytic processing of CDP/Cux is augmented and correlates with increased cysteine protease expression and activity in the nucleus. Taking advantage of an antibody that recognizes the prodomain of human cathepsin L, we showed that human cells express short cathepsin L species that do not contain a signal peptide, do not transit through the endoplasmic reticulum, are not glycosylated, and localize to the nucleus. We also showed that transformation by the ras oncogene causes rapid increases both in the production of short nuclear cathepsin L isoforms and in the processing of CDP/Cux. Using a cell-based assay, we showed that a cell-permeable inhibitor of cysteine proteases is able to delay the progression into S phase and the proliferation in soft agar of ras-transformed cells, whereas the non-cell-permeable inhibitor had no effect. Taken together, these results suggest that the role of cathepsin L in cancer might not be limited to its extracellular activities but may also involve its processing function in the nucleus. PMID:17855659

  16. Role of NF-kappaB-dependent caveolin-1 expression in the mechanism of increased endothelial permeability induced by lipopolysaccharide.

    PubMed

    Tiruppathi, Chinnaswamy; Shimizu, Jun; Miyawaki-Shimizu, Kayo; Vogel, Stephen M; Bair, Angela M; Minshall, Richard D; Predescu, Dan; Malik, Asrar B

    2008-02-15

    We investigated the role of NF-kappaB activation by the bacterial product lipopolysaccharide (LPS) in inducing caveolin-1 (Cav-1) expression and its consequence in contributing to the leakiness of the endothelial barrier. We observed that LPS challenge of human lung microvascular endothelial cells induced concentration- and time-dependent increases in expression of Cav-1 mRNA and protein. The NEMO (NF-kappaB essential modifier binding domain)-binding domain peptide (IkB kinase (IKK)-NEMO-binding domain (NBD) peptide), which prevents NF-kappaB activation by inhibiting the interaction of IKKgamma with the IKK complex, blocked LPS-induced Cav-1 mRNA and protein expression. Knockdown of NF-kappaB subunit p65/RelA expression with small interfering RNA also prevented LPS-induced Cav-1 expression. Caveolae open to the apical and basal plasmalemma of endothelial cells increased 2-4-fold within 4 h of LPS exposure. IKK-NBD peptide markedly reduced the LPS-induced increase in the number of caveolae as well as transendothelial albumin permeability. These observations were recapitulated in mouse studies in which IKK-NBD peptide prevented Cav-1 expression and interfered with the increase in lung microvessel permeability induced by LPS. Thus, LPS mediates NF-kappaB-dependent Cav-1 expression that results in increased caveolae number and thereby contributes to the mechanism of increased transendothelial albumin permeability. PMID:18077459

  17. A Minimalistic Resource Allocation Model to Explain Ubiquitous Increase in Protein Expression with Growth Rate

    PubMed Central

    Keren, Leeat; Segal, Eran; Milo, Ron

    2016-01-01

    Most proteins show changes in level across growth conditions. Many of these changes seem to be coordinated with the specific growth rate rather than the growth environment or the protein function. Although cellular growth rates, gene expression levels and gene regulation have been at the center of biological research for decades, there are only a few models giving a base line prediction of the dependence of the proteome fraction occupied by a gene with the specific growth rate. We present a simple model that predicts a widely coordinated increase in the fraction of many proteins out of the proteome, proportionally with the growth rate. The model reveals how passive redistribution of resources, due to active regulation of only a few proteins, can have proteome wide effects that are quantitatively predictable. Our model provides a potential explanation for why and how such a coordinated response of a large fraction of the proteome to the specific growth rate arises under different environmental conditions. The simplicity of our model can also be useful by serving as a baseline null hypothesis in the search for active regulation. We exemplify the usage of the model by analyzing the relationship between growth rate and proteome composition for the model microorganism E.coli as reflected in recent proteomics data sets spanning various growth conditions. We find that the fraction out of the proteome of a large number of proteins, and from different cellular processes, increases proportionally with the growth rate. Notably, ribosomal proteins, which have been previously reported to increase in fraction with growth rate, are only a small part of this group of proteins. We suggest that, although the fractions of many proteins change with the growth rate, such changes may be partially driven by a global effect, not necessarily requiring specific cellular control mechanisms. PMID:27073913

  18. A Minimalistic Resource Allocation Model to Explain Ubiquitous Increase in Protein Expression with Growth Rate.

    PubMed

    Barenholz, Uri; Keren, Leeat; Segal, Eran; Milo, Ron

    2016-01-01

    Most proteins show changes in level across growth conditions. Many of these changes seem to be coordinated with the specific growth rate rather than the growth environment or the protein function. Although cellular growth rates, gene expression levels and gene regulation have been at the center of biological research for decades, there are only a few models giving a base line prediction of the dependence of the proteome fraction occupied by a gene with the specific growth rate. We present a simple model that predicts a widely coordinated increase in the fraction of many proteins out of the proteome, proportionally with the growth rate. The model reveals how passive redistribution of resources, due to active regulation of only a few proteins, can have proteome wide effects that are quantitatively predictable. Our model provides a potential explanation for why and how such a coordinated response of a large fraction of the proteome to the specific growth rate arises under different environmental conditions. The simplicity of our model can also be useful by serving as a baseline null hypothesis in the search for active regulation. We exemplify the usage of the model by analyzing the relationship between growth rate and proteome composition for the model microorganism E.coli as reflected in recent proteomics data sets spanning various growth conditions. We find that the fraction out of the proteome of a large number of proteins, and from different cellular processes, increases proportionally with the growth rate. Notably, ribosomal proteins, which have been previously reported to increase in fraction with growth rate, are only a small part of this group of proteins. We suggest that, although the fractions of many proteins change with the growth rate, such changes may be partially driven by a global effect, not necessarily requiring specific cellular control mechanisms. PMID:27073913

  19. Chronic neonatal nicotine exposure increases mRNA expression of neurotrophic factors in the postnatal rat hippocampus.

    PubMed

    Son, Jong-Hyun; Winzer-Serhan, Ursula H

    2009-06-30

    Nicotine, the psychoactive ingredient in tobacco, can be neuroprotective but the mechanism is unknown. In the adult hippocampus, chronic nicotine can increase expression of growth factors which could contribute to nicotine's neuroprotective effects. During development, nicotine could also increase expression of neurotrophic factors. Therefore, we determined whether chronic neonatal nicotine (CNN) exposure increased mRNA expression levels of brain-derived neurotrophic factor (BDNF), nerve-growth factor (NGF), neurotrophin-3 (NT-3), fibroblast growth factor-2 (FGF-2), and insulin-like growth factor-1 (IGF-1). Nicotine (6 mg/kg/day in milk formula) or milk formula (controls) were delivered in three daily doses via oral gastric intubation to rat pups from postnatal day (P)1 to P8, and then sacrificed. Brains were processed for in situ hybridization using specific (35)S-labeled cRNA probes. At P8, CNN had a significant stimulant treatment effect on the expression of BDNF, FGF-2, NT-3 and IGF-1 [p<0.01], but not NGF. Specifically, BDNF mRNA expression, detected in CA1, CA3 stratum (s.) pyramidal and granule cell layer of the dentate gyrus (DG), was increased by 27.4%, 23.26% and 27.3%, respectively. FGF-2 mRNA expression, detected in neurons and astrocytes in CA1 s. radiatum, CA2 and CA3 s. pyramidale, and molecular layer of the DG, was increased by 34.0%, 8.9%, 31.0% and 23.1%, respectively. NT-3 mRNA expression in CA2 s. pyramidale was increased by 80.0%, and CNN increased the number of IGF-1-expressing cells in CA1 (18.0%), CA3 (20.9%) and DG (17.7%). Thus, nicotine exposure during early postnatal development differentially up-regulated expression of neurotrophic factor mRNAs in the hippocampus, which could increase neurotrophic tone and alter developmental processes. PMID:19410565

  20. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression.

    PubMed Central

    van der Krol, A R; Mur, L A; Beld, M; Mol, J N; Stuitje, A R

    1990-01-01

    To evaluate the effect of increased expression of genes involved in flower pigmentation, additional dihydroflavonol-4-reductase (DFR) or chalcone synthase (CHS) genes were transferred to petunia. In most transformants, the increased expression had no measurable effect on floral pigmentation. Surprisingly, however, in up to 25% of the transformants, a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression, respectively, was observed. This phenomenon was obtained with both chimeric gene constructs and intact CHS genomic clones. The reduction in gene expression was independent of the promoter driving transcription of the transgene and involved both the endogenous gene and the homologous transgene. The gene-specific collapse in expression was obtained even after introduction of only a single gene copy. The similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits. PMID:2152117

  1. Student Freedom of Expression: A Limited First Amendment Right. Research Bulletin, Vol. 1, No. 3.

    ERIC Educational Resources Information Center

    Mundt, Whitney R.

    The Supreme Court has ruled in several cases that schools must formulate rules and regulations that do not infringe on a student's right to free and unrestricted expression as guaranteed to them under the First Amendment. In two separate cases, the Court decided that students may wear buttons or armbands that express a particular position on an…

  2. Gene expression in bovine mammary gland in response to increased milking frequency as determined by microarray and SAGE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcript profiling was performed by Affymetrix microarray analysis and SAGE to characterize changes in gene expression in the bovine mammary gland in response to 4× versus 2× daily milking during the first week of lactation. These changes in gene expression may contribute to the increased milk pro...

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

    SciTech Connect

    Li, Ruizhao; Zhang, Li; Shi, Wei; Zhang, Bin; Liang, Xinling; Liu, Shuangxin; Wang, Wenjian

    2013-04-15

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

  4. NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression.

    PubMed

    Spina, Anna; Montella, Roberta; Liccardo, Davide; De Rosa, Alfredo; Laino, Luigi; Mitsiadis, Thimios A; La Noce, Marcella

    2016-01-01

    Human dental pulp stem cells (hDPSCs), selected from the stromal-vascular fraction of dental pulp, are ecto-mesenchymal stem cells deriving from neural crests, successfully used in human bone tissue engineering. For their use in human therapy GMP procedures are required. For instance, the use of fetal bovine serum (FBS) is strongly discouraged in clinical practice due to its high risk of prions and other infections for human health. Alternatively, clinical grade sera have been suggested, including the New Zealand FBS (NZ-FBS). Therefore, the aim of this study was to evaluate the behavior of hDPSCs expanded in culture medium containing NZ-FBS. Since it was widely demonstrated hDPSCs display relevant capabilities to differentiate into osteogenic and angiogenic lineages, we performed a comparative study to assess if these features are also retained by cultivating the cells with a safer serum never tested on this cell line. hDPSCs were grown using NZ-FBS and conventional (C-FBS) for 7, 14, and 21 days, in both 2D and 3D cultures. Growth curves, expression of bone-related markers, calcification and angiogenesis were evaluated. NZ-FBS induced significant cell growth with respect to C-FBS and promoted an earlier increase expression of osteogenic markers, in particular of those involved in the formation of mineralized matrix (BSP and OPN) within 14 days. In addition, hDPSCs cultured in presence of NZ-FBS were found to produce higher mRNA levels of the angiogenic factors, such as VEGF and PDGFA. Taken together, our results highlight that hDPSCs proliferate, enhance their osteogenic commitment and increase angiogenic factors in NZ-FBS containing medium. These features have also been found when hDPSC were seeded on the clinical-grade collagen I scaffold (Bio-Gide®), leading to the conclusion that for human therapy some procedures and above all the use of GMP-approved materials have no negative impact. PMID:27594842

  5. NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression

    PubMed Central

    Spina, Anna; Montella, Roberta; Liccardo, Davide; De Rosa, Alfredo; Laino, Luigi; Mitsiadis, Thimios A.; La Noce, Marcella

    2016-01-01

    Human dental pulp stem cells (hDPSCs), selected from the stromal-vascular fraction of dental pulp, are ecto-mesenchymal stem cells deriving from neural crests, successfully used in human bone tissue engineering. For their use in human therapy GMP procedures are required. For instance, the use of fetal bovine serum (FBS) is strongly discouraged in clinical practice due to its high risk of prions and other infections for human health. Alternatively, clinical grade sera have been suggested, including the New Zealand FBS (NZ-FBS). Therefore, the aim of this study was to evaluate the behavior of hDPSCs expanded in culture medium containing NZ-FBS. Since it was widely demonstrated hDPSCs display relevant capabilities to differentiate into osteogenic and angiogenic lineages, we performed a comparative study to assess if these features are also retained by cultivating the cells with a safer serum never tested on this cell line. hDPSCs were grown using NZ-FBS and conventional (C-FBS) for 7, 14, and 21 days, in both 2D and 3D cultures. Growth curves, expression of bone-related markers, calcification and angiogenesis were evaluated. NZ-FBS induced significant cell growth with respect to C-FBS and promoted an earlier increase expression of osteogenic markers, in particular of those involved in the formation of mineralized matrix (BSP and OPN) within 14 days. In addition, hDPSCs cultured in presence of NZ-FBS were found to produce higher mRNA levels of the angiogenic factors, such as VEGF and PDGFA. Taken together, our results highlight that hDPSCs proliferate, enhance their osteogenic commitment and increase angiogenic factors in NZ-FBS containing medium. These features have also been found when hDPSC were seeded on the clinical-grade collagen I scaffold (Bio-Gide®), leading to the conclusion that for human therapy some procedures and above all the use of GMP-approved materials have no negative impact. PMID:27594842

  6. Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

    PubMed

    Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

    2016-06-01

    Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue. PMID:27163741

  7. Cucumber Mosaic Virus Movement Protein Severs Actin Filaments to Increase the Plasmodesmal Size Exclusion Limit in Tobacco[W][OA

    PubMed Central

    Su, Shengzhong; Liu, Zhaohui; Chen, Cheng; Zhang, Yan; Wang, Xu; Zhu, Lei; Miao, Long; Wang, Xue-Chen; Yuan, Ming

    2010-01-01

    Plant viral movement proteins (MPs) enable viruses to pass through cell walls by increasing the size exclusion limit (SEL) of plasmodesmata (PD). Here, we report that the ability of Cucumber mosaic virus (CMV) MP to increase the SEL of the PD could be inhibited by treatment with the actin filament (F-actin)–stabilizing agent phalloidin but not by treatment with the F-actin–destabilizing agent latrunculin A. In vitro studies showed that CMV MP bound globular and F-actin, inhibited actin polymerization, severed F-actin, and participated in plus end capping of F-actin. Analyses of two CMV MP mutants, one with and one without F-actin severing activities, demonstrated that the F-actin severing ability was required to increase the PD SEL. Furthermore, the Tobacco mosaic virus MP also exhibited F-actin severing activity, and its ability to increase the PD SEL was inhibited by treatment with phalloidin. Our data provide evidence to support the hypothesis that F-actin severing is required for MP-induced increase in the SEL of PD. This may have broad implications in the study of the mechanisms of actin dynamics that regulate cell-to-cell transport of viral and endogenous proteins. PMID:20435906

  8. IRAK-M expression limits dendritic cell activation and proinflammatory cytokine production in response to Helicobacter pylori.

    PubMed

    Shiu, Jessica; Czinn, Steven J; Kobayashi, Koichi S; Sun, Yezhou; Blanchard, Thomas G

    2013-01-01

    Helicobacter pylori (H. pylori) infects the gastric mucosa and persists for the life of the host. Bacterial persistence may be due to the induction of regulatory T cells (Tregs) whichmay have protective effects against other diseases such as asthma. It has been shown that H. pylori modulates the T cell response through dendritic cell reprogramming but the molecular pathways involved are relatively unknown. The goal of this study was to identify critical elements of dendritic cell (DC) activation and evaluate potential influence on immune activation. Microarray analysis was used to demonstrate limited gene expression changes in H. pylori stimulated bone marrow derived DCs (BMDCs) compared to the BMDCs stimulated with E. coli. IRAK-M, a negative regulator of TLR signaling, was upregulated and we selectedit for investigation of its role in modulating the DC and T cell responses. IRAK-M(-/-) and wild type BMDC were compared for their response to H. pylori. Cells lacking IRAK-M produced significantly greater amounts of proinflammatory MIP-2 and reduced amounts of immunomodulatory IL-10 than wild type BMDC. IRAK-M(-/-) cells also demonstrated increased MHC II expression upon activation. However, IRAK-M(-/-) BMDCs were comparable to wild type BMDCs in inducing T-helper 17 (TH17) and Treg responses as demonstrated in vitro using BMDC CD4+ T cells co-culture assays,and in vivo though the adoptive transfer of CD4(+) FoxP3-GFP T cells into H. pylori infected IRAK-M(-/-) mice. These results suggest that H. pylori infection leads to the upregulation of anti-inflammatory molecules like IRAK-M and that IRAK-M has a direct impact on innate functions in DCs such as cytokine and costimulation molecule upregulation but may not affect T cell skewing. PMID:23776703

  9. RNase L Attenuates Mitogen-stimulated Gene Expression via Transcriptional and Post-transcriptional Mechanisms to Limit the Proliferative Response*

    PubMed Central

    Brennan-Laun, Sarah E.; Li, Xiao-Ling; Ezelle, Heather J.; Venkataraman, Thiagarajan; Blackshear, Perry J.; Wilson, Gerald M.; Hassel, Bret A.

    2014-01-01

    The cellular response to mitogens is tightly regulated via transcriptional and post-transcriptional mechanisms to rapidly induce genes that promote proliferation and efficiently attenuate their expression to prevent malignant growth. RNase L is an endoribonuclease that mediates diverse antiproliferative activities, and tristetraprolin (TTP) is a mitogen-induced RNA-binding protein that directs the decay of proliferation-stimulatory mRNAs. In light of their roles as endogenous proliferative constraints, we examined the mechanisms and functional interactions of RNase L and TTP to attenuate a mitogenic response. Mitogen stimulation of RNase L-deficient cells significantly increased TTP transcription and the induction of other mitogen-induced mRNAs. This regulation corresponded with elevated expression of serum-response factor (SRF), a master regulator of mitogen-induced transcription. RNase L destabilized the SRF transcript and formed a complex with SRF mRNA in cells providing a mechanism by which RNase L down-regulates SRF-induced genes. TTP and RNase L proteins interacted in cells suggesting that RNase L is directed to cleave TTP-bound RNAs as a mechanism of substrate specificity. Consistent with their concerted function in RNA turnover, the absence of either RNase L or TTP stabilized SRF mRNA, and a subset of established TTP targets was also regulated by RNase L. RNase L deficiency enhanced mitogen-induced proliferation demonstrating its functional role in limiting the mitogenic response. Our findings support a model of feedback regulation in which RNase L and TTP target SRF mRNA and SRF-induced transcripts. Accordingly, meta-analysis revealed an enrichment of RNase L and TTP targets among SRF-regulated genes suggesting that the RNase L/TTP axis represents a viable target to inhibit SRF-driven proliferation in neoplastic diseases. PMID:25301952

  10. RNase L attenuates mitogen-stimulated gene expression via transcriptional and post-transcriptional mechanisms to limit the proliferative response.

    PubMed

    Brennan-Laun, Sarah E; Li, Xiao-Ling; Ezelle, Heather J; Venkataraman, Thiagarajan; Blackshear, Perry J; Wilson, Gerald M; Hassel, Bret A

    2014-11-28

    The cellular response to mitogens is tightly regulated via transcriptional and post-transcriptional mechanisms to rapidly induce genes that promote proliferation and efficiently attenuate their expression to prevent malignant growth. RNase L is an endoribonuclease that mediates diverse antiproliferative activities, and tristetraprolin (TTP) is a mitogen-induced RNA-binding protein that directs the decay of proliferation-stimulatory mRNAs. In light of their roles as endogenous proliferative constraints, we examined the mechanisms and functional interactions of RNase L and TTP to attenuate a mitogenic response. Mitogen stimulation of RNase L-deficient cells significantly increased TTP transcription and the induction of other mitogen-induced mRNAs. This regulation corresponded with elevated expression of serum-response factor (SRF), a master regulator of mitogen-induced transcription. RNase L destabilized the SRF transcript and formed a complex with SRF mRNA in cells providing a mechanism by which RNase L down-regulates SRF-induced genes. TTP and RNase L proteins interacted in cells suggesting that RNase L is directed to cleave TTP-bound RNAs as a mechanism of substrate specificity. Consistent with their concerted function in RNA turnover, the absence of either RNase L or TTP stabilized SRF mRNA, and a subset of established TTP targets was also regulated by RNase L. RNase L deficiency enhanced mitogen-induced proliferation demonstrating its functional role in limiting the mitogenic response. Our findings support a model of feedback regulation in which RNase L and TTP target SRF mRNA and SRF-induced transcripts. Accordingly, meta-analysis revealed an enrichment of RNase L and TTP targets among SRF-regulated genes suggesting that the RNase L/TTP axis represents a viable target to inhibit SRF-driven proliferation in neoplastic diseases. PMID:25301952

  11. Increased metallothionein gene expression, zinc, and zinc-dependent resistance to apoptosis in circulating monocytes during HIV viremia

    PubMed Central

    Raymond, Andrea D.; Gekonge, Bethsebah; Giri, Malavika S.; Hancock, Aidan; Papasavvas, Emmanouil; Chehimi, Jihed; Kossevkov, Andrew V.; Nicols, Calen; Yousef, Malik; Mounzer, Karam; Shull, Jane; Kostman, Jay; Showe, Louise; Montaner, Luis J.

    2010-01-01

    Circulating monocytes exhibit an apoptotic resistance phenotype during HIV viremia in association with increased MT expression. MTs are known to play an important role in zinc metabolism and immune function. We now show, in a cross-sectional study using peripheral monocytes, that expression of MT1 isoforms E, G, H, and X is increased significantly in circulating monocyte cells from HIV+ subjects during chronic viremic episodes as compared with uninfected subjects. This increase in expression is also observed during acute viremia following interruption of suppressive ART. Circulating monocytes from HIV+ donors were also found to have elevated zinc importer gene Zip8 expression in conjunction with elevated intracellular zinc levels in contrast to CD4+T-lymphocytes. In vitro HIV-1 infection studies with elutriated MDM confirm a direct relation between HIV-1 infection and increased MDM MT1 (isoform G) gene expression and increased intracellular zinc levels. A direct link between elevated zinc levels and apoptosis resistance was established using a cell-permeable zinc chelator TPEN, which reversed apoptosis resistance effectively in monocytes from HIV-infected to levels comparable with uninfected controls. Taken together, increases in MT gene expression and intracellular zinc levels may contribute directly to maintenance of an immune-activated monocyte by mediating an increased resistance to apoptosis during active HIV-1 viremia. PMID:20551211

  12. NF kappaB expression increases and CFTR and MUC1 expression decreases in the endometrium of infertile patients with hydrosalpinx: a comparative study

    PubMed Central

    2012-01-01

    Background Hydrosalpinx are associated with infertility, due to reduced rates of implantation and increased abortion rates. The aims of this study were to investigate the expression of cystic fibrosis transmembrane conductance regulator (CFTR), nuclear factor kappa B (NF KappaB) and mucin-1 (MUC-1), and analyze the correlation between the expression of CFTR and NF KappaB or MUC1, in the endometrium of infertile women with and without hydrosalpinx. Methods Thirty-one infertile women with laparoscopy-confirmed unilateral or bilateral hydrosalpinx and 20 infertile women without hydrosalpinx or pelvic inflammatory disease (control group) were recruited. Endometrial biopsy samples were collected and the expression of CFTR, NF KappaB and MUC1 were analyzed using immunohistochemistry and quantitative real-time PCR. Results CFTR, NF KappaB and MUC1 mRNA and protein expression tended to increase in the secretory phase compared to the proliferative phase in both groups; however, these differences were not significantly different. The endometrium of infertile patients with hydrosalpinx had significantly higher NF KappaB mRNA and protein expression, and significantly lower CFTR and MUC1 mRNA and protein expression, compared to control infertile patients. A positive correlation was observed between CFTR and MUC1 mRNA expression (r = 0.65, P < 0.05); a negative correlation was observed between CFTR mRNA and NF KappaB mRNA expression (r = −0.59, P < 0.05). Conclusions Increased NF KappaB expression and decreased CFTR and MUC1 expression in the endometrium of infertile patients with hydrosalpinx reinforce the involvement of a molecular mechanism in the regulation of endometrial receptivity. PMID:23061681

  13. Building an atlas of gene expression driving kidney development: pushing the limits of resolution.

    PubMed

    Potter, S Steven; Brunskill, Eric W

    2014-04-01

    Changing gene expression patterns is the essential driver of developmental processes. Growth factors, micro-RNAs, long intergenic noncoding RNAs, and epigenetic marks, such as DNA methylation and histone modifications, all work by impacting gene expression. The key features of developing cells, including their ability to communicate with others, are defined primarily by their gene-expression profiles. It is therefore clear that a gene-expression atlas of the developing kidney can provide a useful tool for the developmental nephrology research community. Toward this end, the GenitoUrinary Development Molecular Anatomy Project (GUDMAP) consortium has worked to create an atlas of the changing gene-expression patterns that drive kidney development. In this article, the global gene-expression profiling strategies of GUDMAP are reviewed. The initial work used laser-capture microdissection to purify multiple compartments of the developing kidney, including cap mesenchyme, renal vesicle, S-shaped bodies, proximal tubules, and more, which were then gene-expression profiled using microarrays. Resolution of the atlas was then improved by using transgenic mice with specific cell types labeled with green fluorescent protein (GFP), allowing their purification and profiling. In addition, RNA-Seq replaced microarrays. Currently, the atlas is being pushed to the single-cell resolution using microfluidic approaches that allow high-throughput RNA-Seq analysis of hundreds of individual cells. Results can identify novel types of cells and define interesting heterogeneities present within cell populations. PMID:23996451

  14. Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival.

    PubMed

    Olivera, A; Kohama, T; Edsall, L; Nava, V; Cuvillier, O; Poulton, S; Spiegel, S

    1999-11-01

    Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of

  15. Suppression of laminin-5 expression leads to increased motility, tumorigenicity, and invasion

    SciTech Connect

    Yuen Hengwai; Ziober, Amy F.; Gopal, Pallavi; Nasrallah, Ilya; Falls, Erica M.; Meneguzzi, Guerrino; Ang, Hwee-Quan; Ziober, Barry L. . E-mail: bziober@mail.med.upenn.edu

    2005-09-10

    Laminin-5 (Ln-5) is expressed in several human carcinomas and hypothesized to contribute to tumor invasion. To understand the role of Ln-5 in human cancers, we stably delivered small interfering RNAs (siRNAs) directed against the Ln-5 {gamma}2 chain into JHU-022-SCC cells (022), a non-invasive oral squamous cell carcinoma (OSCC) cell line which secretes Ln-5. Lysates from {gamma}2 siRNA cells (022-si{gamma}2) had nearly undetectable levels of the {gamma}2 chain while the {alpha}3 and {beta}3 subunits of Ln-5 remained unchanged compared to parental and control. In conditioned medium from 022-si{gamma}2 cells, the {gamma}2 chain and the Ln-5 heterotrimer were barely detectable, similar to an invasive OSCC cell line. Conditioned medium from 022-si{gamma}2 cells contained less {alpha}3 and {beta}3 subunits than both parental and control. Although the proliferation and adhesive properties of the 022-si{gamma}2 cells remained similar to parental and control cells, 022-si{gamma}2 cells showed increased detachment and a fibroblastic morphology similar to invasive cells. Moreover, migration, in vitro invasion, and in vivo tumorigenicity were enhanced in 022-si{gamma}2 cells. Our results suggest that the Ln-5 {gamma}2 chain regulates the secretion of the {alpha}3 and {beta}3 subunits. More importantly, suppression of Ln-5 results in a phenotype that is representative of invasive tumor cells.

  16. Increased COUP-TFII expression in adult hearts induces mitochondrial dysfunction resulting in heart failure.

    PubMed

    Wu, San-Pin; Kao, Chung-Yang; Wang, Leiming; Creighton, Chad J; Yang, Jin; Donti, Taraka R; Harmancey, Romain; Vasquez, Hernan G; Graham, Brett H; Bellen, Hugo J; Taegtmeyer, Heinrich; Chang, Ching-Pin; Tsai, Ming-Jer; Tsai, Sophia Y

    2015-01-01

    Mitochondrial dysfunction and metabolic remodelling are pivotal in the development of cardiomyopathy. Here, we show that myocardial COUP-TFII overexpression causes heart failure in mice, suggesting a causal effect of elevated COUP-TFII levels on development of dilated cardiomyopathy. COUP-TFII represses genes critical for mitochondrial electron transport chain enzyme activity, oxidative stress detoxification and mitochondrial dynamics, resulting in increased levels of reactive oxygen species and lower rates of oxygen consumption in mitochondria. COUP-TFII also suppresses the metabolic regulator PGC-1 network and decreases the expression of key glucose and lipid utilization genes, leading to a reduction in both glucose and oleate oxidation in the hearts. These data suggest that COUP-TFII affects mitochondrial function, impairs metabolic remodelling and has a key role in dilated cardiomyopathy. Last, COUP-TFII haploinsufficiency attenuates the progression of cardiac dilation and improves survival in a calcineurin transgenic mouse model, indicating that COUP-TFII may serve as a therapeutic target for the treatment of dilated cardiomyopathy. PMID:26356605

  17. Inhibition of PP2A by LIS1 increases HIV-1 gene expression

    PubMed Central

    Epie, Nicolas; Ammosova, Tatyana; Turner, Willie; Nekhai, Sergei

    2006-01-01

    Background Lissencephaly is a severe brain malformation in part caused by mutations in the LIS1 gene. LIS1 interacts with microtubule-associated proteins, and enhances transport of microtubule fragments. Previously we showed that LIS1 interacts with HIV-1 Tat protein and that this interaction was mediated by WD40 domains of LIS1. In the present study, we analyze the effect of LIS1 on Tat-mediated transcription of HIV-1 LTR. Results Tat-mediated HIV-1 transcription was upregulated in 293 cells transfected with LIS1 expression vector. The WD5 but not the N-terminal domain of LIS1 increases Tat-dependent HIV-1 transcription. The effect of LIS1 was similar to the effect of okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). We then analyzed the effect of LIS1 on the activity of PP2A in vitro. We show that LIS1 and its isolated WD5 domain but not the N-terminal domain of LIS1 blocks PP2A activity. Conclusion Our results show that inhibition of PP2A by LIS1 induces HIV-1 transcription. Our results also point to a possibility that LIS1 might function in the cells as a yet unrecognized regulatory subunit of PP2A. PMID:17018134

  18. Increased COUP-TFII expression in adult hearts induces mitochondrial dysfunction resulting in heart failure

    PubMed Central

    Wu, San-Pin; Kao, Chung-Yang; Wang, Leiming; Creighton, Chad J.; Yang, Jin; Donti, Taraka R.; Harmancey, Romain; Vasquez, Hernan G.; Graham, Brett H.; Bellen, Hugo J.; Taegtmeyer, Heinrich; Chang, Ching-Pin; Tsai, Ming-Jer; Tsai, Sophia Y.

    2015-01-01

    Mitochondrial dysfunction and metabolic remodelling are pivotal in the development of cardiomyopathy. Here, we show that myocardial COUP-TFII overexpression causes heart failure in mice, suggesting a causal effect of elevated COUP-TFII levels on development of dilated cardiomyopathy. COUP-TFII represses genes critical for mitochondrial electron transport chain enzyme activity, oxidative stress detoxification and mitochondrial dynamics, resulting in increased levels of reactive oxygen species and lower rates of oxygen consumption in mitochondria. COUP-TFII also suppresses the metabolic regulator PGC-1 network and decreases the expression of key glucose and lipid utilization genes, leading to a reduction in both glucose and oleate oxidation in the hearts. These data suggest that COUP-TFII affects mitochondrial function, impairs metabolic remodelling and has a key role in dilated cardiomyopathy. Last, COUP-TFII haploinsufficiency attenuates the progression of cardiac dilation and improves survival in a calcineurin transgenic mouse model, indicating that COUP-TFII may serve as a therapeutic target for the treatment of dilated cardiomyopathy. PMID:26356605

  19. Increased Acetylcholinesterase Expression in Bumble Bees During Neonicotinoid-Coated Corn Sowing.

    PubMed

    Samson-Robert, Olivier; Labrie, Geneviève; Mercier, Pierre-Luc; Chagnon, Madeleine; Derome, Nicolas; Fournier, Valérie

    2015-01-01

    While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChE) activity in honey bees at sublethal doses. High AChE levels may therefore act as a biomarker of exposure to neonicotinoids. This two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥ 3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoid-treated seeds), and maintained for the duration of corn sowing. As determined by Real Time qPCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. To our knowledge, this is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication. PMID:26223214

  20. Increased Acetylcholinesterase Expression in Bumble Bees During Neonicotinoid-Coated Corn Sowing

    PubMed Central

    Samson-Robert, Olivier; Labrie, Geneviève; Mercier, Pierre-Luc; Chagnon, Madeleine; Derome, Nicolas; Fournier, Valérie

    2015-01-01

    While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChE) activity in honey bees at sublethal doses. High AChE levels may therefore act as a biomarker of exposure to neonicotinoids. This two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoid-treated seeds), and maintained for the duration of corn sowing. As determined by Real Time qPCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. To our knowledge, this is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication. PMID:26223214

  1. Precise digit use increases the expression of handedness in Colombian spider monkeys (Ateles fusciceps rufiventris).

    PubMed

    Nelson, Eliza L; Boeving, Emily R

    2015-12-01

    Decades of research on the hand use patterns of nonhuman primates can be aptly summarized by the following phrase: measurement matter